**4.** *Zymomonas mobilis* **and** *Synechocystis* **alcohol dehydrogenase (ADH)**

In most reports on engineered ethanol cassettes the source of ADH has been *Zymomonas mobilis*. Two ADH isozymes are known to be present within the genome of *Zymomonas mobilis* - ADH I and ADH II (EC 1.1.1.1) [32]. For metabolic engineering of ethanol production the Fe2+ containing ADHII encoded by the *adh*B gene of *Zymomonas* has been utilized [5]. This enzyme has a pH optimum of pH 8.5 (as opposed to pH 6.5 for ADHI) and a cofactor requirement for Nicotinamide adenine dinucleotide (NADH) for the reduction of acetaldehyde to ethanol [33].

Unusually *Synechocystis* also encodes its own native ADH gene via the *adh*A gene (slr1192). This is a medium chain alcohol dehydrogenase, which catalyzes the reversible oxidation of alcohols to aldehydes or ketones [34]. The *Synechocystis* ADH encodes a 140 kDa zinc dependent enzyme with broad alcohol dehydrogenase activity and which interestingly is Nicotinamide adenine dinucleotide phosphate (NADPH) dependent as opposed to the *Zymomonas* activity, which is NADH dependent [34]. Indeed *Synechocystis* has been reported to possess multiple *adh* genes but does not contain an native *pdc* gene [35] suggesting that the native ADH may play an as yet unknown function in the cyanobacterium. Indeed the *Synechocystis adh* gene has been substituted for the *Zymomonas* gene [9] and functions very well. Recently we have reported [36] metabolic engineered cassettes with a copy of the *Zymomonas mobilis adh* gene and the native *Synechocystis adh* gene with increased ethanol producing activity. This may occur because the two activities rely on different co-factors NADH (*Zymomonas*) and NADP (*Synechocystis*) which may spread the co-factor requirement and availability within the cell [36].

## **5. Construction of functional ethanol cassettes in** *Synechocystis*

In general, terms the construction of an ethanol cassette follows the basic components as reported [4, 5]. The *Zymomonas mobilis pdc* gene is amplified and fused with the *Zymomonas mobilis adh*B gene under the control of an inducible promoter. The light inducible P*psbA2* promoter is often utilized but other promoters have also been evaluated [37]. There is then the need for a strong selection of the cassette encoded generally by an antibiotic resistance determinant such as kanamycin or zeocin [36]. Homology sequences are needed at both ends of the cassette to allow homologous integration into a neutral site within the organism (see **Figure 1**). The cassette utilized by Dexter and Fu [5] utilized the *psb*A2 gene as a neutral site for integration but recently a number of other neutral sites have been discovered [38]. Indeed homologous integration has been used as a mechanism of integrating cassettes into functional competing genes, as a knock out mechanism also. This occurs where the cassette is integrated via use of homologous ends into genes such as the *pha* genes whereby integration knocks out the synthesis of polyhydroxyalkanoate (PHA) a competing pathway for pyruvate use [9, 36]. Using the *pha* genes as an integration site effectively increases the flux of pyruvate to ethanol by blocking alternative storage of photosynthetic products.

homology between the two 500 bp ends and the host chromosome leads to integration into the neutral site. In general, because of the polyploid nature of *Synechocystis,* selection for integration requires selection on increasing doses of kanamycin and PCR monitoring using primers across the neutral integration site. Initially many chromosomes will not contain an integrated cassette and this will show as a low molecular weight band (where no integration into the neutral site occurs). Those chromosomes that contain an integrated cassette will possess a higher molecular weight band where the cassette has integrated into the neutral site increasing the band size. At the initial stages, one would observe two bands one without and one with integration (one low and one high band). Following selection all chromosomes should contain a high molecular weight band (and no low molecular weight band) indicating that all chromosomes contain the cassette. This process illustrated in **Figure 2** (below) may take several weeks to segregate and stabilize. In the case of establishing an ethanol cassette, which provides no selective advantage on its host and in fact may be negative in selection terms as it causes diversion of pyruvate towards ethanol rather than biomass, selection and stabilization may take some time. Thus, strong selection

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**Figure 2.** Agarose gel electrophoresis of PCR amplicons analyzing integration into the *psb*A2 neutral site of pUL004. A) Lanes 2 and 3 (and B lanes 2,3,4,5,7) illustrate amplicons using primers to amplify across the neutral integration site which in these cases are all of low molecular weight indicating no integration into the neutral site. A) Lanes 4 and 5 illustrate that two amplicons are detected the lower band with no cassette and the higher band with the cassette integrated. This pattern is detected in strains with partial segregation of the cassette. B) upon selection strains harboring integrated cassettes in all chromosomes should resolve (the cassette is present in all chromosomes of the polyploid genome) as illustrated in lane 8 panel B. This band (lane 8 panel B) can then be removed and sequenced to verify

integration. Lanes 1 a and B illustrate a molecular weight ladder to determine amplicons size.

In attempts to increase ethanol production, gene dosage has been utilized such that two cassettes have been integrated at different sites giving potentially twice the gene copy number and protein expression level of PDC and ADH [9, 36]. While this strategy has been shown to increase the levels of ethanol produced it may be that given the polyploid nature of *Synechocystis* putting in and stabilizing two cassette copies which would be multiplied by some 50 copies (due to polyploidy) may be reaching the very limits of gene dosage with this metabolic engineering strategy.

**Figure 1** illustrates the construction of an ethanol cassette pUL004 Kan. This cassette [36] consists of the Zm*pdc* coupled to the *Synechocystis adh*A gene with a kanamycin resistance determinant from the ICE R391. The genes are controlled via the P*psbA2* light inducible promoter. The cassette contains 500 bp of DNA at each end with homology to a neutral integration site, in this case the *psb*A2 gene. This construct is housed in pUC18 and replicated in *Escherichia coli* and termed pUL004. For integration, the plasmid pUL004 is transformed into *Synechocystis* whereupon

**Figure 1.** Structure of the ethanol cassette pUL004 [36]. The cassette contains the *Zymomonas pdc* gene (Zm*pdc*), the *Synechocystis sp.* PCC6803 *adh* gene (slr1192) and the kanamycin resistance determinant from the ICE R391 all under the control of the P*psbA2* light inducible promoter. There is 500 bp at each end with homology to the neutral integration site and the construct is cloned into pUC18 for replication in *E. coli* prior to purification and transformation into *Synechocystis* PCC6803. Restriction sites within the cassette are also illustrated.

homology between the two 500 bp ends and the host chromosome leads to integration into the neutral site. In general, because of the polyploid nature of *Synechocystis,* selection for integration requires selection on increasing doses of kanamycin and PCR monitoring using primers across the neutral integration site. Initially many chromosomes will not contain an integrated cassette and this will show as a low molecular weight band (where no integration into the neutral site occurs). Those chromosomes that contain an integrated cassette will possess a higher molecular weight band where the cassette has integrated into the neutral site increasing the band size. At the initial stages, one would observe two bands one without and one with integration (one low and one high band). Following selection all chromosomes should contain a high molecular weight band (and no low molecular weight band) indicating that all chromosomes contain the cassette. This process illustrated in **Figure 2** (below) may take several weeks to segregate and stabilize. In the case of establishing an ethanol cassette, which provides no selective advantage on its host and in fact may be negative in selection terms as it causes diversion of pyruvate towards ethanol rather than biomass, selection and stabilization may take some time. Thus, strong selection

cassette to allow homologous integration into a neutral site within the organism (see **Figure 1**). The cassette utilized by Dexter and Fu [5] utilized the *psb*A2 gene as a neutral site for integration but recently a number of other neutral sites have been discovered [38]. Indeed homologous integration has been used as a mechanism of integrating cassettes into functional competing genes, as a knock out mechanism also. This occurs where the cassette is integrated via use of homologous ends into genes such as the *pha* genes whereby integration knocks out the synthesis of polyhydroxyalkanoate (PHA) a competing pathway for pyruvate use [9, 36]. Using the *pha* genes as an integration site effectively increases the flux of pyruvate to ethanol

In attempts to increase ethanol production, gene dosage has been utilized such that two cassettes have been integrated at different sites giving potentially twice the gene copy number and protein expression level of PDC and ADH [9, 36]. While this strategy has been shown to increase the levels of ethanol produced it may be that given the polyploid nature of *Synechocystis* putting in and stabilizing two cassette copies which would be multiplied by some 50 copies (due to polyploidy) may be reaching the very limits of gene dosage with this

**Figure 1** illustrates the construction of an ethanol cassette pUL004 Kan. This cassette [36] consists of the Zm*pdc* coupled to the *Synechocystis adh*A gene with a kanamycin resistance determinant from the ICE R391. The genes are controlled via the P*psbA2* light inducible promoter. The cassette contains 500 bp of DNA at each end with homology to a neutral integration site, in this case the *psb*A2 gene. This construct is housed in pUC18 and replicated in *Escherichia coli* and termed pUL004. For integration, the plasmid pUL004 is transformed into *Synechocystis* whereupon

**Figure 1.** Structure of the ethanol cassette pUL004 [36]. The cassette contains the *Zymomonas pdc* gene (Zm*pdc*), the *Synechocystis sp.* PCC6803 *adh* gene (slr1192) and the kanamycin resistance determinant from the ICE R391 all under the control of the P*psbA2* light inducible promoter. There is 500 bp at each end with homology to the neutral integration site and the construct is cloned into pUC18 for replication in *E. coli* prior to purification and transformation into *Synechocystis*

PCC6803. Restriction sites within the cassette are also illustrated.

by blocking alternative storage of photosynthetic products.

metabolic engineering strategy.

204 Fuel Ethanol Production from Sugarcane

**Figure 2.** Agarose gel electrophoresis of PCR amplicons analyzing integration into the *psb*A2 neutral site of pUL004. A) Lanes 2 and 3 (and B lanes 2,3,4,5,7) illustrate amplicons using primers to amplify across the neutral integration site which in these cases are all of low molecular weight indicating no integration into the neutral site. A) Lanes 4 and 5 illustrate that two amplicons are detected the lower band with no cassette and the higher band with the cassette integrated. This pattern is detected in strains with partial segregation of the cassette. B) upon selection strains harboring integrated cassettes in all chromosomes should resolve (the cassette is present in all chromosomes of the polyploid genome) as illustrated in lane 8 panel B. This band (lane 8 panel B) can then be removed and sequenced to verify integration. Lanes 1 a and B illustrate a molecular weight ladder to determine amplicons size.

and monitoring is required to realize integration and maintenance of such cassettes. To insert a second cassette a different neutral integration site (and hence different homologous sequences within the cassette are required) and a different antibiotic resistance determinant such as zeocin [36] is needed as part of the cassette construction.

the co-factors ThDP, NADH and NADPH for this level of enzyme expression. There may be additional factors such as limitation of pyruvate for other essential cellular functions if high levels of enzyme activity are utilizing it to react to ethanol. This in turn may affect biomass production and synthesis of essential cell components and thus triggering a stress response. In addition, given the negative effect ethanol has on growth there may be the selective pressure to mutate the cassettes selecting for faster growing strains which do not have the burden of ethanol production. The nature of all these possibilities may need to be examined in more

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That gene dosage can have an effect on production has been demonstrated by utilizing the small native *Synechocystis* plasmid pCA2.4 [43]. This plasmid has a copy number of seven per chromosome copy, thus potentially greater than 300 copies per cell. Cloning of the yellow fluorescent protein (YFP) into a neutral site on this highly stable plasmid resulted in >100 fold increase production levels of YFP relative to a chromosomal insert indicating the potential of gene dosage within *Synechocystis* [43] all be it in this case with a non-burdening or non-toxic product.

Most productivity studies for ethanol in *Synechocystis* have been carried out with the light inducible P*psbA2* promoter [4, 5, 9, 36]. However, a number of other promoters have been examined specifically to improve yields (see **Table 1**). Recently heterologous strong promoters P*trc* [44], P*rnpB* [45] and P*lac* [46] have been used for butanol, lactate and ethylene production respectively. Use of the super promoter P*cpc560*, [47] was shown to produce functional proteins at a level of up to 15% of total soluble protein in *Synechocystis sp.* PCC6803, a level comparable to that produced in *E. coli*. This promoter appears to have 14 predicted transcription binding sites, which appear to be key to its high expression level [47]. Many of these promoters are

A number of controllable promoters have also been analyzed [37] with the most useful being the Ni++ Co++ inducible, P*nrsB*, which gives relatively silent expression in the un-induced state and can be induced some 40 fold to approximately the level of the P*psbA2* promoter with inducer. Such promoters may allow tuneable promoter activity for ethanol production. Always on promoters, do not allow biomass to be generated as might happen in the yeast system where removal of aeration during production leads to the switch to anaerobic metabolism and ethanol productivity following adequate biomass production. This decoupling of growth from ethanol production could be achieved by tuneable promoters and has been reported [48] where by a riboswitch was incorporated in an ethanol cassette following the P*psbA2* promoter. Such riboswitches can be induced by theophylline and has been used as a *proof of concept* to

Manipulation of carbon flux within the cell factory *Synechocystis* has been used to increase production of metabolically engineered products. Photoautotrophic growth in the light results in accumulation of a number of storage compounds in *Synechocystis* including the major storage polymers glycogen and polyhydroxyalkanoates (PHA)*,* the best characterized

always on and may not be optimal for controlled expression however.

**6.4. Knockout of competing pathways as an aid to greater production**

decouple biomass from ethanol production [48].

detail to generate optimal strains going forward.

**6.3. Promoter constructs**
