**Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria**

Hitoshi Miyasaka, Hiroshi Okuhata, Satoshi Tanaka, Takuo Onizuka and Hideo Akiyama

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54705

### **1. Introduction**

Global warming is the urgent issue of our time, and the carbon dioxide is a greenhouse gas of the major concern. There are various research activities for carbon dioxide mitigation, such as CO2 recovery from the flue gas of industrial sites, underground and undersea CO2 storage, and also chemical/biological conversion of CO2 into the industrial materials [1].

On-site CO2 fixation by bioprocess is based on the activities of photosynthetic organisms. The fixation of CO2 by photosynthetic microorganisms can be an efficient system for the CO2 mitigation, but one of the major problems of this system is the effective utilization of the fixed biomass. The biomass produced by photosynthetic microorganisms must be utilized as a resource, or it will be easily degraded by microorganisms into CO2 again. Cyanobacteria are procaryotic photosynthetic microorganisms and can provide a simple genetic transformation system for the production of useful materials from CO2. We have established an efficient vector-promoter system for the introduction and expression of foreign genes in the marine cyanobacterium *Synechococcus sp.* PCC7002, and examined the production of biodegradable plastic, polyhydroxyalkanoate (PHA), by genetically engineered cyanobacteria. The PHA is a biopolymer accumulated by various microorganisms as reserves of carbon and reducing equivalents. PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid mono‐ mers (Figure 1), have physical properties similar to those of polyethylene, and can replace the chemical plastics in some applications, such as disposable bulk materials in packing films, containers, and paper coatings [2]. PHA applications as implant biomaterials, drug delivery carrier, and biofuel have also been investigated [3]. The commercial mass production of PHA from corn sugar by using the genetically enigineered microorganism was started in 2009 in the United State and China [3].

© 2013 Miyasaka et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Miyasaka et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Japan

Japan

**Figure 1 Chemical structure of PHAs**

DUMMY TEXT, KOJI NIJE IME KNJIGE

2Toray Research Center, Inc., Kamakura

miyasaka.hitoshi@a4.kepco.co.jp

**1. Introduction** 

**Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria** 

Global warming is the urgent issue of our time, and the carbon dioxide is a greenhouse gas of the major concern. There are various research activities for carbon dioxide mitigation, such as CO2 recovery from the flue gas of industrial sites, underground and

On-site CO2 fixation by bioprocess is based on the activities of photosynthetic organisms. The fixation of CO2 by photosynthetic microorganisms can be an efficient system for the CO2 mitigation, but one of the major problems of this system is the effective utilization of the fixed biomass. The biomass produced by photosynthetic microorganisms must be utilized as a resource, or it will be easily degraded by microorganisms into CO2 again. Cyanobacteria are procaryotic photosynthetic microorganisms and can provide a simple genetic transformation system for the production of useful materials from CO2. We have established an efficient vector-promoter system for the introduction and expression of foreign genes in the marine cyanobacterium *Synechococcus sp.* PCC7002, and examined the production of biodegradable plastic, polyhydroxyalkanoate (PHA), by genetically engineered cyanobacteria. The PHA is a biopolymer accumulated by various microorganisms as reserves of carbon and reducing equivalents.

For the construction of a shuttle-vector between *E. coli* and *Synechococcus sp.* PCC7002, we isolated and characterized the smallest endogenous plasmid pAQ1 of this cyanobacterium [6, 7]. The plasmid pAQ1 is 4809 bp long, and has four open reading frames (ORFs): ORF943, ORF64, ORF71, and ORF93 (numbers show putative amino acid number). The construction of the shuttle-vector was done by digesting pAQ1 plasmid and bacterial pUC19 plasmid with restriction enzymes which cleave each plasmid at a unique site, and by ligating the linearized plasmids. The plasmid pUC19 and the plasmid pAQ1 were linearized by *Sma*I and *Stu*I digestions, respectively, and were ligated to generate the shuttle-vector pAQJ6 (Figure 2; both *Sma*I and *Stu*I are blunt-end forming restriction enzymes). The effect of the four ORFs on the transformation efficiency was examined, and ORF943 was found to be important for the maintenance of the shuttle-vector. From this result, the simplified shuttle-vector pAQJ4 with the full ORF943 was

199

those of polyethylene, and can replace the chemical plastics in some applications, such as disposable bulk materials in packing films, containers, and paper coatings [2]. PHA applications as implant biomaterials, drug delivery carrier, and biofuel have also been investigated [3]. The commercial mass production of PHA from corn sugar by using the genetically enigineered

designed (Figure 2).

*SmaI HindIII*

*SmaI StuI*

*EcoRI SacI*

Ligation

*SacI*

*SacI* (partial digestion)

Ligation

*SacI HindIII*

**pAQJ4** ORF943

Figure 2. Construction of shuttle-vector between

*SalI HindIII*

the shuttle-vector pAQJ4 was about 4 x 105

**Figure 2.** Construction of shuttle-vector between *E. coli* and *Synec hococcus* sp. PCC7002

*EcoRI*

*E. coli* and *Synechococcus*sp. PCC7002

The stability of the prototype shuttle-vector, pAQJ6, in cyanobacterial cells was relatively low, but that of the simplified shuttle-vector, pAQJ4, was much improved; this vector could be stably retained in cyanobacterial cells after 100 generations of growth without antibiotics selection [8]. This is probably because that there are several hot spots for the homologous recombination between endogenous pAQ1 plasmid and pAQJ6 vector, and these hot spots might have been eliminated in the simplified pAQJ4 vector. The transformation efficiency of

*SacI*

*SacI SacI*

*SacI SacI*

**pAQJ6**

*Amp*

*SacI SacI HindIII*

ORF71

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

*StuI*

ORF93

*SacI*

http://dx.doi.org/10.5772/54705

*SacI*

ORF64

*SacI SacI*

*HindIII SacI*

ORF943

**pAQ1**  (4809 bp)

**pUC19**  (2686bp)

*HindIII*

*EcoRI SacI*

Figure 2. Construction of shuttle-vector between *E. coli* and *Synec hococcus* sp. PCC7002

(cfu / μg DNA), when we transformed 4 x 107

cDNA libraries using cyanobacteria as host cells.

cyanobacterial cells with 0.3 μg (0.1 pmol) of pAQJ4 vector in 1 ml solution. This transformation efficiency was 10 to 100 times higher than those of the shuttle-vectors for this cyanobacterium

The stability of the prototype shuttle-vector, pAQJ6, in cyanobacterial cells was relatively low, but that of the simplified shuttlevector, pAQJ4, was much improved; this vector could be stably retained in cyanobacterial cells after 100 generations of growth without antibiotics selection [8]. This is probably because that there are several hot spots for the homologous recombination between endogenous pAQ1 plasmid and pAQJ6 vector, and these hot spots might have been eliminated in the simplified pAQJ4 vector. The transformation efficiency of the shuttle-vector pAQJ4 was about 4 x 105 (cfu / g DNA), when we transformed 4 x 107 of cyanobacterial cells with 0.3 g (0.1 pmol) of pAQJ4 vector in 1 ml solution. This transformation efficiency was 10 to 100 times higher than those of the shuttle-vectors for this cyanobacterium previously reported [4, 9]. With this system, we can obtain several million of cyanobacterial transformant in one experiment, thus this shuttle-vector system can also be applied for the construction of

of

An example of the use of this shuttle-vector for cDNA library is the construction of cDNA library of the halotolerant marine green alga *Chlamydomonas* W80 for the isolation of anti-stress genes [10]. *C.* W80 shows a surprisingly high oxidative stress tolerance caused by methyl viologen (MV), which is reduced by the photosynthetic apparatus generating highly toxic superoxide (O2-

W80 tolerates up to 200 M of MV [11, 12], while other oxygen-evolving photosynthetic organisms such as higher plants, algae and cyanobacteria usually tolerate only less than 5 M of MV. This alga is a prominent genetic resource of anti-stress genes, and various unique anti-stress genes, such as ascorbate peroxidase [13], glutathione peroxidase [14], and the novel salt and cadmium stress related (*scsr*) gene [15], have been isolated from this alga. Using the cDNA library of *C.* W80 constructed in pAQJ4 shuttle-

). *C.*

saving the fossil fuel resource required for the petrochemical plastics production. In addition, the biodegradable plastics reduce

Hitoshi Miyasaka1, Hiroshi Okuhata1, Satoshi Tanaka1, Takuo Onizuka2 and Hideo Akiyama2

undersea CO2 storage, and also chemical/biological conversion of CO2 into the industrial materials [1].

1The Kansai Electric Power Co., Environmental Research Center, Seikacho

microorganism was started in 2009 in the United State and China [3].

**Figure 1.** Chemical structure of PHAs

Figure 1. Chemical structure of PHAs Assimilation and conversion of CO2 into the biodegradable plastics (biopolymers) by photosynthetic microorganisms is an ideal bioprocess because it converts CO2 directly into the useful bioplastics with solar energy. It also contributes to the low carbon society by substituting the environmentally unfriendly petroleum-based plastics with the carbon neutral bioplastics, and also by Assimilation and conversion of CO2 into the biodegradable plastics (biopolymers) by photo‐ synthetic microorganisms is an ideal bioprocess because it converts CO2 directly into the useful bioplastics with solar energy. It also contributes to the low carbon society by substituting the environmentally unfriendly petroleum-based plastics with the carbon neutral bioplastics, and also by saving the fossil fuel resource required for the petrochemical plastics production. In addition, the biodegradable plastics reduce the burden of plastics waste on landfills and the environment.

#### the burden of plastics waste on landfills and the environment. **2. Shuttle-vector construction**

### **2.1. Construction of a shuttle-vector between** *Escherichia coli* **and cyanobacteria**

**2. Shuttle-vector construction 2.1. Construction of a shuttle-vector between** *Escherichia coli* **and cyanobacteria**  Since most of the industrial CO2 emission sites locate in the seashore area in Japan, we choose a marine cyanobacterial strain for the fixation and utilization of CO2. The marine cyanobacterial strain *Synechococcus sp.* PCC7002 (*Agmenellum quadruplicatum* PR-6, ATCC 27264) [4] was obtained from the American Type Culture Collection, and cultured at 30 °C in medium A [5] under continuous illumination (50 μmol photons m-2 s-1) by bubbling with 1% CO2 in air.

Since most of the industrial CO2 emission sites locate in the seashore area in Japan, we choose a marine cyanobacterial strain for the fixation and utilization of CO2. The marine cyanobacterial strain *Synechococcus sp.* PCC7002 (*Agmenellum quadruplicatum* PR-6, ATCC 27264) [4] was obtained from the American Type Culture Collection, and cultured at 30 °C in medium A [5] under continuous illumination (50 mol photons m-2 s-1) by bubbling with 1% CO2 in air. For the construction of a shuttle-vector between *E. coli* and *Synechococcus sp.* PCC7002, we isolated and characterized the smallest endogenous plasmid pAQ1 of this cyanobacterium [6, 7]. The plasmid pAQ1 is 4809 bp long, and has four open reading frames (ORFs): ORF943, ORF64, ORF71, and ORF93 (numbers show putative amino acid number). The construction of the shuttle-vector was done by digesting pAQ1 plasmid and bacterial pUC19 plasmid with restriction enzymes which cleave each plasmid at a unique site, and by ligating the linearized plasmids. The plasmid pUC19 and the plasmid pAQ1 were linearized by *Sma*I and *Stu*I digestions, respectively, and were ligated to generate the shuttle-vector pAQJ6 (Figure 2; both *Sma*I and *Stu*I are blunt-end forming restriction enzymes). The effect of the four ORFs on the transformation efficiency was examined, and ORF943 was found to be important for the maintenance of the shuttle-vector. From this result, the simplified shuttle-vector pAQJ4 with the full ORF943 was designed (Figure 2).

PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), have physical properties similar to those of polyethylene, and can replace the chemical plastics in some applications, such as disposable bulk materials in packing restriction enzymes). The effect of the four ORFs on the transformation efficiency was examined, and ORF943 was found to be important for the maintenance of the shuttle-vector. From this result, the simplified shuttle-vector pAQJ4 with the full ORF943 was Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria http://dx.doi.org/10.5772/54705 199

For the construction of a shuttle-vector between *E. coli* and *Synechococcus sp.* PCC7002, we isolated and characterized the smallest endogenous plasmid pAQ1 of this cyanobacterium [6, 7]. The plasmid pAQ1 is 4809 bp long, and has four open reading frames (ORFs): ORF943, ORF64, ORF71, and ORF93 (numbers show putative amino acid number). The construction of the shuttle-vector was done by digesting pAQ1 plasmid and bacterial pUC19 plasmid with restriction enzymes which cleave each plasmid at a unique site, and by ligating the linearized plasmids. The plasmid pUC19 and the plasmid pAQ1 were linearized by *Sma*I and *Stu*I digestions, respectively, and were ligated to generate the shuttle-vector pAQJ6 (Figure 2; both *Sma*I and *Stu*I are blunt-end forming

cyanobacterial cells with 0.3 g (0.1 pmol) of pAQJ4 vector in 1 ml solution. This transformation efficiency was 10 to 100 times higher than those of the shuttle-vectors for this cyanobacterium previously reported [4, 9]. With this system, we can obtain several million of cyanobacterial transformant in one experiment, thus this shuttle-vector system can also be applied for the construction of

An example of the use of this shuttle-vector for cDNA library is the construction of cDNA library of the halotolerant marine green alga *Chlamydomonas* W80 for the isolation of anti-stress genes [10]. *C.* W80 shows a surprisingly high oxidative stress tolerance caused by methyl viologen (MV), which is reduced by the photosynthetic apparatus generating highly toxic superoxide (O2-

W80 tolerates up to 200 M of MV [11, 12], while other oxygen-evolving photosynthetic organisms such as higher plants, algae and cyanobacteria usually tolerate only less than 5 M of MV. This alga is a prominent genetic resource of anti-stress genes, and various unique anti-stress genes, such as ascorbate peroxidase [13], glutathione peroxidase [14], and the novel salt and cadmium stress related (*scsr*) gene [15], have been isolated from this alga. Using the cDNA library of *C.* W80 constructed in pAQJ4 shuttle-

). *C.*

Since most of the industrial CO2 emission sites locate in the seashore area in Japan, we choose a marine cyanobacterial strain for the Figure 2. Construction of shuttle-vector between **Figure 2.** Construction of shuttle-vector between *E. coli* and *Synec hococcus* sp. PCC7002

designed (Figure 2).

DUMMY TEXT, KOJI NIJE IME KNJIGE

2Toray Research Center, Inc., Kamakura

miyasaka.hitoshi@a4.kepco.co.jp

Figure 1. Chemical structure of PHAs

Assimilation and conversion of CO2 into the biodegradable plastics (biopolymers) by photo‐ synthetic microorganisms is an ideal bioprocess because it converts CO2 directly into the useful bioplastics with solar energy. It also contributes to the low carbon society by substituting the environmentally unfriendly petroleum-based plastics with the carbon neutral bioplastics, and also by saving the fossil fuel resource required for the petrochemical plastics production. In addition, the biodegradable plastics reduce the burden of plastics waste on landfills and the

<sup>O</sup> ( ) n

R

**Figure 1.** Chemical structure of PHAs

**2. Shuttle-vector construction**

the full ORF943 was designed (Figure 2).

environment.

O

**Figure 1 Chemical structure of PHAs**

**2. Shuttle-vector construction** 

Since most of the industrial CO2 emission sites locate in the seashore area in Japan, we choose a marine cyanobacterial strain for the fixation and utilization of CO2. The marine cyanobacterial strain *Synechococcus sp.* PCC7002 (*Agmenellum quadruplicatum* PR-6, ATCC 27264) [4] was obtained from the American Type Culture Collection, and cultured at 30 °C in medium A [5] under continuous illumination (50 μmol photons m-2 s-1) by bubbling with 1% CO2 in air.

For the construction of a shuttle-vector between *E. coli* and *Synechococcus sp.* PCC7002, we isolated and characterized the smallest endogenous plasmid pAQ1 of this cyanobacterium [6, 7]. The plasmid pAQ1 is 4809 bp long, and has four open reading frames (ORFs): ORF943, ORF64, ORF71, and ORF93 (numbers show putative amino acid number). The construction of the shuttle-vector was done by digesting pAQ1 plasmid and bacterial pUC19 plasmid with restriction enzymes which cleave each plasmid at a unique site, and by ligating the linearized plasmids. The plasmid pUC19 and the plasmid pAQ1 were linearized by *Sma*I and *Stu*I digestions, respectively, and were ligated to generate the shuttle-vector pAQJ6 (Figure 2; both *Sma*I and *Stu*I are blunt-end forming restriction enzymes). The effect of the four ORFs on the transformation efficiency was examined, and ORF943 was found to be important for the maintenance of the shuttle-vector. From this result, the simplified shuttle-vector pAQJ4 with

**2.1. Construction of a shuttle-vector between** *Escherichia coli* **and cyanobacteria**

**1. Introduction** 

Japan

Japan

198 Environmental Biotechnology - New Approaches and Prospective Applications

**Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria** 

Global warming is the urgent issue of our time, and the carbon dioxide is a greenhouse gas of the major concern. There are various research activities for carbon dioxide mitigation, such as CO2 recovery from the flue gas of industrial sites, underground and

On-site CO2 fixation by bioprocess is based on the activities of photosynthetic organisms. The fixation of CO2 by photosynthetic microorganisms can be an efficient system for the CO2 mitigation, but one of the major problems of this system is the effective utilization of the fixed biomass. The biomass produced by photosynthetic microorganisms must be utilized as a resource, or it will be easily degraded by microorganisms into CO2 again. Cyanobacteria are procaryotic photosynthetic microorganisms and can provide a simple genetic transformation system for the production of useful materials from CO2. We have established an efficient vector-promoter system for the introduction and expression of foreign genes in the marine cyanobacterium *Synechococcus sp.* PCC7002, and examined the production of biodegradable plastic, polyhydroxyalkanoate (PHA), by genetically engineered cyanobacteria. The PHA is a biopolymer accumulated by various microorganisms as reserves of carbon and reducing equivalents.

films, containers, and paper coatings [2]. PHA applications as implant biomaterials, drug delivery carrier, and biofuel have also

Hitoshi Miyasaka1, Hiroshi Okuhata1, Satoshi Tanaka1, Takuo Onizuka2 and Hideo Akiyama2

undersea CO2 storage, and also chemical/biological conversion of CO2 into the industrial materials [1].

1The Kansai Electric Power Co., Environmental Research Center, Seikacho

microorganism was started in 2009 in the United State and China [3].

the burden of plastics waste on landfills and the environment.

fixation and utilization of CO2. The marine cyanobacterial strain *Synechococcus sp.* PCC7002 (*Agmenellum quadruplicatum* PR-6, ATCC 27264) [4] was obtained from the American Type Culture Collection, and cultured at 30 °C in medium A [5] under continuous illumination (50 mol photons m-2 s-1) by bubbling with 1% CO2 in air. Figure 2. Construction of shuttle-vector between *E. coli* and *Synec hococcus* sp. PCC7002 The stability of the prototype shuttle-vector, pAQJ6, in cyanobacterial cells was relatively low, but that of the simplified shuttlevector, pAQJ4, was much improved; this vector could be stably retained in cyanobacterial cells after 100 generations of growth without antibiotics selection [8]. This is probably because that there are several hot spots for the homologous recombination between endogenous pAQ1 plasmid and pAQJ6 vector, and these hot spots might have been eliminated in the simplified pAQJ4 vector. The transformation efficiency of the shuttle-vector pAQJ4 was about 4 x 105 (cfu / g DNA), when we transformed 4 x 107 of *E. coli* and *Synechococcus*sp. PCC7002 The stability of the prototype shuttle-vector, pAQJ6, in cyanobacterial cells was relatively low, but that of the simplified shuttle-vector, pAQJ4, was much improved; this vector could be stably retained in cyanobacterial cells after 100 generations of growth without antibiotics selection [8]. This is probably because that there are several hot spots for the homologous recombination between endogenous pAQ1 plasmid and pAQJ6 vector, and these hot spots might have been eliminated in the simplified pAQJ4 vector. The transformation efficiency of the shuttle-vector pAQJ4 was about 4 x 105 (cfu / μg DNA), when we transformed 4 x 107 of cyanobacterial cells with 0.3 μg (0.1 pmol) of pAQJ4 vector in 1 ml solution. This transformation efficiency was 10 to 100 times higher than those of the shuttle-vectors for this cyanobacterium

cDNA libraries using cyanobacteria as host cells.

previously reported [4, 9]. With this system, we can obtain several million of cyanobacterial transformant in one experiment, thus this shuttle-vector system can also be applied for the construction of cDNA libraries using cyanobacteria as host cells.

maintained in *S.* PCC7942 cells. The approximate copy numbers of the pAQ-EX1 plasmid in *S.* PCC7002 and *S.* PCC7942 estimated from the yield of plasmid are 15 to 30 copies/cell for *S.* PCC7002, and 5 to 15 copies/cell for *S.* PCC7942, depending on the growth phase of the culture. The *rbc* promoter on pAQ-EX1 vector worked well also in *E. coli* cells, thus the inserted gene on the pAQ-EX1 vector can be efficiently expressed in *E. coli*, in marine cyanobacterium *S.*

*ori*

*Eco*RI (*Eco*RI)

*Sma* I

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

*Bam* HI

 *M L D C R D P R Y H R \**

*Sac* **I**

*Sac* **I**

*Sac* **I** 

*Hin* **cII**

(*Cla*I) *Kpn* I *Sma* I *Bam* HI *Pst*I

*Kpn* I (*Cla*I) (*Eco*RI)

http://dx.doi.org/10.5772/54705

*Hin* **dIII**

*Hin* **cII**

*Hin* **cII**

vector, we isolated anti-stress genes by a functional expression screening in cyanobacteria. The principle of the screening method was based on the acquisition of stress-tolerance of the cyanobacterial cells carrying the genes of *C.* W80, and a unique anti-stress

An effective promoter is important for the expression of the genes on shuttle-vector. The promoter of the RuBisCO (*rbc*) gene of *S.* PCC7002 was chosen for the source of strong promoter, and the *rbc* gene was isolated by screening the genomic library of *S.* PCC7002. RuBisCO is one of the key enzymes of Calvin–Benson cycle (photosynthetic CO2 fixation cycle), and the most abundant protein in photosynthetic organisms. Our genomic clone of the *rbc* gene (DDBJ Accession No. D13971) is 4234 bp long, and has 962 bp of five prime untranslated region (5' UTR) of *rbc* large subunit (*rbcL*). To determine the location of the promoter activity in the 5'-UTR of *rbc* gene, we constructed CAT (chloramphenicol acetyltransferase) reporter gene construct in pAQJ4 vector, and examined the promoter activities of the 5'-UTR of *rbc* gene by introducing various deletions into this region. The promoter activity was found to be located in the region close to the coding region of *rbcL,* and the 304 bp fragment of the 5' UTR containing the promoter region was used for the promoter for pAQJ4 vector. In addition to the promoter, we also introduced multiple cloning site

(MCS) into pAQJ4 vector, and the expression shuttle-vector pAQ-EX1 (DDBJ Accession No. AB071392) was finally developed.

Figure 3 shows the map of the shuttle-vector pAQ-EX1. The transformation efficiency of pAQ-EX1 for *S.* PCC7002 cells was about

The transformation of the fresh water cyanobacterium *S.* PCC7942 with pAQ-EX1 vector was also examined [16], and the *S.* PCC7942 cells were successfully transformed with this vector, although the transformation efficiency (4.0 x 102 cfu/ g DNA ) was much lower than that for *S.* PCC7002. Since *S.* PCC7942 cells do not have the pAQ1 plasmid, which is the origin of pAQ-EX1 vector, there is no possibility of homologous recombination between pAQ-EX1 and pAQ1 in *S.* PCC7942 cells. We can, therefore, expect a higher stability of pAQ-EX1 vector in *S.* PCC7942 cells than in *S.* PCC7002 cells, and actually the pAQ-EX1 plasmid was quite stably maintained in *S.* PCC7942 cells. The approximate copy numbers of the pAQ-EX1 plasmid in *S.* PCC7002 and *S.* PCC7942 estimated from the yield of plasmid are 15 to 30 copies/cell for *S.* PCC7002, and 5 to 15 copies/cell for *S.* PCC7942, depending on the growth phase of the culture. The *rbc* promoter on pAQ-EX1 vector worked well also in *E. coli* cells, thus the inserted gene on the pAQ-EX1 vector can be efficiently expressed in *E. coli*, in marine cyanobacterium *S.* PCC7002, and in fresh

201

 The changes in *rbc* promoter activity in response to CO2 condition were examined because the down-regulation of *rbc* gene expression by elevated CO2 concentration has been reported in several photosynthetic organisms. Table 1 shows the comparison of mRNA levels in *S.* PCC7002 cells cultured under various CO2 conditions (0.03%, 1%, and 15%). The mRNA levels of *rbcL* gene were determined by RT-PCR, and compared to those of the reference gene (*ATPaseA* gene). The mRNA levels of the *rbcL* gene significantly decreased under the higher CO2 conditions, suggesting the presence of some elements, which down regulates the transcription in response to CO2 concentration. To examine the CO2-regulatory element in the *rbc* promoter region, various deletions were introduced into this region, and cyanobacterial CAT assay was done by using pAQJ4-CAT vector [17]. Figure 4

gene (a new member of group 3 late embryogenesis abundant protein genes) was successfully isolated [10].

**2.2. Promoter for the expression of the genes on shuttle-vector** 

PCC7002, and in fresh water cyanobacterium *S.* PCC7942.

rbc promoter (-304 fragment)

*Xba*I

*Eco*RI

**2.3. CO2 response element in** *rbc* **promoter**

water cyanobacterium *S.* PCC7942.

Figure 3. Map of the shuttle-vector pAQ-EX 1

*Stu* **I /***Hin* **dIII**

**Figure 3.** Map of the shuttle-vector pAQ-EX 1

**2.3. CO2 response element in** *rbc* **promoter** 

The changes in *rbc* promoter activity in response to CO2 condition were examined because the down-regulation of *rbc* gene expression by elevated CO2 concentration has been reported in several photosynthetic organisms. Table 1 shows the comparison of mRNA levels in *S.* PCC7002 cells cultured under various CO2 conditions (0.03%, 1%, and 15%). The mRNA levels of *rbcL* gene were determined by RT-PCR, and compared to those of the reference gene (*ATPaseA* gene). The mRNA levels of the *rbcL* gene significantly decreased under the higher CO2 conditions, suggesting the presence of some elements, which down regulates the tran‐ scription in response to CO2 concentration. To examine the CO2-regulatory element in the *rbc* promoter region, various deletions were introduced into this region, and cyanobacterial CAT assay was done by using pAQJ4-CAT vector [17]. Figure 4 shows the tested promoter frag‐ ments and the results of CAT assay. The core promoter region was shown to be located in the

*ORF943*

**pAQ-EX1**

rbc promoter (-304 fragment)

AATTTCTAGaattc....cccc **atg**CTAGACTGCAGGGATCCCGGGTACCATCGATGAATTCGAGCTC...

*Pst*I

**pUC19**

Figure 3 Map of the shuttle-vector pAQ-EX1

*Xba*I

**pAQ1**

6 x 105 cfu / g DNA [16].

An example of the use of this shuttle-vector for cDNA library is the construction of cDNA library of the halotolerant marine green alga *Chlamydomonas* W80 for the isolation of anti-stress genes [10]. *C.* W80 shows a surprisingly high oxidative stress tolerance caused by methyl viologen (MV), which is reduced by the photosynthetic apparatus generating highly toxic superoxide (O2 - ). *C.* W80 tolerates up to 200 μM of MV [11, 12], while other oxygen-evolving photosynthetic organisms such as higher plants, algae and cyanobacteria usually tolerate only less than 5 μM of MV. This alga is a prominent genetic resource of anti-stress genes, and various unique anti-stress genes, such as ascorbate peroxidase [13], glutathione peroxidase [14], and the novel salt and cadmium stress related (*scsr*) gene [15], have been isolated from this alga. Using the cDNA library of *C.* W80 constructed in pAQJ4 shuttle-vector, we isolated anti-stress genes by a functional expression screening in cyanobacteria. The principle of the screening method was based on the acquisition of stress-tolerance of the cyanobacterial cells carrying the genes of *C.* W80, and a unique anti-stress gene (a new member of group 3 late embryo‐ genesis abundant protein genes) was successfully isolated [10].

### **2.2. Promoter for the expression of the genes on shuttle-vector**

An effective promoter is important for the expression of the genes on shuttle-vector. The promoter of the RuBisCO (*rbc*) gene of *S.* PCC7002 was chosen for the source of strong promoter, and the *rbc* gene was isolated by screening the genomic library of *S.* PCC7002. RuBisCO is one of the key enzymes of Calvin–Benson cycle (photosynthetic CO2 fixation cycle), and the most abundant protein in photosynthetic organisms. Our genomic clone of the *rbc* gene (DDBJ Accession No. D13971) is 4234 bp long, and has 962 bp of five prime untranslated region (5' UTR) of *rbc* large subunit (*rbcL*). To determine the location of the promoter activity in the 5'-UTR of *rbc* gene, we constructed CAT (chloramphenicol acetyltransferase) reporter gene construct in pAQJ4 vector, and examined the promoter activities of the 5'-UTR of *rbc* gene by introducing various deletions into this region. The promoter activity was found to be located in the region close to the coding region of *rbcL,* and the 304 bp fragment of the 5' UTR containing the promoter region was used for the promoter for pAQJ4 vector. In addition to the promoter, we also introduced multiple cloning site (MCS) into pAQJ4 vector, and the expression shuttlevector pAQ-EX1 (DDBJ Accession No. AB071392) was finally developed.

Figure 3 shows the map of the shuttle-vector pAQ-EX1. The transformation efficiency of pAQ-EX1 for *S.* PCC7002 cells was about 6 x 105 cfu / μg DNA [16].

The transformation of the fresh water cyanobacterium *S.* PCC7942 with pAQ-EX1 vector was also examined [16], and the *S.* PCC7942 cells were successfully transformed with this vector, although the transformation efficiency (4.0 x 102 cfu/ μg DNA ) was much lower than that for *S.* PCC7002. Since *S.* PCC7942 cells do not have the pAQ1 plasmid, which is the origin of pAQ-EX1 vector, there is no possibility of homologous recombination between pAQ-EX1 and pAQ1 in *S.* PCC7942 cells. We can, therefore, expect a higher stability of pAQ-EX1 vector in *S.* PCC7942 cells than in *S.* PCC7002 cells, and actually the pAQ-EX1 plasmid was quite stably vector, we isolated anti-stress genes by a functional expression screening in cyanobacteria. The principle of the screening method was based on the acquisition of stress-tolerance of the cyanobacterial cells carrying the genes of *C.* W80, and a unique anti-stress

An effective promoter is important for the expression of the genes on shuttle-vector. The promoter of the RuBisCO (*rbc*) gene of *S.* PCC7002 was chosen for the source of strong promoter, and the *rbc* gene was isolated by screening the genomic library of *S.* PCC7002. RuBisCO is one of the key enzymes of Calvin–Benson cycle (photosynthetic CO2 fixation cycle), and the most abundant protein in photosynthetic organisms. Our genomic clone of the *rbc* gene (DDBJ Accession No. D13971) is 4234 bp long, and has 962 bp of five prime untranslated region (5' UTR) of *rbc* large subunit (*rbcL*). To determine the location of the promoter activity in the 5'-UTR of *rbc* gene, we constructed CAT (chloramphenicol acetyltransferase) reporter gene construct in pAQJ4 vector, and examined the promoter activities of the 5'-UTR of *rbc* gene by introducing various deletions into this region. The promoter activity was found to be located in the region close to the coding region of *rbcL,* and the 304 bp fragment of the 5' UTR containing the promoter region was used for the promoter for pAQJ4 vector. In addition to the promoter, we also introduced multiple cloning site

(MCS) into pAQJ4 vector, and the expression shuttle-vector pAQ-EX1 (DDBJ Accession No. AB071392) was finally developed.

Figure 3 shows the map of the shuttle-vector pAQ-EX1. The transformation efficiency of pAQ-EX1 for *S.* PCC7002 cells was about

The transformation of the fresh water cyanobacterium *S.* PCC7942 with pAQ-EX1 vector was also examined [16], and the *S.* PCC7942 cells were successfully transformed with this vector, although the transformation efficiency (4.0 x 102 cfu/ g DNA ) was much lower than that for *S.* PCC7002. Since *S.* PCC7942 cells do not have the pAQ1 plasmid, which is the origin of pAQ-EX1 vector, there is no possibility of homologous recombination between pAQ-EX1 and pAQ1 in *S.* PCC7942 cells. We can, therefore,

were determined by RT-PCR, and compared to those of the reference gene (*ATPaseA* gene). The mRNA levels of the *rbcL* gene significantly decreased under the higher CO2 conditions, suggesting the presence of some elements, which down regulates the transcription in response to CO2 concentration. To examine the CO2-regulatory element in the *rbc* promoter region, various deletions were introduced into this region, and cyanobacterial CAT assay was done by using pAQJ4-CAT vector [17]. Figure 4

gene (a new member of group 3 late embryogenesis abundant protein genes) was successfully isolated [10].

**2.2. Promoter for the expression of the genes on shuttle-vector** 

maintained in *S.* PCC7942 cells. The approximate copy numbers of the pAQ-EX1 plasmid in *S.* PCC7002 and *S.* PCC7942 estimated from the yield of plasmid are 15 to 30 copies/cell for *S.* PCC7002, and 5 to 15 copies/cell for *S.* PCC7942, depending on the growth phase of the culture. The *rbc* promoter on pAQ-EX1 vector worked well also in *E. coli* cells, thus the inserted gene on the pAQ-EX1 vector can be efficiently expressed in *E. coli*, in marine cyanobacterium *S.* PCC7002, and in fresh water cyanobacterium *S.* PCC7942. PCC7942 estimated from the yield of plasmid are 15 to 30 copies/cell for *S.* PCC7002, and 5 to 15 copies/cell for *S.* PCC7942, depending on the growth phase of the culture. The *rbc* promoter on pAQ-EX1 vector worked well also in *E. coli* cells, thus the inserted gene on the pAQ-EX1 vector can be efficiently expressed in *E. coli*, in marine cyanobacterium *S.* PCC7002, and in fresh water cyanobacterium *S.* PCC7942.

Figure 3 Map of the shuttle-vector pAQ-EX1 **Figure 3.** Map of the shuttle-vector pAQ-EX 1

previously reported [4, 9]. With this system, we can obtain several million of cyanobacterial transformant in one experiment, thus this shuttle-vector system can also be applied for the

An example of the use of this shuttle-vector for cDNA library is the construction of cDNA library of the halotolerant marine green alga *Chlamydomonas* W80 for the isolation of anti-stress genes [10]. *C.* W80 shows a surprisingly high oxidative stress tolerance caused by methyl viologen (MV), which is reduced by the photosynthetic apparatus generating highly toxic

photosynthetic organisms such as higher plants, algae and cyanobacteria usually tolerate only less than 5 μM of MV. This alga is a prominent genetic resource of anti-stress genes, and various unique anti-stress genes, such as ascorbate peroxidase [13], glutathione peroxidase [14], and the novel salt and cadmium stress related (*scsr*) gene [15], have been isolated from this alga. Using the cDNA library of *C.* W80 constructed in pAQJ4 shuttle-vector, we isolated anti-stress genes by a functional expression screening in cyanobacteria. The principle of the screening method was based on the acquisition of stress-tolerance of the cyanobacterial cells carrying the genes of *C.* W80, and a unique anti-stress gene (a new member of group 3 late embryo‐

An effective promoter is important for the expression of the genes on shuttle-vector. The promoter of the RuBisCO (*rbc*) gene of *S.* PCC7002 was chosen for the source of strong promoter, and the *rbc* gene was isolated by screening the genomic library of *S.* PCC7002. RuBisCO is one of the key enzymes of Calvin–Benson cycle (photosynthetic CO2 fixation cycle), and the most abundant protein in photosynthetic organisms. Our genomic clone of the *rbc* gene (DDBJ Accession No. D13971) is 4234 bp long, and has 962 bp of five prime untranslated region (5' UTR) of *rbc* large subunit (*rbcL*). To determine the location of the promoter activity in the 5'-UTR of *rbc* gene, we constructed CAT (chloramphenicol acetyltransferase) reporter gene construct in pAQJ4 vector, and examined the promoter activities of the 5'-UTR of *rbc* gene by introducing various deletions into this region. The promoter activity was found to be located in the region close to the coding region of *rbcL,* and the 304 bp fragment of the 5' UTR containing the promoter region was used for the promoter for pAQJ4 vector. In addition to the promoter, we also introduced multiple cloning site (MCS) into pAQJ4 vector, and the expression shuttle-

Figure 3 shows the map of the shuttle-vector pAQ-EX1. The transformation efficiency of pAQ-

The transformation of the fresh water cyanobacterium *S.* PCC7942 with pAQ-EX1 vector was also examined [16], and the *S.* PCC7942 cells were successfully transformed with this vector,

*S.* PCC7002. Since *S.* PCC7942 cells do not have the pAQ1 plasmid, which is the origin of pAQ-EX1 vector, there is no possibility of homologous recombination between pAQ-EX1 and pAQ1 in *S.* PCC7942 cells. We can, therefore, expect a higher stability of pAQ-EX1 vector in *S.* PCC7942 cells than in *S.* PCC7002 cells, and actually the pAQ-EX1 plasmid was quite stably

cfu / μg DNA [16].

cfu/ μg DNA ) was much lower than that for

). *C.* W80 tolerates up to 200 μM of MV [11, 12], while other oxygen-evolving

construction of cDNA libraries using cyanobacteria as host cells.

200 Environmental Biotechnology - New Approaches and Prospective Applications

genesis abundant protein genes) was successfully isolated [10].

**2.2. Promoter for the expression of the genes on shuttle-vector**

vector pAQ-EX1 (DDBJ Accession No. AB071392) was finally developed.

EX1 for *S.* PCC7002 cells was about 6 x 105

although the transformation efficiency (4.0 x 102

superoxide (O2


### **2.3. CO2 response element in** *rbc* **promoter**

6 x 105 cfu / g DNA [16].

Figure 3. Map of the shuttle-vector pAQ-EX 1 **2.3. CO2 response element in** *rbc* **promoter**  The changes in *rbc* promoter activity in response to CO2 condition were examined because the down-regulation of *rbc* gene expression by elevated CO2 concentration has been reported in several photosynthetic organisms. Table 1 shows the comparison of mRNA levels in *S.* PCC7002 cells cultured under various CO2 conditions (0.03%, 1%, and 15%). The mRNA levels of *rbcL* gene The changes in *rbc* promoter activity in response to CO2 condition were examined because the down-regulation of *rbc* gene expression by elevated CO2 concentration has been reported in several photosynthetic organisms. Table 1 shows the comparison of mRNA levels in *S.* PCC7002 cells cultured under various CO2 conditions (0.03%, 1%, and 15%). The mRNA levels of *rbcL* gene were determined by RT-PCR, and compared to those of the reference gene (*ATPaseA* gene). The mRNA levels of the *rbcL* gene significantly decreased under the higher CO2 conditions, suggesting the presence of some elements, which down regulates the tran‐ scription in response to CO2 concentration. To examine the CO2-regulatory element in the *rbc* promoter region, various deletions were introduced into this region, and cyanobacterial CAT assay was done by using pAQJ4-CAT vector [17]. Figure 4 shows the tested promoter frag‐ ments and the results of CAT assay. The core promoter region was shown to be located in the



<sup>a</sup> mRNA level is shown as the relative value to the mRNA level at 0.03% CO2.

Table 1 Transcript levels of *rbcL* and reference (*ATPase A*) genes of *S.*

amRNA level is shown as the relative value to the mRNA level at 0.03% CO2

*rbcL rbcS* 300bp

Table 1. Transcript levels of *rbc*L and reference (ARPase A) genes of S. PCC7002 under various CO2 conditions

a mRNA level is shown as the relative value to the mRNA level at 0.03% CO2

(CO2 %)


PCC7002 under various CO2 conditions

Relative mRNA level a

amRNA level is shown as the relative value to the mRNA level at 0.03% CO2

**3.** *recA* **complementation as a selection pressure for plasmid stability** 

recombination. The advantages of the plasmid system are i) the higher copy numbers of the foreign genes in cyanobacterial cells compared to the genome integration method, ii) the well established procedure for the modification of the genes on plasmid, such as point mutation, insertion and deletion, and iii) the wide range of expression host with a shuttle vector system. On the other hand the limitation of plasmid system is the necessity for antibiotics for the maintenance of plasmid. Especially when the genes on plasmid cause a heavy metabolic load, such as PHA production, to the host cells, the plasmids are easily excluded from the cells in the absence of antibiotics pressure. The use of antibiotics is, however, not realistic for the large scale cyanobacterial culture for CO2 mitigation with respect to its cost. In *E. coli* cells, the *parB* (*hok/sok*) locus of plasmid R which mediates stabilization *via* postsegregational killing of plasmid-free cells is effective for the antibiotics-independent stable maintenance of the plasmid [20], but in

There are two foreign gene expression systems for cyanobacteria [18, 19]; one is the plasmid vector system, as we describe in this chapter, and another one is the integration of the foreign DNA into the cyanobacterial genome through homologous recombination. The advantages of the plasmid system are i) the higher copy numbers of the foreign genes in cyanobacterial cells compared to the genome integration method, ii) the well established procedure for the modification of the genes on plasmid, such as point mutation, insertion and deletion, and iii) the wide range of expression host with a shuttle vector system. On the other hand the limitation of plasmid system is the necessity for antibiotics for the maintenance of plasmid. Especially when the genes on plasmid cause a heavy metabolic load, such as PHA production, to the host cells, the plasmids are easily excluded from the cells in the absence of antibiotics pressure. The use of antibiotics is, however, not realistic for the large scale cyanobacterial culture for CO2 mitigation with respect to its cost. In *E. coli* cells, the *parB* (*hok/sok*) locus of plasmid R which mediates stabilization *via* postsegregational killing of plasmid-free cells is effective for the antibiotics-independent stable maintenance of the plasmid [20], but in

shows the tested promoter fragments and the results of CAT assay. The core promoter region was shown to be located in the -228

region. The promoter fragment, designated as R7, which contains whole -304 through -1 region, showed down regulation in promoter activity by elevated CO2 condition (1% CO2), while this down regulation was not observed in the R6 fragment lacking the

**3.** *recA* **complementation as a selection pressure for plasmid stability**

mid which causes a significant metabolic burden to the host cells.

There are two foreign gene expression systems for cyanobacteria [18, 19]; one is the plasmid vec‐ tor system, as we describe in this chapter, and another one is the integration of the foreign DNA into the cyanobacterial genome through homologous recombination. The advantages of the plasmid system are i) the higher copy numbers of the foreign genes in cyanobacterial cells com‐ pared to the genome integration method, ii) the well established procedure for the modification of the genes on plasmid, such as point mutation, insertion and deletion, and iii) the wide range of expression host with a shuttle vector system. On the other hand the limitation of plasmid sys‐ tem is the necessity for antibiotics for the maintenance of plasmid. Especially when the genes on plasmid cause a heavy metabolic load, such as PHA production, to the host cells, the plasmids are easily excluded from the cells in the absence of antibiotics pressure. The use of antibiotics is, however, not realistic for the large scale cyanobacterial culture for CO2 mitigation with respect to its cost. In *E. coli* cells, the *parB* (*hok/sok*) locus of plasmid R which mediates stabilization *via* post-segregational killing of plasmid-free cells is effective for the antibiotics-independent sta‐ ble maintenance of the plasmid [20], but in cyanobacteria there has been no such a practical plas‐ mid stabilization system reported. We developed a practical plasmid stabilization system by utilizing the *recA* complementation mechanism. RecA is a multifunctional protein that plays key roles in various cellular processes, such as recombination and DNA repair in bacteria [21, 22]. The amino acid sequences of RecA proteins from the different microorganisms are well con‐ served, and there are several reports on the complementation of *recA* mutation in some bacteria by *E. coli recA* gene. Murphy et al. [23] reported that a *recA* null mutation is lethal in the cyano‐ bacterium, but the *E. colirecA* gene in trans on a plasmid can complement the function of *recA* re‐ sulting in segregation of cyanobacterial *recA* null mutant. We expected that this complementation mechanism can be used as a selection pressure to prevent the loss of the plas‐

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

http://dx.doi.org/10.5772/54705

203

Figure 5 shows the principles of the selective pressure for the maintainance of plasmid in con‐ ventional antibiotics selection (Figure 5A) and our *recA* complementation (Figure 5B) systems. In the conventional antibiotics selection system, the antibiotics-resistant (Ab-R) gene casette is introduced in plasmid, and the loss of the plasmid makes the host cells sensitive to antibiotics. In the *recA* complementation system, the *recA* gene in the genome of the host cells is inactivated by homologous recombination, and the function of genomic *recA* gene is complemented by the *re‐ cA* gene on the plasmid. In cyanobacteria, *recA* null mutation is lethal, and the host cell without plasmid can not survive. Generally, cyanobacteria have several copies of genome [24] and *recA* null mutant cells carrying a plasmid with *E. colirecA* gene were generated by three steps as fol‐ lows (Figure 6); i) generation of *recA* partial mutant of *S.* PCC7002 by homologous recombina‐ tion with kanamycin resistance gene (*km*) cassette, ii) introduction of shuttle-vector with *E. coli recA* gene into the *recA* partial mutant cells, and iii) conversion of the partial *recA* mutant into the *recA* null mutant by further homologous recombination. The use of *recA* complementation as a selection pressure is a simple and versatile method; only the *E. colirecA* gene on the plasmid, and the partial *recA* mutant host are required, and the *recA* null mutant can easily be obtained by subculturing the cells in the medium with kanamycin (Km). Unlike cyanobacteria, *recA* null mutation is not lethal in *E. coli*, thus this complementation system is not applicable for *E. coli*.

There are two foreign gene expression systems for cyanobacteria [18, 19]; one is the plasmid vector system, as we describe in this chapter, and another one is the integration of the foreign DNA into the cyanobacterial genome through homologous **Figure 4.** Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp*. PCC7002

Figure 4. Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp*. PCC7002

Figure 4 Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp.* PCC7002

**3.** *recA* **complementation as a selection pressure for plasmid stability** 

#### -304 through -250 region. These results indicate that a CO2-regulatory *cis* element exists in the -304 through -250 region, and a high expression level can be retained with R6 promoter fragment even under high CO2 condition. The -304 through -250 region is quite **3.** *recA* **complementation as a selection pressure for plasmid stability**


shows the tested promoter fragments and the results of CAT assay. The core promoter region was shown to be located in the -228 through -132 region, because the promoter activities were drastically decreased in the R3, R4, and R5 fragments, which lack this region. The promoter fragment, designated as R7, which contains whole -304 through -1 region, showed down regulation in promoter activity by elevated CO2 condition (1% CO2), while this down regulation was not observed in the R6 fragment lacking the -304 through -250 region. These results indicate that a CO2-regulatory *cis* element exists in the -304 through -250 region, and a high expression level can be retained with R6 promoter fragment even under high CO2 condition. The -304 through -250 region is quite A/T rich, and we also identified, by a DNA affinity precipitation assay, the 16-kDa protein which acts as a *trans*-element in CO2

shows the tested promoter fragments and the results of CAT assay. The core promoter region was shown to be located in the -228 through -132 region, because the promoter activities were drastically decreased in the R3, R4, and R5 fragments, which lack this region. The promoter fragment, designated as R7, which contains whole -304 through -1 region, showed down regulation in promoter activity by elevated CO2 condition (1% CO2), while this down regulation was not observed in the R6 fragment lacking the

Table 1. Transcript levels of *rbc*L and reference (ARPase A) genes of S. PCC7002 under various CO2 conditions

**CAT activity (µmol/min/mg)**

*rbcL*

30

+1

**CAT activity (µmol/min/mg)**

0

10

20

30

amRNA level is shown as the relative value to the mRNA level at 0.03% CO2

*rbcL rbcS* 300bp

Table 1. Transcript levels of *rbc*L and reference (ARPase A) genes of S. PCC7002 under various CO2 conditions

15 0.57 2.2 0.26 1 0.80 1.0 0.80 0.03 1.0 1.0 1.0

<sup>a</sup> mRNA level is shown as the relative value to the mRNA level at 0.03% CO2.

Culture condition *rbcL ATPase A rbcL* / *ATPase A*

Relative mRNA level a

1.0

Table 1 Transcript levels of *rbcL* and reference (*ATPase A*) genes of *S.*

Figure 4. Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp*. PCC7002

Figure 4 Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp.* PCC7002

10

20

0

**3.** *recA* **complementation as a selection pressure for plasmid stability** 

There are two foreign gene expression systems for cyanobacteria [18, 19]; one is the plasmid vector system, as we describe in this chapter, and another one is the integration of the foreign DNA into the cyanobacterial genome through homologous recombination. The advantages of the plasmid system are i) the higher copy numbers of the foreign genes in cyanobacterial cells compared to the genome integration method, ii) the well established procedure for the modification of the genes on plasmid, such as point mutation, insertion and deletion, and iii) the wide range of expression host with a shuttle vector system. On the other hand the limitation of plasmid system is the necessity for antibiotics for the maintenance of plasmid. Especially when the genes on plasmid cause a heavy metabolic load, such as PHA production, to the host cells, the plasmids are easily excluded from the cells in the absence of antibiotics pressure. The use of antibiotics is, however, not realistic for the large scale cyanobacterial culture for CO2 mitigation with respect to its cost. In *E. coli* cells, the *parB* (*hok/sok*) locus of plasmid R which mediates stabilization *via* postsegregational killing of plasmid-free cells is effective for the antibiotics-independent stable maintenance of the plasmid [20], but in

There are two foreign gene expression systems for cyanobacteria [18, 19]; one is the plasmid vector system, as we describe in this chapter, and another one is the integration of the foreign DNA into the cyanobacterial genome through homologous recombination. The advantages of the plasmid system are i) the higher copy numbers of the foreign genes in cyanobacterial cells compared to the genome integration method, ii) the well established procedure for the modification of the genes on plasmid, such as point mutation, insertion and deletion, and iii) the wide range of expression host with a shuttle vector system. On the other hand the limitation of plasmid system is the necessity for antibiotics for the maintenance of plasmid. Especially when the genes on plasmid cause a heavy metabolic load, such as PHA production, to the host cells, the plasmids are easily excluded from the cells in the absence of antibiotics pressure. The use of antibiotics is, however, not realistic for the large scale cyanobacterial culture for CO2 mitigation with respect to its cost. In *E. coli* cells, the *parB* (*hok/sok*) locus of plasmid R which mediates stabilization *via* postsegregational killing of plasmid-free cells is effective for the antibiotics-independent stable maintenance of the plasmid [20], but in

R1 R2 R3 R4 R5 R6 R7 *trc* pAQJ4-

R1 R2 R3 R4 R5 R6 R7 *trc* pAQJ4-

CAT7

CAT7

which acts as a *trans*-element in CO2 regulation [17].

(CO2 %)

mRNA level is shown as the relative value to the mRNA level at 0.03% CO2

**R1 R2 R3 R4 R5 R6 R7**

Core promoter region

*rbc* gene


Promoter region

a

**R1 R2 R3 R4 R5 R6 R7**

*rbc* gene



CO2 response element

regulation [17].

(CO2 %)




CO2 response element

PCC7002 under various CO2 conditions

Culture condition *rbcL ATPase A rbcL* / *ATPase A*

Relative mRNA level a

Table 1 Transcript levels of *rbcL* and reference (*ATPase A*) genes of *S.*

15 0.57 2.2 0.26 1 0.80 1.0 0.80 0.03 1.0 1.0 1.0

<sup>a</sup> mRNA level is shown as the relative value to the mRNA level at 0.03% CO2.


Promoter region


Core promoter region



*rbcL rbcS* 300bp

**Table 1.** Transcript levels of *rbc*L and reference (ARPase A) genes of S. PCC7002 under various CO2 conditions

amRNA level is shown as the relative value to the mRNA level at 0.03% CO2


+1

Figure 4. Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp*. PCC7002

**Figure 4.** Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp*. PCC7002

Figure 4 Promoter activities of various fragments of *rbc* promoter of *Synechococcus sp.* PCC7002

**3.** *recA* **complementation as a selection pressure for plasmid stability** 


*rbcL*

regulation [17].

202 Environmental Biotechnology - New Approaches and Prospective Applications

PCC7002 under various CO2 conditions

A/T rich, and we also identified, by a DNA affinity precipitation assay, the 16-kDa protein which acts as a *trans*-element in CO2 There are two foreign gene expression systems for cyanobacteria [18, 19]; one is the plasmid vec‐ tor system, as we describe in this chapter, and another one is the integration of the foreign DNA into the cyanobacterial genome through homologous recombination. The advantages of the plasmid system are i) the higher copy numbers of the foreign genes in cyanobacterial cells com‐ pared to the genome integration method, ii) the well established procedure for the modification of the genes on plasmid, such as point mutation, insertion and deletion, and iii) the wide range of expression host with a shuttle vector system. On the other hand the limitation of plasmid sys‐ tem is the necessity for antibiotics for the maintenance of plasmid. Especially when the genes on plasmid cause a heavy metabolic load, such as PHA production, to the host cells, the plasmids are easily excluded from the cells in the absence of antibiotics pressure. The use of antibiotics is, however, not realistic for the large scale cyanobacterial culture for CO2 mitigation with respect to its cost. In *E. coli* cells, the *parB* (*hok/sok*) locus of plasmid R which mediates stabilization *via* post-segregational killing of plasmid-free cells is effective for the antibiotics-independent sta‐ ble maintenance of the plasmid [20], but in cyanobacteria there has been no such a practical plas‐ mid stabilization system reported. We developed a practical plasmid stabilization system by utilizing the *recA* complementation mechanism. RecA is a multifunctional protein that plays key roles in various cellular processes, such as recombination and DNA repair in bacteria [21, 22]. The amino acid sequences of RecA proteins from the different microorganisms are well con‐ served, and there are several reports on the complementation of *recA* mutation in some bacteria by *E. coli recA* gene. Murphy et al. [23] reported that a *recA* null mutation is lethal in the cyano‐ bacterium, but the *E. colirecA* gene in trans on a plasmid can complement the function of *recA* re‐ sulting in segregation of cyanobacterial *recA* null mutant. We expected that this complementation mechanism can be used as a selection pressure to prevent the loss of the plas‐ mid which causes a significant metabolic burden to the host cells.

> Figure 5 shows the principles of the selective pressure for the maintainance of plasmid in con‐ ventional antibiotics selection (Figure 5A) and our *recA* complementation (Figure 5B) systems. In the conventional antibiotics selection system, the antibiotics-resistant (Ab-R) gene casette is introduced in plasmid, and the loss of the plasmid makes the host cells sensitive to antibiotics. In the *recA* complementation system, the *recA* gene in the genome of the host cells is inactivated by homologous recombination, and the function of genomic *recA* gene is complemented by the *re‐ cA* gene on the plasmid. In cyanobacteria, *recA* null mutation is lethal, and the host cell without plasmid can not survive. Generally, cyanobacteria have several copies of genome [24] and *recA* null mutant cells carrying a plasmid with *E. colirecA* gene were generated by three steps as fol‐ lows (Figure 6); i) generation of *recA* partial mutant of *S.* PCC7002 by homologous recombina‐ tion with kanamycin resistance gene (*km*) cassette, ii) introduction of shuttle-vector with *E. coli recA* gene into the *recA* partial mutant cells, and iii) conversion of the partial *recA* mutant into the *recA* null mutant by further homologous recombination. The use of *recA* complementation as a selection pressure is a simple and versatile method; only the *E. colirecA* gene on the plasmid, and the partial *recA* mutant host are required, and the *recA* null mutant can easily be obtained by subculturing the cells in the medium with kanamycin (Km). Unlike cyanobacteria, *recA* null mutation is not lethal in *E. coli*, thus this complementation system is not applicable for *E. coli*.

which causes a significant metabolic burden to the host cells.

not lethal in *E. coli*, thus this complementation system is not applicable for *E. coli*.

cyanobacteria there has been no such a practical plasmid stabilization system reported. We developed a practical plasmid stabilization system by utilizing the *recA* complementation mechanism. RecA is a multifunctional protein that plays key roles in various cellular processes, such as recombination and DNA repair in bacteria [21, 22]. The amino acid sequences of RecA proteins from the different microorganisms are well conserved, and there are several reports on the complementation of *recA* mutation in some bacteria by *E. coli recA* gene. Murphy et al. [23] reported that a *recA* null mutation is lethal in the cyanobacterium, but the *E. coli recA* gene in trans on a plasmid can complement the function of *recA* resulting in segregation of cyanobacterial *recA* null mutant. We expected that this complementation mechanism can be used as a selection pressure to prevent the loss of the plasmid

Figure 5 shows the principles of the selective pressure for the maintainance of plasmid in conventional antibiotics selection (Figure 5A) and our *recA* complementation (Figure 5B) systems. In the conventional antibiotics selection system, the antibiotics-resistant (Ab-R) gene casette is introduced in plasmid, and the loss of the plasmid makes the host cells sensitive to antibiotics. In the *recA* complementation system, the *recA* gene in the genome of the host cells is inactivated by homologous recombination, and the function of genomic *recA* gene is complemented by the *recA* gene on the plasmid. In cyanobacteria, *recA* null mutation is lethal, and the host cell without plasmid can not survive. Generally, cyanobacteria have several copies of genome [24] and *recA* null mutant cells carrying a plasmid with *E. coli recA* gene were generated by three steps as follows (Figure 6); i) generation of *recA* partial mutant of *S.* PCC7002 by homologous recombination with kanamycin resistance gene (*km*) cassette, ii) introduction of shuttle-vector with *E. coli recA* gene into the *recA* partial mutant cells, and iii) conversion of the partial *recA* mutant into the *recA* null mutant by further homologous recombination. The use of *recA* complementation as a selection pressure is a simple and versatile method; only the *E. coli recA* gene on the plasmid, and the partial *recA* mutant host are required, and the *recA* null mutant

**4. PHA production by recombinant cyanobacteria**

*recA*

*recA*

Step 1

Homologous recombination with *recA* gene containing *km* cassette

Step 3

*recA*

*RecA* null mutant carrying shuttle-vector with *recA* gene

*recA*

*recA*

*recA*

with *E. coli recA* gene

*recA*

Genomic DNA

Wild type cell

*hirsutum* (cotton), and *Zea mays* (corn) [30].

O O O

3-ketothiolase (*phaA*)

SCoA

(Acetyl-CoA)

(A)

(B)

[33, 34].

**4.1. Vector construct with** *recA* **complementation system for PHA production**

Figure 6 Procedure for generation of *recA* null mutant cells carrying plasmids

**4. PHA production by recombinant cyanobacteria** 

*thaliana*, *Gossypium hirsutum* (cotton), and *Zea mays* (corn) [30].

SCoA

acetoacetyl-CoA reductase (*phaB*)

*Bam***HI** *Eco***RI** *phaC phaA phaB*

There are several cyanobacterial strains which can naturally accumulate PHAs, but generally the PHA productivity in these strains are low [31, 32]. Several attempts have also been made

We investigated the production of PHA by the recombinant cyanobacteria with the *recA*

Figure 7. Biosynthetic pathway for PHA (A) and structure of pha genes (B)

to introduce PHA genes into non-PHA-producing cyanobacterial strains [33, 34].

complementation antibiotics-free cyanobacterial expression system [35].

**Figure 7.** Biosynthetic pathway for PHA (A) and structure of pha genes (B)

Figure 7 Biosynthetic pathway for PHA (A) and structure of *pha* genes (B)

PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), and there are at least 100 different 3-hydroxy alkanoic acids among the PHA constituents [25]. The first PHA discovered was poly(3-hydroxy-butyrate) (PHB). It is a highly crystalline thermoplastic sharing many properties with polypropylene, and the most abundant of the PHAs in nature. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes (Figure 7A), 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). These three enzymes are encoded by the genes of the *phbCAB* operon (Figure 7B). There are several well established PHA production systems using natural microorganisms such as *Wautersia eutropha*, *Methylobacterium*, and *Pseudmonas*, and also using recombinant bacteria such as *E. coli* [2, 26], and intracellular accumulation of PHA of over 90% of the cell dry weight has been reported. The use of agroindustrial by-products [27], forest biomass [28], and glycerol (by-product of bio-diesel production) [29] for the substrates of microbial PHA production has also been reported. The production of PHAs in transgenic plants carrying bacterial *phb* genes has also been investigated in *Arabidopsis thaliana*, *Gossypium*

Figure 6. Procedure for generation of *rec*A null mutant cells carrying plasmids with *E. coli rec*A gene

**4.1. Vector construct with** *recA* **complementation system for PHA production** 

OH O

SCoA

*recA*

*RecA* partial mutant with shuttlevector carrying *recA* gene

*recA*

Step 2

(Transformation)

*recA*

*recA*

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

*recA*

*recA*

*RecA* partial mutant

PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), and there are at least 100 different 3-hydroxy alkanoic acids among the PHA constituents [25]. The first PHA discovered was poly(3-hydroxy-butyrate) (PHB). It is a highly crystalline thermoplastic sharing many properties with polypropylene, and the most abundant of the PHAs in nature. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes (Figure 7A), 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC). These three enzymes are encoded by the genes of the *phbCAB* operon (Figure 7B). There are several well established PHA production systems using natural microorganisms such as *Wautersia eutropha*, *methylobacterium*, and *Pseudmonas*, and also using recombinant bacteria such as *E. coli* [2, 26], and intracellular accumulation of PHA of over 90% of the cell dry weight has been reported. The use of agroindustrial by-products [27], forest biomass [28], and glycerol (by-product of bio-diesel production) [29] for the substrates of microbial PHA production has also been reported. The production of PHAs in transgenic plants carrying bacterial *phb* genes has also been investigated in *Arabidopsis* 

> Polyhydroxyalkanoate (PHA) synthase

(*phaC*) <sup>O</sup>

<sup>O</sup> ( ) n PHB

*recA*

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205

There are several cyanobacterial strains which can naturally accumulate PHAs, but generally the PHA productivity in these strains are low [31, 32]. Several attempts have also been made to introduce PHA genes into non-PHA-producing cyanobacterial strains

Figure 5 *recA* complementation as a selection pressure for plasmid stability **Figure 5.** recA complementation as a selection pressure for plasmid stability

Figure 6. Procedure for generation of *rec*A null mutant cells carrying plasmids with *E. coli rec*A gene

**4.1. Vector construct with** *recA* **complementation system for PHA production** 

OH O

SCoA

PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), and there are at least 100 different 3-hydroxy alkanoic acids among the PHA constituents [25]. The first PHA discovered was poly(3-hydroxy-butyrate) (PHB). It is a highly crystalline thermoplastic sharing many properties with polypropylene, and the most abundant of the PHAs in nature. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes (Figure 7A), 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC). These three enzymes are encoded by the genes of the *phbCAB* operon (Figure 7B). There are several well established PHA production systems using natural microorganisms such as *Wautersia eutropha*, *methylobacterium*, and *Pseudmonas*, and also using recombinant bacteria such as *E. coli* [2, 26], and intracellular accumulation of PHA of over 90% of the cell dry weight has been reported. The use of agroindustrial by-products [27], forest biomass [28], and glycerol (by-product of bio-diesel production) [29] for the substrates of microbial PHA production has also been reported. The production of PHAs in transgenic plants carrying bacterial *phb* genes has also been investigated in *Arabidopsis* 

> Polyhydroxyalkanoate (PHA) synthase

(*phaC*) <sup>O</sup>

<sup>O</sup> ( ) n PHB

There are several cyanobacterial strains which can naturally accumulate PHAs, but generally the PHA productivity in these strains are low [31, 32]. Several attempts have also been made to introduce PHA genes into non-PHA-producing cyanobacterial strains

Figure 6 Procedure for generation of *recA* null mutant cells carrying plasmids with *E. coli recA* gene CoA reductase**Figure 6.** Procedure for generation of *rec*A null mutant cells carrying plasmids with *E. coli rec*A gene

**4. PHA production by recombinant cyanobacteria** 

*thaliana*, *Gossypium hirsutum* (cotton), and *Zea mays* (corn) [30].

SCoA

acetoacetyl-CoA reductase (*phaB*)

*Bam***HI** *Eco***RI** *phaC phaA phaB*

Figure 7. Biosynthetic pathway for PHA (A) and structure of pha genes (B)

Figure 7 Biosynthetic pathway for PHA (A) and structure of *pha* genes (B)

[33, 34].

SCoA

(Acetyl-CoA)

(A)

(B)

O O O

3-ketothiolase (*phaA*)

*recA*

*recA*

*RecA* partial mutant with shuttlevector carrying *recA* gene

*recA*

Step 2

(Transformation)

*recA*

*RecA* partial mutant

biomass [28], and glycerol (by-product of bio-diesel production) [29] for the substrates of microbial PHA production has also been reported. The production of PHAs in transgenic plants carrying bacterial *phb* genes has also been investigated in *Arabidopsis* 

#### **4. PHA production by recombinant cyanobacteria** *recA recA*

*recA*

Step 1

Homologous recombination with *recA* gene containing *km* cassette

Step 3

*recA*

*RecA* null mutant carrying shuttle-vector with *recA* gene

*recA*

*recA*

Genomic DNA

Wild type cell

cyanobacteria there has been no such a practical plasmid stabilization system reported. We developed a practical plasmid stabilization system by utilizing the *recA* complementation mechanism. RecA is a multifunctional protein that plays key roles in various cellular processes, such as recombination and DNA repair in bacteria [21, 22]. The amino acid sequences of RecA proteins from the different microorganisms are well conserved, and there are several reports on the complementation of *recA* mutation in some bacteria by *E. coli recA* gene. Murphy et al. [23] reported that a *recA* null mutation is lethal in the cyanobacterium, but the *E. coli recA* gene in trans on a plasmid can complement the function of *recA* resulting in segregation of cyanobacterial *recA* null mutant. We expected that this complementation mechanism can be used as a selection pressure to prevent the loss of the plasmid

Figure 5 shows the principles of the selective pressure for the maintainance of plasmid in conventional antibiotics selection (Figure 5A) and our *recA* complementation (Figure 5B) systems. In the conventional antibiotics selection system, the antibiotics-resistant (Ab-R) gene casette is introduced in plasmid, and the loss of the plasmid makes the host cells sensitive to antibiotics. In the *recA* complementation system, the *recA* gene in the genome of the host cells is inactivated by homologous recombination, and the function of genomic *recA* gene is complemented by the *recA* gene on the plasmid. In cyanobacteria, *recA* null mutation is lethal, and the host cell without plasmid can not survive. Generally, cyanobacteria have several copies of genome [24] and *recA* null mutant cells carrying a plasmid with *E. coli recA* gene were generated by three steps as follows (Figure 6); i) generation of *recA* partial mutant of *S.* PCC7002 by homologous recombination with kanamycin resistance gene (*km*) cassette, ii) introduction of shuttle-vector with *E. coli recA* gene into the *recA* partial mutant cells, and iii) conversion of the partial *recA* mutant into the *recA* null mutant by further homologous recombination. The use of *recA* complementation as a selection pressure is a simple and versatile method; only the *E. coli recA* gene on the plasmid, and the partial *recA* mutant host are required, and the *recA* null mutant can easily be obtained by subculturing the cells in the medium with kanamycin (Km). Unlike cyanobacteria, *recA* null mutation is

> Cell death caused by antibiotics

Cell death caused by *recA* mutation

Antibiotics pressure

which causes a significant metabolic burden to the host cells.

Ab-R

204 Environmental Biotechnology - New Approaches and Prospective Applications

(A) Selection by antibiotics pressure (conventional method)

Plasmid

Cyanobacteria cell

Ab-R: antibioticsresistant gene

Genomic DNA

*recA*

*recA*

Plasmid

*RecA* lethal mutation on genomic DNA is complemented by *recA* on plasmid.

*recA*

*RecA* null mutant carrying shuttle-vector with *recA* gene

*recA*

*recA*

*recA*

with *E. coli recA* gene

*recA*

Genomic DNA

Wild type cell

**Figure 5.** recA complementation as a selection pressure for plasmid stability

*recA*

*recA*

(B) Selection by *recA* complementation

not lethal in *E. coli*, thus this complementation system is not applicable for *E. coli*.

Loss of plasmid

Loss of plasmid

*recA*

Figure 6. Procedure for generation of *rec*A null mutant cells carrying plasmids with *E. coli rec*A gene

**4.1. Vector construct with** *recA* **complementation system for PHA production** 

OH O

SCoA

*recA*

*RecA* partial mutant with shuttlevector carrying *recA* gene

*recA*

Step 2

(Transformation)

*recA*

*recA*

*recA*

*recA*

*RecA* partial mutant

PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), and there are at least 100 different 3-hydroxy alkanoic acids among the PHA constituents [25]. The first PHA discovered was poly(3-hydroxy-butyrate) (PHB). It is a highly crystalline thermoplastic sharing many properties with polypropylene, and the most abundant of the PHAs in nature. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes (Figure 7A), 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC). These three enzymes are encoded by the genes of the *phbCAB* operon (Figure 7B). There are several well established PHA production systems using natural microorganisms such as *Wautersia eutropha*, *methylobacterium*, and *Pseudmonas*, and also using recombinant bacteria such as *E. coli* [2, 26], and intracellular accumulation of PHA of over 90% of the cell dry weight has been reported. The use of agroindustrial by-products [27], forest biomass [28], and glycerol (by-product of bio-diesel production) [29] for the substrates of microbial PHA production has also been reported. The production of PHAs in transgenic plants carrying bacterial *phb* genes has also been investigated in *Arabidopsis* 

> Polyhydroxyalkanoate (PHA) synthase

(*phaC*) <sup>O</sup>

<sup>O</sup> ( ) n PHB

*recA*

There are several cyanobacterial strains which can naturally accumulate PHAs, but generally the PHA productivity in these strains are low [31, 32]. Several attempts have also been made to introduce PHA genes into non-PHA-producing cyanobacterial strains

**4. PHA production by recombinant cyanobacteria** 

CoA reductase**Figure 6.** Procedure for generation of *rec*A null mutant cells carrying plasmids with *E. coli rec*A gene

*thaliana*, *Gossypium hirsutum* (cotton), and *Zea mays* (corn) [30].

SCoA

acetoacetyl-CoA reductase (*phaB*)

*Bam***HI** *Eco***RI** *phaC phaA phaB*

Figure 7. Biosynthetic pathway for PHA (A) and structure of pha genes (B)

Figure 7 Biosynthetic pathway for PHA (A) and structure of *pha* genes (B)

[33, 34].

SCoA

(Acetyl-CoA)

(A)

(B)

O O O

3-ketothiolase (*phaA*)

Figure 5. recA complementation as a selection pressure for plasmid stability

Step 1

Homologous recombination with *recA* gene containing *km* cassette

Step 3

Figure 6 Procedure for generation of *recA* null mutant cells carrying plasmids

Figure 5 *recA* complementation as a selection pressure for plasmid stability

#### **4.1. Vector construct with** *recA* **complementation system for PHA production** Figure 6 Procedure for generation of *recA* null mutant cells carrying plasmids with *E. coli recA* gene

PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), and there are at least 100 different 3-hydroxy alkanoic acids among the PHA constituents [25]. The first PHA discovered was poly(3-hydroxy-butyrate) (PHB). It is a highly crystalline thermoplastic sharing many properties with polypropylene, and the most abundant of the PHAs in nature. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes (Figure 7A), 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). These three enzymes are encoded by the genes of the *phbCAB* operon (Figure 7B). There are several well established PHA production systems using natural microorganisms such as *Wautersia eutropha*, *Methylobacterium*, and *Pseudmonas*, and also using recombinant bacteria such as *E. coli* [2, 26], and intracellular accumulation of PHA of over 90% of the cell dry weight has been reported. The use of agroindustrial by-products [27], forest biomass [28], and glycerol (by-product of bio-diesel production) [29] for the substrates of microbial PHA production has also been reported. The production of PHAs in transgenic plants carrying bacterial *phb* genes has also been investigated in *Arabidopsis thaliana*, *Gossypium hirsutum* (cotton), and *Zea mays* (corn) [30]. Figure 6. Procedure for generation of *rec*A null mutant cells carrying plasmids with *E. coli rec*A gene **4. PHA production by recombinant cyanobacteria 4.1. Vector construct with** *recA* **complementation system for PHA production**  PHAs are linear head to tail polyesters composed of 3-hydroxy fatty acid monomers (Figure 1), and there are at least 100 different 3-hydroxy alkanoic acids among the PHA constituents [25]. The first PHA discovered was poly(3-hydroxy-butyrate) (PHB). It is a highly crystalline thermoplastic sharing many properties with polypropylene, and the most abundant of the PHAs in nature. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes (Figure 7A), 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC). These three enzymes are encoded by the genes of the *phbCAB* operon (Figure 7B). There are several well established PHA production systems using natural microorganisms such as *Wautersia eutropha*, *methylobacterium*, and *Pseudmonas*, and also using recombinant bacteria such as *E. coli* [2, 26], and intracellular accumulation of PHA of over 90% of the cell dry weight has been reported. The use of agroindustrial by-products [27], forest

*thaliana*, *Gossypium hirsutum* (cotton), and *Zea mays* (corn) [30].

**Figure 7.** Biosynthetic pathway for PHA (A) and structure of pha genes (B)

Figure 7. Biosynthetic pathway for PHA (A) and structure of pha genes (B) There are several cyanobacterial strains which can naturally accumulate PHAs, but generally the PHA productivity in these strains are low [31, 32]. Several attempts have also been made to introduce PHA genes into non-PHA-producing cyanobacterial strains There are several cyanobacterial strains which can naturally accumulate PHAs, but generally the PHA productivity in these strains are low [31, 32]. Several attempts have also been made to introduce PHA genes into non-PHA-producing cyanobacterial strains [33, 34].

Figure 7 Biosynthetic pathway for PHA (A) and structure of *pha* genes (B)

[33, 34]. We investigated the production of PHA by the recombinant cyanobacteria with the *recA* complementation antibiotics-free cyanobacterial expression system [35].

cyanobacterial expression system [35].

Figure 8 DNA constructs on pAQJ4 vector for PHA production

We investigated the production of PHA by the recombinant cyanobacteria with the *recA* complementation antibiotics-free

Figure 9. Integrity of the genomic *rec*A gene in transformant cyanobacteria. The genomic DNA was isolated from the wild type *S*. PCC7002, Synpha/B transformant, and Syn-pha/Ac transformant, and the *rec*A gene and *rec*A with a *km* cassette (*recA::km*) where amplified by PCR. The PCR products were analyzed with 1% agarose gel electrophoresis. M: Molecular weight marker, lane 1: wild type *S*. PCC7002, lane 2: transformant Syn-

http://dx.doi.org/10.5772/54705

207

To obtain the *recA* null mutant of Syn-pha/B and Syn-pha/Ac transformants, the transformant cells were subcultured in the liquid medium with carbenicillin (4 g / ml) and Km (200 g /ml). Each liquid culture was allowed to grow into the late stationary phase prior to subculturing to enhance the efficiency of homologous recombination. After five times subculturing, the integrity of the genomic *recA* gene was examined with PCR (Figure 9). In the Syn-pha/Ac transformant cells, only the DNA fragment corresponding to the *recA* with km cassette was amplified, and no DNA fragment of wild type *recA* gene was detected on the agarose gel, indicating that the Syn-pha/Ac transformant was changed to a *recA* null mutant. On the other hand, in the Syn-pha/B transformant cells, both *recA* with km cassette and wild type *recA* fragments were amplified, thus the *recA* gene in cyanobacterial genome was not completely inactivated. The reason for the failure of *recA* null mutant segregation in the Syn-pha/B transformant is not clear, but a possible explanation is the insufficient complementation of RecA protein by the *E. coli recA* gene on the plasmid.

The Syn-pha/Ac transformant was used for the following experiments for the *pha* gene stability and PHA production.

pha/B (*rec*A partial mutant), and lane 3: transformant Syn-pha/ac (*rec*A null mutant)

**Figure 9.** Integrity of the genomic *rec*A gene in transformant cyanobacteria. The genomic DNA was isolated from the wild type *S*. PCC7002, Syn-pha/B transformant, and Syn-pha/Ac transformant, and the *rec*A gene and *rec*A with a *km* cassette (*recA::km*) where amplified by PCR. The PCR products were analyzed with 1% agarose gel electrophoresis. M: Molecular weight marker, lane 1: wild type *S*. PCC7002, lane 2: transformant Syn-pha/B (*rec*A partial mutant), and

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

To obtain the *recA* null mutant of Syn-pha/B and Syn-pha/Ac transformants, the transformant cells were subcultured in the liquid medium with carbenicillin (4 μg / ml) and Km (200 μg / ml). Each liquid culture was allowed to grow into the late stationary phase prior to subcul‐ turing to enhance the efficiency of homologous recombination. After five times subculturing, the integrity of the genomic *recA* gene was examined with PCR (Figure 9). In the Syn-pha/Ac transformant cells, only the DNA fragment corresponding to the *recA* with *km* cassette was amplified, and no DNA fragment of wild type *recA* gene was detected on the agarose gel, indicating that the Syn-pha/Ac transformant was changed to a *recA* null mutant. On the other hand, in the Syn-pha/B transformant cells, both *recA* with *km* cassette and wild type *recA* fragments were amplified, thus the *recA* gene in cyanobacterial genome was not completely inactivated. The reason for the failure of *recA* null mutant segregation in the Syn-pha/B transformant is not clear, but a possible explanation is the insufficient complementation of RecA protein by the *E. coli recA* gene on the plasmid. The Syn-pha/Ac transformant was used

for the following experiments for the *pha* gene stability and PHA production.

lane 3: transformant Syn-pha/ac (*rec*A null mutant)

**Figure 8.** DNA constructs on pAQJ4 vector for PHA production

Figure 8. DNA constructs on pAQJ4 vector for PHA production

The *pha* genes and the *E. coli recA* gene were integrated on the shuttle vector pAQJ4, and the genomic *recA* genes of the transformant cyanobacteria were inactivated by an homologous recombination with the cyanobacterial *recA* gene containing a kanamycin resistance (*km*) cassette insertion. Figure 8 shows the *pha* and *E. coli recA* genes constructs on the pAQJ4 vector. For the *pha* genes, the *phaCAB* operon of *Wautersia eutropha* was used, and the *E. coli recA* gene (1.66 kb) with its upstream promoter region was connected to the *pha* genes in the same or opposite directions. For the expression of *pha* genes, the R6 promoter fragment (Figure 4), which lacks the CO2-down-regulating element, was used. These two constructs were introduced into the pAQJ4-MCS (MCS: multiple cloning site) and pAQJ4-MCS(c) vectors (GenBank accession numbers AB480231 and AB480232, each contains the MCS in a different orientation), generating the four kinds of vector constructs A, B, A-complementary (Ac) and B-complementary (Bc) as shown in Figure 8. The partial *recA* mutant cells of *S.* PCC7002 was used for the transformation. The *recA* genes of the host cyanobacteria were partially inactivated by a homologous recombination with the *recA* gene containing a *km* cassette, and the partial *recA* mutant cells were transformed with the four kinds of pha-*recA* constructs (A, B, Ac, and Bc). The transformant cyanobacteria were obtained only for constructs B, and Ac, and these transformants were designated as Syn-pha/B and Synpha/Ac, respectively. The reason for the failure in the isolation of the transformants in the other constructs is not clear, but we speculate that the expression efficiency of *pha* genes might be too high in these constructs, and as a result the transformants could not gain enough energy and/or cellular metabolites for growth. The *pha* genes and the *E. coli recA* gene were integrated on the shuttle vector pAQJ4, and the genomic *recA* genes of the transformant cyanobacteria were inactivated by an homologous recombination with the cyanobacterial *recA* gene containing a kanamycin resistance (*km*) cassette insertion. Figure 8 shows the *pha* and *E. colirecA* genes constructs on the pAQJ4 vector. For the *pha* genes, the *phaCAB* operon of *Wautersia eutropha* was used, and the *E. coli recA* gene (1.66 kb) with its upstream promoter region was connected to the *pha* genes in the same or opposite directions. For the expression of *pha* genes, the R6 promoter fragment (Figure 4), which lacks the CO2-down-regulating element, was used. These two constructs were intro‐ duced into the pAQJ4-MCS (MCS: multiple cloning site) and pAQJ4-MCS(c) vectors (GenBank accession numbers AB480231 and AB480232, each contains the MCS in a different orientation), generating the four kinds of vector constructs A, B, A-complementary (Ac) and B-comple‐ mentary (Bc) as shown in Figure 8. The partial *recA* mutant cells of *S.* PCC7002 was used for the transformation. The *recA* genes of the host cyanobacteria were partially inactivated by a homologous recombination with the *recA* gene containing a *km* cassette, and the partial *recA* mutant cells were transformed with the four kinds of *pha-recA* constructs (A, B, Ac, and Bc). The transformant cyanobacteria were obtained only for constructs B, and Ac, and these transformants were designated as Syn-pha/B and Syn-pha/Ac, respectively. The reason for the failure in the isolation of the transformants in the other constructs is not clear, but we speculate that the expression efficiency of *pha* genes might be too high in these constructs, and as a result the transformants could not gain enough energy and/or cellular metabolites for growth.

We investigated the production of PHA by the recombinant cyanobacteria with the *recA* complementation antibiotics-free

**R6 promoter**

**R6 promoter**

*Bam***HI** *Bam***HI** *Eco***RI**

*Bam***HI** *Bam***HI** *Eco***RI**

*Eco***RI** *Bam***HI** *Bam***HI**

*Eco***RI** *Bam***HI** *Bam***HI**

**pAQJ4-MCS**

**MCS**

(5878bp)

Ampr

Figure 8 DNA constructs on pAQJ4 vector for PHA production

The *pha* genes and the *E. coli recA* gene were integrated on the shuttle vector pAQJ4, and the genomic *recA* genes of the transformant cyanobacteria were inactivated by an homologous recombination with the cyanobacterial *recA* gene containing a kanamycin resistance (*km*) cassette insertion. Figure 8 shows the *pha* and *E. colirecA* genes constructs on the pAQJ4 vector. For the *pha* genes, the *phaCAB* operon of *Wautersia eutropha* was used, and the *E. coli recA* gene (1.66 kb) with its upstream promoter region was connected to the *pha* genes in the same or opposite directions. For the expression of *pha* genes, the R6 promoter fragment (Figure 4), which lacks the CO2-down-regulating element, was used. These two constructs were intro‐ duced into the pAQJ4-MCS (MCS: multiple cloning site) and pAQJ4-MCS(c) vectors (GenBank accession numbers AB480231 and AB480232, each contains the MCS in a different orientation), generating the four kinds of vector constructs A, B, A-complementary (Ac) and B-comple‐ mentary (Bc) as shown in Figure 8. The partial *recA* mutant cells of *S.* PCC7002 was used for the transformation. The *recA* genes of the host cyanobacteria were partially inactivated by a homologous recombination with the *recA* gene containing a *km* cassette, and the partial *recA* mutant cells were transformed with the four kinds of *pha-recA* constructs (A, B, Ac, and Bc). The transformant cyanobacteria were obtained only for constructs B, and Ac, and these transformants were designated as Syn-pha/B and Syn-pha/Ac, respectively. The reason for the failure in the isolation of the transformants in the other constructs is not clear, but we speculate that the expression efficiency of *pha* genes might be too high in these constructs, and as a result the transformants could not gain enough energy and/or cellular metabolites for growth.

ORF943

*phaB phaA phaC E. coli recA*

*phaB phaA phaC E. coli recA*

*E. coli recA phaC phaA phaB*

*E. coli recA phaC phaA phaB*

**R6 promoter**

**R6 promoter**

cyanobacterial expression system [35].

206 Environmental Biotechnology - New Approaches and Prospective Applications

**Construct A**

**Construct B**

**Construct Ac**

**Construct Bc**

Figure 8. DNA constructs on pAQJ4 vector for PHA production

**Figure 8.** DNA constructs on pAQJ4 vector for PHA production

not gain enough energy and/or cellular metabolites for growth.

The *pha* genes and the *E. coli recA* gene were integrated on the shuttle vector pAQJ4, and the genomic *recA* genes of the transformant cyanobacteria were inactivated by an homologous recombination with the cyanobacterial *recA* gene containing a kanamycin resistance (*km*) cassette insertion. Figure 8 shows the *pha* and *E. coli recA* genes constructs on the pAQJ4 vector. For the *pha* genes, the *phaCAB* operon of *Wautersia eutropha* was used, and the *E. coli recA* gene (1.66 kb) with its upstream promoter region was connected to the *pha* genes in the same or opposite directions. For the expression of *pha* genes, the R6 promoter fragment **Figure 9.** Integrity of the genomic *rec*A gene in transformant cyanobacteria. The genomic DNA was isolated from the wild type *S*. PCC7002, Syn-pha/B transformant, and Syn-pha/Ac transformant, and the *rec*A gene and *rec*A with a *km* cassette (*recA::km*) where amplified by PCR. The PCR products were analyzed with 1% agarose gel electrophoresis. M: Molecular weight marker, lane 1: wild type *S*. PCC7002, lane 2: transformant Syn-pha/B (*rec*A partial mutant), and lane 3: transformant Syn-pha/ac (*rec*A null mutant)

(Figure 4), which lacks the CO2-down-regulating element, was used. These two constructs were introduced into the pAQJ4-MCS (MCS: multiple cloning site) and pAQJ4-MCS(c) vectors (GenBank accession numbers AB480231 and AB480232, each contains the MCS in a different orientation), generating the four kinds of vector constructs A, B, A-complementary (Ac) and B-complementary (Bc) as shown in Figure 8. The partial *recA* mutant cells of *S.* PCC7002 was used for the transformation. The *recA* genes of the host cyanobacteria were partially inactivated by a homologous recombination with the *recA* gene containing a *km* cassette, and the partial *recA* mutant cells were transformed with the four kinds of pha-*recA* constructs (A, B, Ac, and Bc). The transformant cyanobacteria were obtained only for constructs B, and Ac, and these transformants were designated as Syn-pha/B and Synpha/Ac, respectively. The reason for the failure in the isolation of the transformants in the other constructs is not clear, but we speculate that the expression efficiency of *pha* genes might be too high in these constructs, and as a result the transformants could Figure 9. Integrity of the genomic *rec*A gene in transformant cyanobacteria. The genomic DNA was isolated from the wild type *S*. PCC7002, Synpha/B transformant, and Syn-pha/Ac transformant, and the *rec*A gene and *rec*A with a *km* cassette (*recA::km*) where amplified by PCR. The PCR products were analyzed with 1% agarose gel electrophoresis. M: Molecular weight marker, lane 1: wild type *S*. PCC7002, lane 2: transformant Synpha/B (*rec*A partial mutant), and lane 3: transformant Syn-pha/ac (*rec*A null mutant) To obtain the *recA* null mutant of Syn-pha/B and Syn-pha/Ac transformants, the transformant cells were subcultured in the liquid medium with carbenicillin (4 g / ml) and Km (200 g /ml). Each liquid culture was allowed to grow into the late stationary phase prior to subculturing to enhance the efficiency of homologous recombination. After five times subculturing, the integrity of the genomic *recA* gene was examined with PCR (Figure 9). In the Syn-pha/Ac transformant cells, only the DNA fragment corresponding to the *recA* with km cassette was amplified, and no DNA fragment of wild type *recA* gene was detected on the agarose gel, indicating that the Syn-pha/Ac transformant was changed to a *recA* null mutant. On the other hand, in the Syn-pha/B transformant cells, both *recA* with km cassette and wild type *recA* fragments were amplified, thus the *recA* gene in cyanobacterial genome was not completely inactivated. The reason for the failure of *recA* null mutant segregation in the Syn-pha/B transformant is not clear, but a possible explanation is the insufficient complementation of RecA protein by the *E. coli recA* gene on the plasmid. To obtain the *recA* null mutant of Syn-pha/B and Syn-pha/Ac transformants, the transformant cells were subcultured in the liquid medium with carbenicillin (4 μg / ml) and Km (200 μg / ml). Each liquid culture was allowed to grow into the late stationary phase prior to subcul‐ turing to enhance the efficiency of homologous recombination. After five times subculturing, the integrity of the genomic *recA* gene was examined with PCR (Figure 9). In the Syn-pha/Ac transformant cells, only the DNA fragment corresponding to the *recA* with *km* cassette was amplified, and no DNA fragment of wild type *recA* gene was detected on the agarose gel, indicating that the Syn-pha/Ac transformant was changed to a *recA* null mutant. On the other hand, in the Syn-pha/B transformant cells, both *recA* with *km* cassette and wild type *recA* fragments were amplified, thus the *recA* gene in cyanobacterial genome was not completely inactivated. The reason for the failure of *recA* null mutant segregation in the Syn-pha/B transformant is not clear, but a possible explanation is the insufficient complementation of RecA protein by the *E. coli recA* gene on the plasmid. The Syn-pha/Ac transformant was used for the following experiments for the *pha* gene stability and PHA production.

The Syn-pha/Ac transformant was used for the following experiments for the *pha* gene stability and PHA production.

*recA* null mutant transformant (A), wild type (nonrecA-mutant) transformant (B), and *recA* partial mutant transformant (C) All the transformants (*recA* null mutant, wild type, and *recA* partial mutant) carry the construct Ac **Figure 10.** The stability of PHA productivity in the *recA* null mutant transformant (A), wild type (non-*recA*-mutant) transformant (B), and *recA* partial mutant transformant (C). All the transformants (*recA* null mutant, wild type, and *recA* partial mutant) carry the construct Ac plasmid of figure 8. The cells were subcultured in the antibiotics free medi‐ um for five times at one week intervals, and the PHA contents in the cells were determinated at the end of each cul‐ ture. The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as black bars).

Figure 10 The stability of PHA productivity in the

plasmid of Figure 8. The cells were subcultured in

the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determined at the end of each culture. The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as black bars). The stability of PHA productivity in the *recA* null mutant of Syn-pha/Ac transformant was examined in comparison to the wild type (non- *recA* -mutant) transformant cells carrying the construct Ac plasmid of Figure 8, and *recA* partial mutant of Syn-pha/Ac transformant. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determined at the end of each culture. The cell densities at the end of cultures were 7.5 x 108 to 1 x 109 /ml, and the passage of culture was done by diluting the culture into a fresh medium at a dilution ratio of 1:1,000. Figure 10 shows the changes in the PHA productivities in the transformant cells of the *recA* null mutant (Figure 10A), wild type (non- *recA* -mutant) (Figure 10B), and *recA* partial mutant (Figure 10C). The PHA

antibiotics (passage number 0, shown as black bars).

Figure 10.The stability of PHA productivity in the *recA* null mutant transformant (A), wild type (non-*recA*-mutant) transformant (B), and *recA* partial mutant transformant (C). All the transformants (*recA* null mutant, wild type, and *recA* partial mutant) carry the construct Ac plasmid of figure 8. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determinated at the end of each culture. The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with

productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as black bars in Figure 10). The PHA productivities in the *recA* null mutant (Figure 10A) were kept at the approximately same level with that of passage number 0 at the passage numbers 1 through 4 in the antibiotics free medium, but suddenly decreased to 45% of the passage number 0 at the passage number 5. On the other hand, the PHA productivities in the wild type (non- *recA* -mutant) transformant significantly decreased at the passage number 1 (45% of the passage number 0), and no PHA production was detected at the end of passage number 2 (Figure 10B). Interestingly a partial positive effect for the PHA productivity was observed in the *recA* partial mutant (Figure 10C); the PHA productivity decreased gradually during the consecutive culture passages to a trace level of PHA production at the passage number 6. These results indicate that the *recA* complementation effectively acted as a selection pressure in the *recA* null mutant for the maintenance of the plasmid carrying the *pha* genes, at least for 3 to 4 passages at a dilution rate of 1:1,000. The cell

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

dilution rate of 1:1,000, and therefore this antibiotics-free PHA production system is applicable to the large scale PHA production. The reason for the sudden decrease in PHA productivity in the *recA* null mutant at the passage 5 is not clear, but this might not be caused by the loss of plasmid in the cyanobacterial cells because the cells of passage numbers 4 (high PHA produc‐ tivity) and 5 (low productivity) did not show any difference in colony forming ability on the antibiotics (carbenicillin) plates. The decrease in the PHA productivity at the passage numbers 4 and 5 might, therefore, be attributed to the other reasons, such as the mutation in *pha* genes

Figure 11 shows the electron micrograph of the control wild type *S.* PCC7002 (A), and the PHA accumulating *recA* null mutant transformant (Syn-pha/Ac) (B) cells. The small PHA granules aligning along the thilacoid membrane were observed in the Syn-phaAc transformant cell. The molecular mass distribution of the PHA was estimated with the gel permeation chromatog‐ raphy (GPC). The molecular mass distribution of the PHA from the Syn-pha/Ac transformant was a little shifted to the higher side compared to that of the standard PHA from *W. eutropha* (Figure 12), but in the range previously reported for various microbial PHAs. The main component of the hydroxyalkanoic acid of the PHA from the Syn-phaAc transformant was hydroxybutyric acid (more than 98%), and a small amount of lactic acid (0.5 to 1.5 %), and a

To obtain a higher PHA accumulation in the cyanobacterial cells, the nutrient condition of the culture was also examined. Since it is reported that the nitrogen and phosphorus supplies, much affect the PHA accumulation in microorganisms [36, 37], the Syn-phaAc transformant cells were cultured in the medium containing various concentrations of nitrogen (NaNO3) and phosphorus (KH2PO4) sources, and the cell growth and PHA accumulation were compared (Figure 13). There was a clear negative relationship between cell growth and PHA accumula‐ tion, and nitrogen limitation seemed to be effective for the accumulation of PHA although the cell growth was significantly suppressed in the nitrogen limited medium. The maximum PHA

times with three culture passages at a

http://dx.doi.org/10.5772/54705

209

number (and also culture scale) can be increased 109

and/or the inhibition of the expression of *pha* genes on the plasmid.

**4.2. PHA production by transformant cyanobacteria cells**

trace amount of hydroxyvaleric acid were also contained.

The stability of PHA productivity in the *recA* null mutant of Syn-pha/Ac transformant was examined in comparison to the wild type (non- *recA* -mutant) transformant cells carrying the construct Ac plasmid of Figure 8, and *recA* partial mutant of Syn-pha/Ac transformant. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determined at the end of each culture. The cell densities at the end of cultures were 7.5 x 108 to 1 x 109 /ml, and the passage of culture was done by diluting the culture into a fresh medium at a dilution ratio of 1:1,000. Figure 10 shows the changes in the PHA productivities in the transformant cells of the *recA* null mutant (Figure 10A), wild type (non- *recA* -mutant) (Figure 10B), and *recA* partial mutant (Figure 10C). The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as black bars in Figure 10). The PHA productivities in the *recA* null mutant (Figure 10A) were kept at the approximately same level with that of passage number 0 at the passage numbers 1 through 4 in the antibiotics free medium, but suddenly decreased to 45% of the passage number 0 at the passage number 5. On the other hand, the PHA productivities in the wild type (non- *recA* -mutant) transformant significantly decreased at the passage number 1 (45% of the passage number 0), and no PHA production was detected at the end of passage number 2 (Figure 10B). Interestingly a partial positive effect for the PHA productivity was observed in the *recA* partial mutant (Figure 10C); the PHA productivity decreased gradually during the consecutive culture passages to a trace level of PHA production at the passage number 6. These results indicate that the *recA* complementation effectively acted as a selection pressure in the *recA* null mutant for the maintenance of the plasmid carrying the *pha* genes, at least for 3 to 4 passages at a dilution rate of 1:1,000. The cell number (and also culture scale) can be increased 109 times with three culture passages at a dilution rate of 1:1,000, and therefore this antibiotics-free PHA productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as black bars in Figure 10). The PHA productivities in the *recA* null mutant (Figure 10A) were kept at the approximately same level with that of passage number 0 at the passage numbers 1 through 4 in the antibiotics free medium, but suddenly decreased to 45% of the passage number 0 at the passage number 5. On the other hand, the PHA productivities in the wild type (non- *recA* -mutant) transformant significantly decreased at the passage number 1 (45% of the passage number 0), and no PHA production was detected at the end of passage number 2 (Figure 10B). Interestingly a partial positive effect for the PHA productivity was observed in the *recA* partial mutant (Figure 10C); the PHA productivity decreased gradually during the consecutive culture passages to a trace level of PHA production at the passage number 6. These results indicate that the *recA* complementation effectively acted as a selection pressure in the *recA* null mutant for the maintenance of the plasmid carrying the *pha* genes, at least for 3 to 4 passages at a dilution rate of 1:1,000. The cell number (and also culture scale) can be increased 109 times with three culture passages at a dilution rate of 1:1,000, and therefore this antibiotics-free PHA production system is applicable to the large scale PHA production. The reason for the sudden decrease in PHA productivity in the *recA* null mutant at the passage 5 is not clear, but this might not be caused by the loss of plasmid in the cyanobacterial cells because the cells of passage numbers 4 (high PHA produc‐ tivity) and 5 (low productivity) did not show any difference in colony forming ability on the antibiotics (carbenicillin) plates. The decrease in the PHA productivity at the passage numbers 4 and 5 might, therefore, be attributed to the other reasons, such as the mutation in *pha* genes and/or the inhibition of the expression of *pha* genes on the plasmid.

### **4.2. PHA production by transformant cyanobacteria cells**

Figure 10.The stability of PHA productivity in the *recA* null mutant transformant (A), wild type (non-*recA*-mutant) transformant (B), and *recA* partial mutant transformant (C). All the transformants (*recA* null mutant, wild type, and *recA* partial mutant) carry the construct Ac plasmid of figure 8. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determinated at the end of each culture. The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with

The stability of PHA productivity in the *recA* null mutant of Syn-pha/Ac transformant was examined in comparison to the wild type (non- *recA* -mutant) transformant cells carrying the construct Ac plasmid of Figure 8, and *recA* partial mutant of Syn-pha/Ac transformant. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determined at the end of each culture. The cell densities at the end of cultures were 7.5 x 108 to 1 x 109 /ml, and the passage of culture was done by diluting the culture into a fresh medium at a dilution ratio of 1:1,000. Figure 10 shows the changes in the PHA productivities in the transformant cells of the *recA* null mutant (Figure 10A), wild type (non- *recA* -mutant) (Figure 10B), and *recA* partial mutant (Figure 10C). The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as black bars in Figure 10). The PHA productivities in the *recA* null mutant (Figure 10A) were kept at the approximately same level with that of passage number 0 at the passage numbers 1 through 4 in the antibiotics free medium, but suddenly decreased to 45% of the passage number 0 at the passage number 5. On the other hand, the PHA productivities in the wild type (non- *recA* -mutant) transformant significantly decreased at the passage number 1 (45% of the passage number 0), and no PHA production was detected at the end of passage number 2 (Figure 10B). Interestingly a partial positive effect for the PHA productivity was observed in the *recA* partial mutant (Figure 10C); the PHA productivity decreased gradually during the consecutive culture passages to a trace level of PHA production at the passage number 6. These results indicate that the *recA* complementation effectively acted as a selection pressure in the *recA* null mutant for the maintenance of the plasmid carrying the *pha* genes, at least for 3 to 4 passages at a dilution rate of 1:1,000. The cell number (and also culture scale) can be increased 109 times with three culture passages at a dilution rate of 1:1,000, and therefore this antibiotics-free PHA

antibiotics (passage number 0, shown as black bars).

0

100

0

black bars).

mutant transformant (C)

to 1 x 109

100

Productivity

(passage number 0, shown as black bars).

the end of cultures were 7.5 x 108

C

Productivity

B

Productivity

A

(% of control)

(% of control)

(% of control)

0

0123456

Passage number

0123456

Passage number

Figure 10 The stability of PHA productivity in the *recA* null mutant transformant (A), wild type (nonrecA-mutant) transformant (B), and *recA* partial

**Figure 10.** The stability of PHA productivity in the *recA* null mutant transformant (A), wild type (non-*recA*-mutant) transformant (B), and *recA* partial mutant transformant (C). All the transformants (*recA* null mutant, wild type, and *recA* partial mutant) carry the construct Ac plasmid of figure 8. The cells were subcultured in the antibiotics free medi‐ um for five times at one week intervals, and the PHA contents in the cells were determinated at the end of each cul‐ ture. The PHA productivities were expressed as the percentage to the PHA content in the cells cultured with antibiotics

The stability of PHA productivity in the *recA* null mutant of Syn-pha/Ac transformant was examined in comparison to the wild type (non- *recA* -mutant) transformant cells carrying the construct Ac plasmid of Figure 8, and *recA* partial mutant of Syn-pha/Ac transformant. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determined at the end of each culture. The cell densities at

the culture into a fresh medium at a dilution ratio of 1:1,000. Figure 10 shows the changes in the PHA productivities in the transformant cells of the *recA* null mutant (Figure 10A), wild type (non- *recA* -mutant) (Figure 10B), and *recA* partial mutant (Figure 10C). The PHA

All the transformants (*recA* null mutant, wild type, and *recA* partial mutant) carry the construct Ac plasmid of Figure 8. The cells were subcultured in the antibiotics free medium for five times at one week intervals, and the PHA contents in the cells were determined at the end of each culture. The PHA productivities were expressed as the

percentage to the PHA content in the cells cultured with antibiotics (passage number 0, shown as

/ml, and the passage of culture was done by diluting

0123456

Passage number

100

208 Environmental Biotechnology - New Approaches and Prospective Applications

Figure 11 shows the electron micrograph of the control wild type *S.* PCC7002 (A), and the PHA accumulating *recA* null mutant transformant (Syn-pha/Ac) (B) cells. The small PHA granules aligning along the thilacoid membrane were observed in the Syn-phaAc transformant cell. The molecular mass distribution of the PHA was estimated with the gel permeation chromatog‐ raphy (GPC). The molecular mass distribution of the PHA from the Syn-pha/Ac transformant was a little shifted to the higher side compared to that of the standard PHA from *W. eutropha* (Figure 12), but in the range previously reported for various microbial PHAs. The main component of the hydroxyalkanoic acid of the PHA from the Syn-phaAc transformant was hydroxybutyric acid (more than 98%), and a small amount of lactic acid (0.5 to 1.5 %), and a trace amount of hydroxyvaleric acid were also contained.

To obtain a higher PHA accumulation in the cyanobacterial cells, the nutrient condition of the culture was also examined. Since it is reported that the nitrogen and phosphorus supplies, much affect the PHA accumulation in microorganisms [36, 37], the Syn-phaAc transformant cells were cultured in the medium containing various concentrations of nitrogen (NaNO3) and phosphorus (KH2PO4) sources, and the cell growth and PHA accumulation were compared (Figure 13). There was a clear negative relationship between cell growth and PHA accumula‐ tion, and nitrogen limitation seemed to be effective for the accumulation of PHA although the cell growth was significantly suppressed in the nitrogen limited medium. The maximum PHA accumulation was 52% of cell dry weight, the highest among the ever reported PHA accumu‐ lation in cyanobacteria. Accordingly the two-staged culture system consisting of cell growth and PHA production phases should be applied to increase the total productivity (g per liter culture) of PHA. Asada et al. reported that acetyl-CoA flux is the limiting factor in PHA production by genetically engineered cyanobacterium [32], and the high PHA productivity in Syn-phaAc transformant cells suggests the abundant intracellular supply of acetyl-CoA in *S.* PCC7002.

Our study is the first practical approach for the antibiotics-free maintenance of plasmid in cyanobacteria, and with this system the fixation and direct conversion of CO2 into the useful

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

http://dx.doi.org/10.5772/54705

211

The future research subjects to realize the on-site CO2 fixation and utilization system with

**1.** Although the promoter derived from the *rbc* gene was found to be quite effective both in cyanobacterial and bacterial cells, and the PHA production by the transformed cyano‐ bacterial cells was also quite successful with this promoter, the use of a switchable

**2.** Method and system for the efficient harvesting of cyanobacteria at low energy and low

**3.** Photosynthetic CO2 assimilation only occurs during the day, and the productivity of biomaterials is much influenced by light condition. The use of cyanobacteria capable of growing photoautotrophic and also heterotrophic (with waste water) is one possible

**4.** Generally biomaterials, such as fuel and plastic, produced from CO2 are low price. Simultaneous prodcuction of higher value products, such as fine chemicals, can lower the

(1, 10)

the ratio of each nutritient to the standard concentration is shown in the parenthesis.

0123 Cell dry weight (g/l)

Figure 13 Effects of nitrogen and phosphorus concentrations in medium on cell

**Figure 13.** Effect of nitrogen and phosphorus concentrations in medium on cell growth and PHA accumulation in Transformant cyanovacteria. The cells (initial cell density; 5x106 cells/ml) were cultured in the 50 ml medium contain‐ ing various concentration of NaNO3 and KH2PO4. The standard concentration of NaNO3 and KH2PO4 are 1g/l and 50 mg/l, respectively. The standard concentrations of NaNO3 and KH2PO4 are shown as (1,1), and the ratio of each nutri‐

The cells (initial cell density; 5 x 106 cells / ml) were cultured in the 50 ml medium

containing various concentrations of NaNO3 and KH2PO4. The standard concentration of NaNO3 and KH2PO4 are 1 g/l and 50 mg/l, respectively. The standard concentrations of NaNO3 and KH2PO4 are shown as (1, 1), and the ratio of

each nutrient to the standard concentration is shown in the parentheses.

(1, 0.1) (1, 1)

Standard (1, 1):

NaNO3 1g/l, KH2PO4 50mg/l

(5, 1)

[1] Lal R. Carbon sequestration. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2008; 363 815-830.

engineered marine cyanobacterium. Stud. Surf. Sci. Catal. 1998; 114 237-242.

plasmids and characterization of their transformation properties. J. Bacteriol. 1983; 154 1446-1450.

simple functional expression screening with *E. coli*. World J. Microbiol. Biotechnol. 2000; 16 23-29.

Figure 13.Effect of nitrogen and phosphorus concentrations in medium on cell growth and PHA accumulation in Transformant cyanovacteria. The cells (initial cell density; 5x106 cells/ml) were cultured in the 50 ml medium containing various concentration of NaNO3 and KH2PO4. The standard concentration of NaNO3 and KH2PO4 are 1g/l and 50 mg/l, respectively. The standard concentrations of NaNO3 and KH2PO4 are shown as (1,1), and

The fixation and direct conversion of CO2 into the useful biomaterials by the transgenic cyanobacteria are two processes of a promising technology for the coming low carbon economy. We have developed an efficient shuttle-vector between the marine cyanobacterium *Synechococcus sp.* PCC7002 and *E. coli*, and also a practical antibiotics-free cyanobacterial plasmid expression system by using the complementation of the cyanobacterial *recA* null mutation with the *E. coli recA* gene on the plasmid. Although considerable researches are still required to realize the practical on-site applications of the present system to the industrial emission sites, such as thermal power plants, this technology can be a promising option for the biological conversion of CO2 into useful

[2] Madison, L. L., Huisman, G. W. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol.

[3] Chen G. Q. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem. Soc. Rev. 2009; 38 2434-2446. [4] Buzby J. S., Porter R. D., Stevens S. E. Jr. Plasmid transformation in *Agmenellum quadruplicatum* PR-6: construction of biphasic

[5] Tabita R. F., Stevens S. E. Jr., Quijano R. D-ribulose 1, 5-diphosphate carboxylase from blue-green algae. Biochem. Biophys.

[6] Akiyama H., Kanai S., Hirano M., Miyasaka H. Nucleotide sequences of plasmid pAQ1 of marine cyanobacterium

[7] Miyasaka H., Nakano H., Akiyama H., Kanai S., Hirano M. Production of PHA (poly hydroxyalkanoate) by the genetically

[8] Akiyama H., Kanai S., Hirano M., Miyasaka H. A novel plasmid recombination mechanism of the marine cyanobacterium

[9] Lorimier R., Guglielmi G., Bryant D. A., Stevens S. E. Jr. Functional expression of plastid allophycocyanin genes in a

[10] Tanaka S., Ikeda K. Miyasaka H. Isolation of a new member of group 3 late embryogenesis abundant (LEA) protein gene from a halotorelant green alga by a functional expression screening with cyanobacterial cells. FEMS Microbiol. Lett. 2004; 236 41-45. [11] Miyasaka H., Kanaboshi H., Ikeda K. Isolation of several anti-stress genes from the halotolerant green alga *Chlamydomonas* by

[12] Tanaka S., Ikeda K., Miyasaka H., Shioi Y., Suzuki Y., Tamoi M., Takeda T., Shigeoka S., Harada K., Hirata K. Comparison of

three Chlamydomonas strains which show distinctive oxidative stress tolerance. J. Biosci. Bioeng. 2011; 112 462-468.

promoter (ON/OFF type promoter) might further enhance the PHA production.

bioplastics can be realized under low maintenance and low cost conditions.

recombinant cyanobacteria are the followings.

cost should be developed.

solution to this limitation.

**6. Conclusion** 

0 10

tient to the standard concentration is shown in the parenthesis.

20 30

40 50

PHA (% of dry weight)

60

industrial materials.

Biol. Rev. 1999; 63 21-53.

Res. Commun. 1974; 61 45-52.

*Synechococcus* sp. PCC7002. DNA Res. 1998; 5 127-129.

*Synechococcus* sp. PCC7002. DNA Res. 1998; 5 327-334.

cyanobacterium. J. Bacteriol. 1987; 169 1830-1835.

**References** 

cost for the production of biomaterials from CO2.

(0.2, 1)

(5, 10)

(0.2, 0.1)

growth and PHA accumulation in transformant cyanobacteria

**Figure 11.** Electron micrograph of wild type *S.* PCC7002 (A) and PHA accumulating Syn-pha/Ac transformant cells (B) in the early exponential growth phase (OD550=2). The PHA content in the Syn-pha/Ac transformant cell is approxi‐ metly 10% of the dry weight. Scale bars represent 0.5 μm.

Figure 12 Molecular mass distribution of the PHA from *recA* null mutant Syn-pha/Ac transformant cells (solid line), and *Wautersia eutropha* H16 (broken line) **Figure 12.** Molecular distribution of the PHA from *recA* null mutant Syn-pha/Ac transformant cells (solid line), and *Wautersia eutropha* H16 (broken line). Molecular weight of PHA samples was determined by gel permeation chroma‐ tography (GPC)

Molecular weight of PHA samples was determined by gel permeation

followings.

**should be developed** 

chromatography (GPC).

solution to this limitation.

line). Molecular weight of PHA samples was determined by gel permeation chromatography (GPC)

promoter (ON/OFF type promoter) might further enhance the PHA production.

products, such as fine chemicals, can lower the cost for the production of biomaterials from CO2.

Figure 12.Molecular distribution of the PHA from *recA* null mutant Syn-pha/Ac transformant cells (solid line), and *Wautersia eutropha* H16 (broken

Our study is the first practical approach for the antibiotics-free maintenance of plasmid in cyanobacteria, and with this system the fixation and direct conversion of CO2 into the useful bioplastics can be realized under low maintenance and low cost conditions.

The future research subjects to realize the on-site CO2 fixation and utilization system with recombinant cyanobacteria are the

Although the promoter derived from the *rbc* gene was found to be quite effective both in cyanobacterial and bacterial cells, and the PHA production by the transformed cyanobacterial cells was also quite successful with this promoter, the use of a switchable

**5. Method and system for the efficient harvesting of cyanobacteria at low energy and low cost** 

Photosynthetic CO2 assimilation only occurs during the day, and the productivity of biomaterials is much influenced by light condition. The use of cyanobacteria capable of growing photoautotrophic and also heterotrophic (with waste water) is one possible

Generally biomaterials, such as fuel and plastic, produced from CO2 are low price. Simultaneous prodcuction of higher value

Our study is the first practical approach for the antibiotics-free maintenance of plasmid in cyanobacteria, and with this system the fixation and direct conversion of CO2 into the useful bioplastics can be realized under low maintenance and low cost conditions.

accumulation was 52% of cell dry weight, the highest among the ever reported PHA accumu‐ lation in cyanobacteria. Accordingly the two-staged culture system consisting of cell growth and PHA production phases should be applied to increase the total productivity (g per liter culture) of PHA. Asada et al. reported that acetyl-CoA flux is the limiting factor in PHA production by genetically engineered cyanobacterium [32], and the high PHA productivity in Syn-phaAc transformant cells suggests the abundant intracellular supply of acetyl-CoA in *S.*

210 Environmental Biotechnology - New Approaches and Prospective Applications

**Figure 11.** Electron micrograph of wild type *S.* PCC7002 (A) and PHA accumulating Syn-pha/Ac transformant cells (B) in the early exponential growth phase (OD550=2). The PHA content in the Syn-pha/Ac transformant cell is approxi‐

Figure 12.Molecular distribution of the PHA from *recA* null mutant Syn-pha/Ac transformant cells (solid line), and *Wautersia eutropha* H16 (broken

Our study is the first practical approach for the antibiotics-free maintenance of plasmid in cyanobacteria, and with this system the fixation and direct conversion of CO2 into the useful bioplastics can be realized under low maintenance and low cost conditions.

The future research subjects to realize the on-site CO2 fixation and utilization system with recombinant cyanobacteria are the

Although the promoter derived from the *rbc* gene was found to be quite effective both in cyanobacterial and bacterial cells, and the PHA production by the transformed cyanobacterial cells was also quite successful with this promoter, the use of a switchable

**5. Method and system for the efficient harvesting of cyanobacteria at low energy and low cost** 

Photosynthetic CO2 assimilation only occurs during the day, and the productivity of biomaterials is much influenced by light condition. The use of cyanobacteria capable of growing photoautotrophic and also heterotrophic (with waste water) is one possible

Generally biomaterials, such as fuel and plastic, produced from CO2 are low price. Simultaneous prodcuction of higher value

line). Molecular weight of PHA samples was determined by gel permeation chromatography (GPC)

**Molecular Weight**

Figure 12 Molecular mass distribution of the PHA from *recA* null mutant Syn-pha/Ac transformant cells (solid line), and *Wautersia eutropha* H16

**Figure 12.** Molecular distribution of the PHA from *recA* null mutant Syn-pha/Ac transformant cells (solid line), and *Wautersia eutropha* H16 (broken line). Molecular weight of PHA samples was determined by gel permeation chroma‐

Molecular weight of PHA samples was determined by gel permeation

*Wautersia eutropha* **H16**

**10 3 10 4 10 5 10 6 10 7 10 8**

**cyanobacteria**

promoter (ON/OFF type promoter) might further enhance the PHA production.

products, such as fine chemicals, can lower the cost for the production of biomaterials from CO2.

metly 10% of the dry weight. Scale bars represent 0.5 μm.

**2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2**

**d (Weight)** 

**d log (Molecular Weight)**

(broken line)

tography (GPC)

chromatography (GPC).

followings.

**should be developed** 

solution to this limitation.

PCC7002.

The future research subjects to realize the on-site CO2 fixation and utilization system with recombinant cyanobacteria are the followings.


growth and PHA accumulation in transformant cyanobacteria The cells (initial cell density; 5 x 106 cells / ml) were cultured in the 50 ml medium containing various concentrations of NaNO3 and KH2PO4. The standard concentration of NaNO3 and KH2PO4 are 1 g/l and 50 mg/l, respectively. The standard concentrations of NaNO3 and KH2PO4 are shown as (1, 1), and the ratio of **Figure 13.** Effect of nitrogen and phosphorus concentrations in medium on cell growth and PHA accumulation in Transformant cyanovacteria. The cells (initial cell density; 5x106 cells/ml) were cultured in the 50 ml medium contain‐ ing various concentration of NaNO3 and KH2PO4. The standard concentration of NaNO3 and KH2PO4 are 1g/l and 50 mg/l, respectively. The standard concentrations of NaNO3 and KH2PO4 are shown as (1,1), and the ratio of each nutri‐ tient to the standard concentration is shown in the parenthesis.

each nutrient to the standard concentration is shown in the parentheses.

Figure 13 Effects of nitrogen and phosphorus concentrations in medium on cell

the ratio of each nutritient to the standard concentration is shown in the parenthesis.

[1] Lal R. Carbon sequestration. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2008; 363 815-830.

engineered marine cyanobacterium. Stud. Surf. Sci. Catal. 1998; 114 237-242.

plasmids and characterization of their transformation properties. J. Bacteriol. 1983; 154 1446-1450.

simple functional expression screening with *E. coli*. World J. Microbiol. Biotechnol. 2000; 16 23-29.

**6. Conclusion** 

industrial materials.

Biol. Rev. 1999; 63 21-53.

Res. Commun. 1974; 61 45-52.

*Synechococcus* sp. PCC7002. DNA Res. 1998; 5 127-129.

*Synechococcus* sp. PCC7002. DNA Res. 1998; 5 327-334.

cyanobacterium. J. Bacteriol. 1987; 169 1830-1835.

**References** 

Figure 13.Effect of nitrogen and phosphorus concentrations in medium on cell growth and PHA accumulation in Transformant cyanovacteria. The cells (initial cell density; 5x106 cells/ml) were cultured in the 50 ml medium containing various concentration of NaNO3 and KH2PO4. The standard concentration of NaNO3 and KH2PO4 are 1g/l and 50 mg/l, respectively. The standard concentrations of NaNO3 and KH2PO4 are shown as (1,1), and

The fixation and direct conversion of CO2 into the useful biomaterials by the transgenic cyanobacteria are two processes of a promising technology for the coming low carbon economy. We have developed an efficient shuttle-vector between the marine cyanobacterium *Synechococcus sp.* PCC7002 and *E. coli*, and also a practical antibiotics-free cyanobacterial plasmid expression system by using the complementation of the cyanobacterial *recA* null mutation with the *E. coli recA* gene on the plasmid. Although considerable researches are still required to realize the practical on-site applications of the present system to the industrial emission sites, such as thermal power plants, this technology can be a promising option for the biological conversion of CO2 into useful

[2] Madison, L. L., Huisman, G. W. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol.

[3] Chen G. Q. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem. Soc. Rev. 2009; 38 2434-2446. [4] Buzby J. S., Porter R. D., Stevens S. E. Jr. Plasmid transformation in *Agmenellum quadruplicatum* PR-6: construction of biphasic

[5] Tabita R. F., Stevens S. E. Jr., Quijano R. D-ribulose 1, 5-diphosphate carboxylase from blue-green algae. Biochem. Biophys.

[6] Akiyama H., Kanai S., Hirano M., Miyasaka H. Nucleotide sequences of plasmid pAQ1 of marine cyanobacterium

[7] Miyasaka H., Nakano H., Akiyama H., Kanai S., Hirano M. Production of PHA (poly hydroxyalkanoate) by the genetically

[8] Akiyama H., Kanai S., Hirano M., Miyasaka H. A novel plasmid recombination mechanism of the marine cyanobacterium

[9] Lorimier R., Guglielmi G., Bryant D. A., Stevens S. E. Jr. Functional expression of plastid allophycocyanin genes in a

[10] Tanaka S., Ikeda K. Miyasaka H. Isolation of a new member of group 3 late embryogenesis abundant (LEA) protein gene from a halotorelant green alga by a functional expression screening with cyanobacterial cells. FEMS Microbiol. Lett. 2004; 236 41-45. [11] Miyasaka H., Kanaboshi H., Ikeda K. Isolation of several anti-stress genes from the halotolerant green alga *Chlamydomonas* by

[12] Tanaka S., Ikeda K., Miyasaka H., Shioi Y., Suzuki Y., Tamoi M., Takeda T., Shigeoka S., Harada K., Hirata K. Comparison of

three Chlamydomonas strains which show distinctive oxidative stress tolerance. J. Biosci. Bioeng. 2011; 112 462-468.

### **5. Conclusion**

The fixation and direct conversion of CO2 into the useful biomaterials by the transgenic cyanobacteria are two processes of a promising technology for the coming low carbon economy. We have developed an efficient shuttle-vector between the marine cyanobacterium *Synechococcus sp.* PCC7002 and *E. coli*, and also a practical antibiotics-free cyanobacterial plasmid expression system by using the complementation of the cyanobacterial recA null mutation with the E. coli recA gene on the plasmid. Although considerable researches are still required to realize the practical on-site applications of the present system to the industrial emission sites, such as thermal power plants, this technology can be a promising option for the biological conversion of CO2 into useful industrial materials.

[6] Akiyama H., Kanai S., Hirano M., Miyasaka H. Nucleotide sequences of plasmid pAQ1 of marine cyanobacterium *Synechococcus* sp. PCC7002. DNA Res. 1998; 5 127-129.

Polyhydroxyalkanoate (PHA) Production from Carbon Dioxide by Recombinant Cyanobacteria

http://dx.doi.org/10.5772/54705

213

[7] Miyasaka H., Nakano H., Akiyama H., Kanai S., Hirano M. Production of PHA (poly hydroxyalkanoate) by the genetically engineered marine cyanobacterium. Stud. Surf.

[8] Akiyama H., Kanai S., Hirano M., Miyasaka H. A novel plasmid recombination mechanism of the marine cyanobacterium *Synechococcus* sp. PCC7002. DNA Res. 1998;

[9] Lorimier R., Guglielmi G., Bryant D. A., Stevens S. E. Jr. Functional expression of plastid allophycocyanin genes in a cyanobacterium. J. Bacteriol. 1987; 169 1830-1835.

[10] Tanaka S., Ikeda K. Miyasaka H. Isolation of a new member of group 3 late embryo‐ genesis abundant (LEA) protein gene from a halotorelant green alga by a functional expression screening with cyanobacterial cells. FEMS Microbiol. Lett. 2004; 236 41-45.

[11] Miyasaka H., Kanaboshi H., Ikeda K. Isolation of several anti-stress genes from the halotolerant green alga *Chlamydomonas* by simple functional expression screening with

[12] Tanaka S., Ikeda K., Miyasaka H., Shioi Y., Suzuki Y., Tamoi M., Takeda T., Shigeoka S., Harada K., Hirata K. Comparison of three Chlamydomonas strains which show

[13] Takeda T., Yoshimura K., Yoshii M., Kanaboshi H., Miyasaka H., Shigeoka S. Molecular characterization and physiological role of ascorbate peroxidase from halotolerant

[14] Takeda T., Miyao K., Tamoi M., Kanaboshi H., Miyasaka H., Shigeoka S. Molecular characterization of glutathione peroxidase-like protein in halotolerant *Chlamydomo‐*

[15] Tanaka S., Suda Y., Ikeda K., Ono M., Miyasaka H., Watanabe M., Sasaki K., Miyamoto K., Hirata K. A novel gene with anti-salt and anti-cadmium stress activites from a halotolerant marine green alga *Chlamydomonas* sp. W80. FEMS Microbiol. Lett. 2007;

[16] Ikeda K., Ono M., Akiyama H., Onizuka M., Tanaka S., Miyasaka H. Transformation of the fresh water cyanobacterium *Synechococcus* PCC7942 with the shuttle-vector pAQ-EX1 developed for the marine cyanobacterium *Synechococcus* PCC7002. World J.

[17] Onizuka T., Akiyama H., Endo S., Kanai S., Hirano M., Tanaka S., Miyasaka H. CO2 response element and corresponding trans-acting factor of the promoter for ribu‐ lose-1,5-bisphosphate carboxylase/oxygenase genes in *Synechococcus* sp. PCC7002 found by an improved electrophoretic mobility shift assay. Plant Cell Physiol. 2002; 43

distinctive oxidative stress tolerance. J. Biosci. Bioeng. 2011; 112 462-468.

*Chlamydomonas* sp. W80 strain. Arch. Biochem. Biophys. 2000; 376 82-90.

*E. coli*. World J. Microbiol. Biotechnol. 2000; 16 23-29.

*nas* sp. W80. Physiol. Plant 2003; 117 467-475.

Microbiol. Biotechnol. 2002; 18 55-56.

Sci. Catal. 1998; 114 237-242.

5 327-334.

271 48-52.

660-667.

### **Author details**

Hitoshi Miyasaka1 , Hiroshi Okuhata1 , Satoshi Tanaka1 , Takuo Onizuka2 and Hideo Akiyama2

\*Address all correspondence to: miyasaka.hitoshi@a4.kepco.co.jp

1 The Kansai Electric Power Co., Environmental Research Center, Seikacho, Japan

2 Toray Research Center, Inc., Kamakura, Japan

Current affiliation for Hideo Akiyama: New Projects Development Division, Toray Indus‐ tries, Inc., Kamakura, Japan

### **References**


[6] Akiyama H., Kanai S., Hirano M., Miyasaka H. Nucleotide sequences of plasmid pAQ1 of marine cyanobacterium *Synechococcus* sp. PCC7002. DNA Res. 1998; 5 127-129.

**5. Conclusion**

**Author details**

Hitoshi Miyasaka1

tries, Inc., Kamakura, Japan

Hideo Akiyama2

**References**

The fixation and direct conversion of CO2 into the useful biomaterials by the transgenic cyanobacteria are two processes of a promising technology for the coming low carbon economy. We have developed an efficient shuttle-vector between the marine cyanobacterium *Synechococcus sp.* PCC7002 and *E. coli*, and also a practical antibiotics-free cyanobacterial plasmid expression system by using the complementation of the cyanobacterial recA null mutation with the E. coli recA gene on the plasmid. Although considerable researches are still required to realize the practical on-site applications of the present system to the industrial emission sites, such as thermal power plants, this technology can be a promising option for

, Satoshi Tanaka1

Current affiliation for Hideo Akiyama: New Projects Development Division, Toray Indus‐

[1] Lal R. Carbon sequestration. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2008; 363 815-830.

[2] Madison, L. L., Huisman, G. W. Metabolic engineering of poly(3-hydroxyalkanoates):

[3] Chen G. Q. A microbial polyhydroxyalkanoates (PHA) based bio- and materials

[4] Buzby J. S., Porter R. D., Stevens S. E. Jr. Plasmid transformation in *Agmenellum quadruplicatum* PR-6: construction of biphasic plasmids and characterization of their

[5] Tabita R. F., Stevens S. E. Jr., Quijano R. D-ribulose 1, 5-diphosphate carboxylase from

from DNA to plastic. Microbiol. Mol. Biol. Rev. 1999; 63 21-53.

transformation properties. J. Bacteriol. 1983; 154 1446-1450.

blue-green algae. Biochem. Biophys. Res. Commun. 1974; 61 45-52.

industry. Chem. Soc. Rev. 2009; 38 2434-2446.

1 The Kansai Electric Power Co., Environmental Research Center, Seikacho, Japan

, Takuo Onizuka2

and

the biological conversion of CO2 into useful industrial materials.

\*Address all correspondence to: miyasaka.hitoshi@a4.kepco.co.jp

, Hiroshi Okuhata1

212 Environmental Biotechnology - New Approaches and Prospective Applications

2 Toray Research Center, Inc., Kamakura, Japan


[18] Koksharova O. A., Wolk C. P. Genetic tools for cyanobacteria. Appl. Microbiol. Biotechnol. 2002; 58 123-137.

[32] Asada Y., Miyake M., Miyake J., Kurane R., Tokiwa Y. Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2

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[33] Sudesh K., Taguchi K., Doi Y. Effect of increased PHA synthase activity on polyhy‐ droxyalkanoates biosynthesis in Synechocystis sp. PCC6803. Int. J. Biol. Macromol.

[34] Takahashi H., Miyake M., Tokiwa Y., Asada Y. Improved accumulation of poly-3 hydroxybutyrate by a recombinant cyanobacterium. Biotechnol. Lett. 1998; 20 183-186.

[35] Akiyama H., Okuhata H., Onizuka T., Kanai S., Hirano M., Tanaka S., Sasaki K., Miyasaka H. Antibiotics-free stable polyhydroxyalkanoate (PHA) production from carbon dioxide by recombinant cyanobacteria. Bioresour. Technol. 2011; 102 11039–

[36] Hankermeyer C. R., Tjeerdema R. S. Polyhydroxybutyrate: plastic made and degraded

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by microorganisms. Rev. Environ. Contam. Toxicol. 1999; 159 1-24.

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[32] Asada Y., Miyake M., Miyake J., Kurane R., Tokiwa Y. Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling. Int. J. Biol. Macromol. 1999; 25 37-42.

[18] Koksharova O. A., Wolk C. P. Genetic tools for cyanobacteria. Appl. Microbiol.

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**Chapter 10**

**The Extracellular Indolic Compounds of** *Lentinus*

In macrobasidial fungi, the properties of compounds of a phytohormonal nature, which are well known in higher plants and are intensively studied in soil associative microorganisms, are only described in an unsystematic manner and, apparently, to an insufficient degree. Auxins are the most studied group of phytohormonal substances. As an object of research, along with other mycological objects of industrial cultivation, the higher fungus–xylotrophic basidiomycete *Lentinus edodes* (Berk.) Sing (*Lentinula edodes* (Berk.) Pegler or shiitake), which is of high practical importance and the physiological and biochemical characteristics of

For long enough, there have been speculations that phytohormones, including representa‐ tives of the auxin group, are involved in the processes of cell growth and cytodifferentiation not only in plants, but also in fungi. Nevertheless, this issue still remains practically unstudied.

Of particular interest are the effects and mechanisms of the action of biologically active sub‐ stances at low doses. At small and ultrasmall concentrations (10–20 – 10–13 M), there is a mani‐ festation of the activity of many natural chemomediators - toxins and antidotes, substances warning of danger, pheromones, cryoprotectants, and other compounds, including phyto‐ hormones [1]. There is a description of the paradoxical nature of the effect of low concentra‐ tions of toxic substances and drugs, which is particularly expressed in the bimodal or polymodal dependence dose–effect. It is noted in [2] that the consequences of the effects of small doses of xenobiotics may be no less serious than the consequences of high single doses: under their influence, essential links may change and some adaptation systems may fail, because the body is only able to adapt to effects, which are in the usual range of action.

> © 2013 M. Tsivileva et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 M. Tsivileva et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Olga M. Tsivileva, Ekaterina A. Loshchinina and

Additional information is available at the end of the chapter

which are obviously insufficiently studied, is of particular interest.

*edodes*

Valentina E. Nikitina

**1. Introduction**

http://dx.doi.org/10.5772/53262
