**2. The model strain and production of key intermediates**

The first *Synechocystis* strain was originally isolated in Oakland, California in 1968 [12] and placed in the Pasteur Culture Collection as *Synechocystis sp*. PCC6803 and the American Type Culture Collection as *Synechocystis sp.* ATCC27184. Over the years, many sub-strains emerged from the original strain, such as the *Synechocystis sp.* GT (Glucose Tolerant) strain. This GT strain was sent to the Kazusa Research Institute in Japan and became known as the 'Kazusa' strain. Other sub-strain are known as the 'Vermaas' strain [13], the 'China' strain [5] and indeed others sub-strains have been reported [14] such as 'Moscow', 'Amsterdam' and 'New Zealand' depending on the location of the research laboratories using the so-called original *Synechocystis* sp. PCC6803 strain. Many of these strains have undergone microevolution, which may be a feature of cyanobacterial strains growing in high light conditions under laboratory conditions [15] with such genetic changes being detected by genome sequencing. Many sub-strains have interesting variations, which may be of biotechnological interest such as low transformation rates, buoyancy and variation in growth rate.

Yields of product such as ethanol are highly dependent on the biomass produced during growth of engineered strains. When growing photoautotrophically at 30°C doubling times of *Synechocystis sp.* PCC6803 can vary between 10 and 15 h, with optimal conditions observed at light intensities of 40–70 μE.m−2.s−1 [16]. In controlled photobioreactors higher growth rates can be achieved when optimal conditions are provided throughout a growth cycle. Because the flux of carbon is diverted in metabolic engineered strains, from pyruvate via many of the engineered pathways, this has the effect of lowering biomass yields and indeed the more ethanol as a product that is produced the greater the effect on biomass yield will be. In general, the relatively slow growth rates of *Synechocystis* may be attributed to many reasons, its photoautotrophic metabolism or its polyploid (multi-copy) genome; however, one of the key issues is its encoding genome optimized for photoautotrophy. *Synechocystis sp.* PCC6803 was the first cyanobacterium to have its genome sequenced [17] and since then many further sub-strains have been sequenced [15, 18, 19]. Analysis of genome data reveals that *Synechocystis* in the main does not possess transporters for vitamins, co-factors, amino acids or nucleotides and must encode synthesis pathways and synthesize essential building blocks from the energy of photosynthesis. The needs therefore for complex synthetic machinery for its photoautotrophic lifestyle coupled to polyploidy are key aspects of its relatively slow growth rate. This then may be exacerbated when this organism is used as a cell factory for products such as ethanol.

can evolve oxygen. Model species such as *Synechocystis sp.* PCC6803 have received considerable attention because they can be relatively easily manipulated genetically and metabolically engineered to produce a wide range of potentially valuable products of biotechnological interest

**Table 1.** Ethanol yields (g.L−1.day−1) as reported for various constructs using the Zymomonas mobilis (Zm) *pdc* gene, a

**Genetic construct Strain Rate per day (g.L−1.day−1) References**

ZmPDC and ADH1 P*rbLS Synechococcus* PCC7942 0.0082 [4] ZmPDC and ADH1 P*psbA2 Synechocystis* PCC6803 0.0766 [5] ZmPDC and slr1192 *Synechocystis* PCC6803 0.097 [6] JCC1581 B Isolate *Synechococcus* PCC7002 0.41 [7] ZmPDC and slr1192 PziaA *Synechocystis* PCC6803 0.236 [8] ZmPDC and slr1192 PcorT *Synechococcus* PCC7002 0.235 [8] ZmPDC and slr1192 Prbc *Synechocystis* PCC6803 0.202 [9] ZmPDC and slr1192 PpetJ *Synechocystis* PCC6803 0.261 [10] TK504 Plasmid Pco ABICyanol1 0.552 [11]

ethanol as a biofuel at yields comparable to other biological production systems. Although there have been reports of natural ethanol production during dark metabolism, reported levels

The interest in utilizing cyanobacteria as cell factories for ethanol production has been stimulated via flux balance analysis on ethanol yields, which estimate that the stoichiometric energy yield for ethanol compares well with other potential fuel metabolites [3]. The earliest reports of photoautotrophic metabolically engineered ethanol production came in *Synechococcus elongatus* PCC 7942 [4] where heterologous genes encoding pyruvate decarboxylase and alcohol dehydrogenase were expressed from the ethanol producer *Zymomonas mobilis.* This was followed by expression of the same constructs in *Synechocystis sp.* PCC6803 [5] with reported higher yields (**Table 1**).

This was followed by reports in several patents from the US biotechnology companies Algenol and Joule Unlimited who further manipulated the system to improve yields (**Table 1**). The reported yields are represented as a daily yield and often the production cycle can last up to 20 days such that the yields would be multiplied by the production days. However, with potential evaporative loss and degradation of ethanol by contaminants in non-axenic culture these yields are lower than would be needed for commercial production. Thus, much effort

The first *Synechocystis* strain was originally isolated in Oakland, California in 1968 [12] and placed in the Pasteur Culture Collection as *Synechocystis sp*. PCC6803 and the American Type

has been focusing on improving this yield level by metabolic and strain engineering.

**2. The model strain and production of key intermediates**

to produce

[1]. Considerable attention has focused on the potential to utilize sunlight and CO<sup>2</sup>

variety of ADH genes and various promoter constructs to express these genes.

are far too low for exploitation [2].

200 Fuel Ethanol Production from Sugarcane

During photoautotrophic metabolism in *Synechocystis* an intermediate of the Calvin cycle, Ribulose 1,5-bisphosphate, is used to fix carbon dioxide to 3-phosphoglycerate. This can be converted to phosphoenolpyruvate (PEP) from 2-phosphoglycerate via enolase or travel back through the Calvin cycle. Pyruvate kinase (pk) is then used to convert PEP to pyruvate [20]. This central intermediate, pyruvate, can then be diverted via metabolic engineering to a number of potential biotechnological products including ethanol [1].
