Cloning and expression of Zymobacter palmae pyruvate decarboxylase for enhanced ethanol production in the cyanobacterium Synechocystis sp. PCC 6803

Recently, photoautotrophic cyanobacteria have gained much attention due to their ability to convert carbon dioxide using light energy into carbon metabolites, some with potential biofuel applications. This is significant due to the sustainable nature of production and the global limitation of fossil fuels in the future. Model organisms such as Synechocystis sp. PCC 6803 have been described as “cell factories” for this reason and can be genetically modified and manipulated to produce compounds such as ethanol. It was decided to construct a recombinant strain of Synechocystis 6803 expressing a Zymomonas mobilis pyruvate decarboxylase (pdc) and an alcohol dehydrogenase (adh) native to Synechocystis 6803 which is capable of autotrophic conversion of pyruvate to ethanol. In an attempt to improve ethanol yield it was hypothesised that utilising a PDC with a reported lower Km might improve flux to ethanol and have identified and utilised a novel pdc gene from Zymobacter palmae in this respect. A pET22b (+) expression vector with a strong T7 promoter was primarily used to overexpress the ZpPDC for enzymatic analysis and purification purposes for potential structural studies. The pdc gene from Zymobacter palmae was amplified and a codon optimised version was also synthesised. Two constructs were created which contained the native/codon optimised Zppdc and the adh native to Synechocystis 6803 under the control of the strong light driven pPSBAII promoter. These cassette constructs were transformed into wildtype Synechocystis 6803 and integration of the cassettes into the polyploidy chromosome at the PSBAII neutral site was confirmed via DNA sequencing and PCR screening. Strains confirmed as verified recombinants were then characterised for growth, biomass and ethanol productivity relative to UL004 (expressing the Zymomonas mobilis pdc). Unfortunately and to our surprise, no increase in ethanol production was observed in the fully segregated strains in comparison to UL004, the original production strain expressing the ZmPDC despite testing a number of different segregants and utilising repeated assays. Although there may in fact be several reasons for this it was hypothesised that the ZpPDC may be less compatible than the ZmPDC in terms of its pH optimum, functional linkage to the native ADH or just sensitivity to ethanol. Going forward, it may be worthwhile focusing on other PDC enzymes from organisms such as Gluconacetobacter diazotrophicus or Gluconobacter oxydans which have recently been reported to display even lower Km values than the PDC from Zymobacter palmae.