The rapidly growing demand for energy and the environmental concerns about carbon dioxide emissions make the development of renewable biofuels more and more attractive. Tremendous academic and industrial efforts have been made to produce bioethanol, which is one major type of biofuel. The current production of bioethanol is limited for commercialization because of issues with food competition (from food-based biomass) or cost effectiveness (from lignocellulose-based biomass). In this report we applied a consolidated bioprocessing strategy to integrate photosynthetic biomass production and microbial conversion producing ethanol together into the photosynthetic bacterium, Synechocystis sp. PCC6803, which can directly convert carbon dioxide to ethanol in one single biological system. A Synechocystis sp. PCC6803 mutant strain with significantly higher ethanol-producing efficiency (5.50 g L⁻¹, 212 mg L⁻¹ day⁻¹) compared to previous research was constructed by genetically introducing pyruvate decarboxylase from Zymomonas mobilis and overexpressing endogenous alcohol dehydrogenase through homologous recombination at two different sites of the chromosome, and disrupting the biosynthetic pathway of poly-β-hydroxybutyrate. In total, nine alcohol dehydrogenases from different cyanobacterial strains were cloned and expressed in E. coli to test ethanol-producing efficiency. The effects of different culturing conditions including tap water, metal ions, and anoxic aeration on ethanol production were evaluated.

Background: Cyanobacteria can utilize solar energy and convert carbon dioxide into biofuel molecules in one single biological system. Synechocystis sp. PCC 6803 is a model cyanobacterium for basic and applied research. Alkanes are the major constituents of gasoline, diesel and jet fuels. A two-step alkane biosynthetic pathway was identified in cyanobacteria recently. It opens a door to achieve photosynthetic production of alka(e)nes with high efficiency by genetically engineering cyanobacteria. Results: A series of Synechocystis sp. PCC6803 mutant strains have been constructed and confirmed. Overexpression of both acyl-acyl carrier protein reductase and aldehyde-deformylating oxygenase from several cyanobacteria strains led to a doubled alka(e)ne production. Redirecting the carbon flux to acyl- ACP can provide larger precursor pool for further conversion to alka(e)nes. In combination with the overexpression of alkane biosynthetic genes, alka(e)ne production was significantly improved in these engineered strains. Alka(e)ne content in a Synechocystis mutant harboring alkane biosynthetic genes over-expressed in both slr0168 and slr1556 gene loci (LX56) was 1.3% of cell dry weight, which was enhanced by 8.3 times compared with wildtype strain (0.14% of cell dry weight) cultivated in shake flasks. Both LX56 mutant and the wildtype strain were cultivated in column photo-bioreactors, and the alka(e)ne production in LX56 mutant was 26 mg/L (1.1% of cell dry weight), which was enhanced by 8 times compared with wildtype strain (0.13% of cell dry weight). Conclusions: The extent of alka(e)ne production could correlate positively with the expression level of alkane biosynthetic genes. Redirecting the carbon flux to acyl-ACP and overexpressing alkane biosynthetic genes simultaneously can enhance alka(e)ne production in cyanobacteria effectively.

Cytochrome P450 OleTJE from Jeotgalicoccus sp. ATCC 8456, a new member of the CYP152 peroxygenase family, was recently found to catalyze the unusual decarboxylation of long-chain fatty acids to form α-alkenes using H2O2 as the sole electron and oxygen donor. Because aliphatic α-alkenes are important chemicals that can be used as biofuels to replace fossil fuels, or for making lubricants, polymers and detergents, studies on OleTJE fatty acid decarboxylase are significant and may lead to commercial production of biogenic α-alkenes in the future, which are renewable and more environmentally friendly than petroleum-derived equivalents.