| Summary: | To produce bioethanol from model cyanobacteria such as <i>Synechocystis</i>, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in <i>Synechocystis</i> are of interest. PDC from <i>Zymobacter palmae</i> (ZpPDC) has been reported to have a lower Km then the <i>Zymomonas mobilis</i> PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The <i>Zppdc</i> gene was combined with the native <i>slr1192</i> alcohol dehydrogenase gene (<i>adh</i>A) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium <i>Synechocystis</i> sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (<i>ZpOpdc</i>) versions of the ZpPDC were cloned into a construct where <i>pdc</i> expression was controlled via the <i>psb</i>A2 light inducible promoter from <i>Synechocystis</i> sp. PCC 6803. These constructs were transformed into wildtype <i>Synechocystis</i> sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the <i>Zmpdc</i>. While strains with the <i>Zppdc</i> (UL071) and <i>ZpOpdc</i> (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from <i>Zymomonas mobilis</i>. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from <i>Zymobacter palmae</i> unusually did not result in enhanced ethanol production in <i>Synechocystis</i> sp. PCC 6803.
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