**6. Issues and methodologies to enhance ethanol production levels**

#### **6.1. Ploidy as an issue in cloning in** *Synechocystis*

Strains of *Synechocystis* are polyploid with the chromosome number varying as a function of growth phase [39]. This causes issues with metabolic engineering and stabilization of engineered genes into chromosomal locations as one must select for integration into all chromosomal copies. In addition, the high polyploid level causes problems with generation and selection of mutants, which again must be fixed into all chromosomal copies.

Griese et al. using a real time PCR method demonstrated that the motile 'Moscow strain' of *Synechocystis* contained 58 genome copies per cell at both the log and stationary phases, while the GT 'Vermass strain' contained somewhat less with 42 chromosome copies during the same time period [39]. The 'Kazuza' strain had been reported to possess 12 copies but may have altered its ploidy because of laboratory growth over extended periods [40]. High copy number during growth has also been seen in several archaeal and other bacterial species [41, 42] but the levels reported in *Synechocystis* are amongst the highest chromosome copy numbers recorded for any cyanobacterial species or indeed prokaryote. Given the high chromosome number, the energy expended on its maintenance is high and contributes significantly to the slow growth rate of many polyploid cyanobacteria. Equally, this high chromosome number poses added difficulties in genetic and metabolic engineering in requiring many rounds of selection and screening to achieve stable integration of cloned genes. One possibility of limiting the polyploidy may be to culture production strains in limited phosphate containing media as this may have the effect of limiting phosphate availability for DNA synthesis and can limit the chromosome number, making it easier to establish recombinants and ease stabilization of chromosomally inserted cassettes.

#### **6.2. Gene dosage**

The initial cloning strategies [4, 5] used one copy of the ethanol cassette inserted into a chromosomal neutral site. To enhance productivity two copies of the cassette where then utilized [9, 36]. This had the effect of increasing productivity all be it at the expense of biomass and indeed stability during production. Attempts in our laboratory to generate strains with three cassette copies per cell have thus far failed. This suggests there could be a limit to the gene dosage that can be utilized for ethanol cassettes at least. This limit may be due to several factors and many of these factors may combine to limit production. There is the ploidy issue such that during growth if the ploidy level is some 50 copies [39] then with one cassette the copy number is already 50, two copies would mean it would be approximately 100 and the cell may not be able to tolerate more. There may also be instability issues with recombination events between similar cassette sequences. In addition, there may be the issue of ability to supply 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 detail to generate optimal strains going forward.

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.

#### **6.3. Promoter constructs**

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

Strains of *Synechocystis* are polyploid with the chromosome number varying as a function of growth phase [39]. This causes issues with metabolic engineering and stabilization of engineered genes into chromosomal locations as one must select for integration into all chromosomal copies. In addition, the high polyploid level causes problems with generation and

Griese et al. using a real time PCR method demonstrated that the motile 'Moscow strain' of *Synechocystis* contained 58 genome copies per cell at both the log and stationary phases, while the GT 'Vermass strain' contained somewhat less with 42 chromosome copies during the same time period [39]. The 'Kazuza' strain had been reported to possess 12 copies but may have altered its ploidy because of laboratory growth over extended periods [40]. High copy number during growth has also been seen in several archaeal and other bacterial species [41, 42] but the levels reported in *Synechocystis* are amongst the highest chromosome copy numbers recorded for any cyanobacterial species or indeed prokaryote. Given the high chromosome number, the energy expended on its maintenance is high and contributes significantly to the slow growth rate of many polyploid cyanobacteria. Equally, this high chromosome number poses added difficulties in genetic and metabolic engineering in requiring many rounds of selection and screening to achieve stable integration of cloned genes. One possibility of limiting the polyploidy may be to culture production strains in limited phosphate containing media as this may have the effect of limiting phosphate availability for DNA synthesis and can limit the chromosome number, making it easier to establish recombinants and ease stabi-

The initial cloning strategies [4, 5] used one copy of the ethanol cassette inserted into a chromosomal neutral site. To enhance productivity two copies of the cassette where then utilized [9, 36]. This had the effect of increasing productivity all be it at the expense of biomass and indeed stability during production. Attempts in our laboratory to generate strains with three cassette copies per cell have thus far failed. This suggests there could be a limit to the gene dosage that can be utilized for ethanol cassettes at least. This limit may be due to several factors and many of these factors may combine to limit production. There is the ploidy issue such that during growth if the ploidy level is some 50 copies [39] then with one cassette the copy number is already 50, two copies would mean it would be approximately 100 and the cell may not be able to tolerate more. There may also be instability issues with recombination events between similar cassette sequences. In addition, there may be the issue of ability to supply

**6. Issues and methodologies to enhance ethanol production levels**

selection of mutants, which again must be fixed into all chromosomal copies.

[36] is needed as part of the cassette construction.

206 Fuel Ethanol Production from Sugarcane

**6.1. Ploidy as an issue in cloning in** *Synechocystis*

lization of chromosomally inserted cassettes.

**6.2. Gene dosage**

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

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 decouple biomass from ethanol production [48].

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

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 being polyhydroxybutyrate (PHB) [49]. Mutants deficient in accumulation of such storage compounds have been used to express metabolic engineered pathways such as in the production of lactate [50]. Here diverting flux away from storage has been demonstrated to have positive effects on production. In a similar way, inserting the ethanol cassette directly into the *pha* genes has also been shown to increase production of ethanol [9, 36].

strategies also show potential in *Synechocystis.* Manipulating the Calvin-Benson-Bassham cycle, has been proposed as a strategy for improving cyanobacterial growth and product metabolites [58]. Four enzymes of the cycle, ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1, 6/sedoheptulose-1, 7-bisphosphatase, transketolase and aldolase were co-expressed with an ethanol cassette (containing *pdc* and *adh*) expressed with the P*nrsB* promoter in *Synechocystis*. In all cases there was a 55, 67, 37 and 69% increase respectively in ethanol production and also a relative increase in biomass [58], indicating the potential of

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increasing carbon flux within the cell factory as a means of improving product yield.

As integrative vectors, which utilize homologous recombination into the chromosome, are widespread when metabolic engineering *Synechocystis* [59] the characterization of integration sites, termed neutral sites, is important, particularly in terms of functionality and stability of insert. Pinto et al. carried out a systematic study of neutral sites using insertion and deletion at the site and expression of the green fluorescent protein module [38]. Although a large number and variety of sites were chosen based on genomic and sequence analysis further analysis revealed that many of these were unsuitable. Location of genuine neutral sites appears to be complicated by the possibility that non-coding regions may possess cis-acting sites thus a systematic assay using trials was needed [38]. Investigation indicated that not all neutral sites were the same and that insertion in some caused some growth defects relative to wild type. This systematic review points to a new and validated set of potential sites that can be utilized going forward which is important given the popularity of integration as the preferred tool for

Replicative plasmids have been utilized for genetic engineering in *Synechocystis* [60] and many are based on the broad host range chassis of the IncQ plasmid, RSF1010, which functions in *Synechocystis*. While this chassis allows ease of construction of inserts and can replicate in *E. coli* for generating transformation material [61] they are not widely utilized. Replicative plasmids in *Synechocystis* suffer from a number of drawbacks including recombination back into the chromosome, loss of the vector without selective pressure and unusual effects on transcription within the vector [38]. Also given the polyploid nature of the organism, segre-

For high level, production of ethanol within *Synechocystis* it has been estimated that yields would need to be above 15 g.L−1 but indeed as high as possible from a commercial perspective [62]. Currently levels of production are nowhere near these levels. However were such levels, approaching 15 g.L−1, to be reached the tolerance of the organism to ethanol could become an issue, as this would stress the cell factory. In competitive yeast fermentation systems up to 20% ethanol (v/v) has been reported [63], which is far from the current production capacity of metabolically engineered *Synechocystis* (**Table 1**). However, in preparation for strains that

**6.7. Neutral sites for integration**

metabolic engineering in *Synechocystis*.

gation and maintenance of such vectors can be problematic.

**6.8. Replicative plasmids**

**6.9. Tolerance to ethanol**

Increasing levels of substrate, in this case pyruvate, have also been used to increase yield in metabolic engineered strains. Expressing the enzyme pyruvate kinase (PK), which transfers a phosphate group from PEP to ADP forming Pyruvate [51], has been shown to increase flux to product [52]. Thus, there appears to be some potential for manipulating the flux pathways to and from pyruvate as a means of increasing product yield, which may prove useful when coupled to ethanol production.

#### **6.5. Mutagenesis strategies**

Mutagenesis and mutant selection has been developed in *Synechocystis* however; the use of random mutagenesis is difficult. This stems from the polyploid nature of the organism and the need to establish the mutant genotype in all chromosomes before the phenotype is apparent. A novel microfluidics strategy has been developed as an aid to select mutants with higher ethanol production levels [53]. This micro-droplet technique can detect increased ethanol from single cells of engineered *Synechocystis* in micro-droplets. The technique is based on an enzymatic assay, which couples ethanol levels produced within the micro-droplet directly to resorufin, a fluorescent compound. The extra fluorescence apparent with a high ethanol producer can be detected and the droplet containing the higher producer collected [53]. Passage of large quantities of metabolic engineered ethanol producing *Synechocystis*, through the system coupled to laser detection and separation of high fluorescent strains facilitates separation of higher ethanol producers [53]. Such a technique could be used to rapidly screen a large library of transposon insertion mutants, a cloned library of genes potentially enhancing ethanol production (such as PK) or directed insertion libraries (such as PHA) to select higher producers.

#### **6.6. Improving carbon capture**

Several mechanisms of carbon accumulation have been described to operate in *Synechocystis*, which include both bicarbonate and CO<sup>2</sup> transporters [54, 55]. These systems include the high affinity bicarbonate transporter BCT1 (locus slr0040–44), the sodium dependent bicarbonate transporter *Sbt*A (slr1512), the medium affinity bicarbonate transporter *Bic*A (locus sll0834) and the multi component CO<sup>2</sup> transporters NDH [56]. Theoretically, manipulation of transporters could provide more carbon for fixation and conversion to products such as ethanol. While some of the transporters are multi subunit complexes and could be difficult to express to functional activities, some are single gene encoded activities and more easily amenable to metabolic engineering. The *Bic*A protein was expressed in *Synechocystis* [57] by engineering the strain to contain additional inducible copies. Studies revealed that this strain resulted in enhanced biomass yields. We confirmed that expressing *Bic*A did in fact increase biomass whereas expressing *sbt*A in our hands did not (O'Riordan, Armshaw and Pembroke, unpublished 2018). This offers a proof of concept that increasing carbon flux can affect productivity and may have applications in enhancement of product yield. Other strategies also show potential in *Synechocystis.* Manipulating the Calvin-Benson-Bassham cycle, has been proposed as a strategy for improving cyanobacterial growth and product metabolites [58]. Four enzymes of the cycle, ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1, 6/sedoheptulose-1, 7-bisphosphatase, transketolase and aldolase were co-expressed with an ethanol cassette (containing *pdc* and *adh*) expressed with the P*nrsB* promoter in *Synechocystis*. In all cases there was a 55, 67, 37 and 69% increase respectively in ethanol production and also a relative increase in biomass [58], indicating the potential of increasing carbon flux within the cell factory as a means of improving product yield.

#### **6.7. Neutral sites for integration**

being polyhydroxybutyrate (PHB) [49]. Mutants deficient in accumulation of such storage compounds have been used to express metabolic engineered pathways such as in the production of lactate [50]. Here diverting flux away from storage has been demonstrated to have positive effects on production. In a similar way, inserting the ethanol cassette directly into the

Increasing levels of substrate, in this case pyruvate, have also been used to increase yield in metabolic engineered strains. Expressing the enzyme pyruvate kinase (PK), which transfers a phosphate group from PEP to ADP forming Pyruvate [51], has been shown to increase flux to product [52]. Thus, there appears to be some potential for manipulating the flux pathways to and from pyruvate as a means of increasing product yield, which may prove useful when

Mutagenesis and mutant selection has been developed in *Synechocystis* however; the use of random mutagenesis is difficult. This stems from the polyploid nature of the organism and the need to establish the mutant genotype in all chromosomes before the phenotype is apparent. A novel microfluidics strategy has been developed as an aid to select mutants with higher ethanol production levels [53]. This micro-droplet technique can detect increased ethanol from single cells of engineered *Synechocystis* in micro-droplets. The technique is based on an enzymatic assay, which couples ethanol levels produced within the micro-droplet directly to resorufin, a fluorescent compound. The extra fluorescence apparent with a high ethanol producer can be detected and the droplet containing the higher producer collected [53]. Passage of large quantities of metabolic engineered ethanol producing *Synechocystis*, through the system coupled to laser detection and separation of high fluorescent strains facilitates separation of higher ethanol producers [53]. Such a technique could be used to rapidly screen a large library of transposon insertion mutants, a cloned library of genes potentially enhancing ethanol production (such as PK) or directed insertion libraries (such as PHA) to select higher producers.

Several mechanisms of carbon accumulation have been described to operate in *Synechocystis*,

high affinity bicarbonate transporter BCT1 (locus slr0040–44), the sodium dependent bicarbonate transporter *Sbt*A (slr1512), the medium affinity bicarbonate transporter *Bic*A (locus

of transporters could provide more carbon for fixation and conversion to products such as ethanol. While some of the transporters are multi subunit complexes and could be difficult to express to functional activities, some are single gene encoded activities and more easily amenable to metabolic engineering. The *Bic*A protein was expressed in *Synechocystis* [57] by engineering the strain to contain additional inducible copies. Studies revealed that this strain resulted in enhanced biomass yields. We confirmed that expressing *Bic*A did in fact increase biomass whereas expressing *sbt*A in our hands did not (O'Riordan, Armshaw and Pembroke, unpublished 2018). This offers a proof of concept that increasing carbon flux can affect productivity and may have applications in enhancement of product yield. Other

transporters [54, 55]. These systems include the

transporters NDH [56]. Theoretically, manipulation

*pha* genes has also been shown to increase production of ethanol [9, 36].

coupled to ethanol production.

**6.6. Improving carbon capture**

which include both bicarbonate and CO<sup>2</sup>

sll0834) and the multi component CO<sup>2</sup>

**6.5. Mutagenesis strategies**

208 Fuel Ethanol Production from Sugarcane

As integrative vectors, which utilize homologous recombination into the chromosome, are widespread when metabolic engineering *Synechocystis* [59] the characterization of integration sites, termed neutral sites, is important, particularly in terms of functionality and stability of insert. Pinto et al. carried out a systematic study of neutral sites using insertion and deletion at the site and expression of the green fluorescent protein module [38]. Although a large number and variety of sites were chosen based on genomic and sequence analysis further analysis revealed that many of these were unsuitable. Location of genuine neutral sites appears to be complicated by the possibility that non-coding regions may possess cis-acting sites thus a systematic assay using trials was needed [38]. Investigation indicated that not all neutral sites were the same and that insertion in some caused some growth defects relative to wild type. This systematic review points to a new and validated set of potential sites that can be utilized going forward which is important given the popularity of integration as the preferred tool for metabolic engineering in *Synechocystis*.

#### **6.8. Replicative plasmids**

Replicative plasmids have been utilized for genetic engineering in *Synechocystis* [60] and many are based on the broad host range chassis of the IncQ plasmid, RSF1010, which functions in *Synechocystis*. While this chassis allows ease of construction of inserts and can replicate in *E. coli* for generating transformation material [61] they are not widely utilized. Replicative plasmids in *Synechocystis* suffer from a number of drawbacks including recombination back into the chromosome, loss of the vector without selective pressure and unusual effects on transcription within the vector [38]. Also given the polyploid nature of the organism, segregation and maintenance of such vectors can be problematic.

#### **6.9. Tolerance to ethanol**

For high level, production of ethanol within *Synechocystis* it has been estimated that yields would need to be above 15 g.L−1 but indeed as high as possible from a commercial perspective [62]. Currently levels of production are nowhere near these levels. However were such levels, approaching 15 g.L−1, to be reached the tolerance of the organism to ethanol could become an issue, as this would stress the cell factory. In competitive yeast fermentation systems up to 20% ethanol (v/v) has been reported [63], which is far from the current production capacity of metabolically engineered *Synechocystis* (**Table 1**). However, in preparation for strains that would have this capacity it is important to determine the level of tolerance to ethanol in engineered strains and examine toxic or stress related effects. Proteomic analysis has been used to determine the response of engineered strains [64] with current ethanol production levels and the response of strains with ethanol added up to projected or expected ethanol production levels [65]. In the case of added ethanol incubation with 1.5% (v/v) of ethanol for 24 h reduced growth of *Synechocystis* by 50% with cell aggregation visible [65]. Proteome analysis revealed some 32 unique proteins up-regulated and some 42 down-regulated after 24 h. This number of altered proteins increased after 48 h. incubation. Many of these proteins were demonstrated to be involved in the common stress response such as those associated with oxidative stress [65], transporters, cell-membrane modifying proteins and proteins associated with the photosystems. Many of the altered protein observed in the proteome response in *Synechocystis* were similar to those observed in the tolerance response of *Zymomonas mobilis* [66]. Proteomic analysis has also been observed on metabolic engineered *Synechocystis* producing levels as outlined in **Table 1** [64]. Here some 60–70% of the carbon fixed was converted to ethanol via a single ethanol cassette. At this rate of ethanol production there was no significant stress response observed rather there was a realignment of systems. Some upregulation of carbon concentrating mechanisms were observed, as were enzymes of the Calvin cycle and photosynthesis antennae proteins [64]. Interestingly the t*hi*C gene, encoding phosphomethylpyrimidine synthase involved in thDP synthesis (the PDC-cofactor), was also up-regulated suggesting that even at this low level of ethanol production that co-factor availability was limited [64].

Development of the downstream aspects of production will require optimization of several parameters and a more favorable economic outlook. Capital expenditure (CapEx) will be a key driving force with many components needing to be considered. Chief amongst these is the nature of the producing organisms being a recombinant strain. This poses potential safety and containment considerations, which would add to the economics of plant construction and operation. The need for sunlight (which may limit location of production facilities) or continuous LCD exposure again adds costs with either cyclic day exposure in high light climates or continuous growth with added light, which would come with an added energy cost. Equally, calculations of volumes that would be needed suggest large CapEx expenditure on plant, large water requirements and effluent processing costs. Many geographical areas that have high sunlight with marginal land, such as desert areas, at first sight might seem suitable but will suffer from water limitations. Other issues that are related to CapEx relate to the growth of the production strains themselves and the provision of optimal conditions for growth and production. Currently as one diverts photosynthetic intermediates to ethanol, one is affecting the flux to biomass. The more ethanol that is produced the slower the growth and the less biomass that can be produced. This impinges significantly on the growth rate and hence competitiveness of production strains. Given that, growth under sterile conditions in photobioreactors would be economically unsustainable (due to cost and the low value of the product ethanol); competitor contamination would need to be built into the growth cycle. Thus, slow growth of producers would have two major potential consequences that could affect the process. Firstly, there may be mutational selection for faster growers, which have lost the engineered ethanol cassette reducing the yield during production, and secondly given that axenic conditions could not be maintained during aseptic but non-sterile culturing, contaminants could easily outgrow the engineered strains. Strategies that might mitigate this could be the addition of mutualistic consortia, which might stimulate the production strains by providing vitamins or co-factors while limiting the growth of contaminants [68]. Thus, strategies that would aid production at large scale would need to be factored in at the initial

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The need for significant scale up of photoautotrophic ethanol production in a high light environment can add significantly to initial CapEx. Within the reactor system itself, several components may need significant attention. It is impractical for low value ethanol products, at least in comparison to current fuel costs, for growth and production to be carried out in sterile photobioreactors (PBRs) with full control over light, and key physiological conditions. Although the technologies for such photobioreactors are well developed their practicality can reasonably only be considered suitable for high value products [69]. In addition to containment issues, there are issues with inoculum development for non-axenic culturing to insure that initial inoculum is stable, productive and clonal. Depending on the plant size, this may

The most frequent types of PBRs proposed are non-sterile horizontal tubular or vertical flat panel PBRs, which have several limitations including: (a) cost, which have been estimated at

for small scale, reducing in cost slightly with scale [70]. This would result in a cost

stages of metabolic engineering.

require significant CapEx.

€2400 m2

**7.2. Reactor design for large-scale economic production**

Tolerance has also been examined via transcriptomic analysis following exogenous ethanol addition [67] with 1.2–3% ethanol addition to wild type *Synechocystis*. Addition of 1.5% caused a 50% reduction in growth rate with visible aggregation suggesting stress. Many of the genes up-regulated in the transcriptomic study were associated with energy metabolism particularly photosynthesis. The results observed were broadly in line those observed in the proteomic studies [64].
