Abstract
To produce bioethanol from model cyanobacteria such as Synechocystis, 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 Synechocystis are of interest. PDC from Zymobacter palmae (ZpPDC) has been reported to have a lower Km then the Zymomonas mobilis PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The Zppdc gene was combined with the native slr1192 alcohol dehydrogenase gene (adhA) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (ZpOpdc) versions of the ZpPDC were cloned into a construct where pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis sp. PCC 6803. These constructs were transformed into wildtype Synechocystis sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from Zymomonas mobilis. 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 Zymobacter palmae unusually did not result in enhanced ethanol production in Synechocystis sp. PCC 6803.
Highlights
Much effort has focused on the development of alternative sources of energy that are environmentally friendly and sustainable [1] in comparison to fossil fuels [2,3]
US biofuel companies Algenol and Joule Unlimited have since worked towards the development of industrial ethanol producing cyanobacteria using an alcohol dehydrogenase gene (adhA) native to Synechocystis PCC 6803 coupled to the pdc gene from Zymomonas mobilis allowing overexpression of these genes and enhanced ethanol production [10]
To enhance ethanol production further, gene dosage has been used, which employed two copies of the Zmpdc and slr1192 adhA genes coupled with the knockout of the PHB storage compound pathway [11] leading to further increased ethanol yields
Summary
Much effort has focused on the development of alternative sources of energy that are environmentally friendly and sustainable [1] in comparison to fossil fuels [2,3]. In the late 1980s, Escherichia coli was initially engineered with these genes [7] to produce ethanol as a proof of concept This was followed by the metabolic engineering of the first cyanobacterium Synechococcus elongatus PCC 7942 [8] and soon afterwards Synechocystis PCC 6803 was used to produce ethanol using the same pdc and adhA genes from Zymomonas mobilis using a strong light driven native psbA2 promoter. We hypothesized that the Zymomonas mobilis pdc gene could be replaced with a pdc gene from Zymobacter palmae that possesses a PDC with a reported lower Km value This could potentially increase flux from pyruvate to ethanol in engineered Synechocystis PCC 6803 strains. This possibility was examined via cloning and expression of the Zppdc gene, both native (Zppdc) and codon optimized (ZpOpdc), with these cassettes compared to those expressing the Zmpdc with respect to ethanol
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