Improved bioethanol production from metabolic engineering of Enterobacter aerogenes ATCC 29007

• Lactate dehydrogenase gene was successfully deleted from E. aerogenes ATCC 29007.
• Glycerol was used as a carbon source by E. aerogenes to produce bioethanol.
• E. aerogenes SUMI014 produced one and half fold higher bioethanol than wild strain.
• Deletion of ldhA and expression of adhE gene, bioethanol production was maximized.
• Deletion of ldhA gene increased the fermentation life time of E. aerogenes.

This study investigates the enhancement of bioethanol production using a genetic engineering approach. The bioethanol-producing strain, E. aerogenes ATCC 29007, was engineered by deleting the D-lactate dehydrogenase (ldhA) gene to block the production of lactic acid. The Open-reading frame coding region of ldhA gene was replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method to inactivate chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. The colony PCR was used to confirm the deleted gene. Glycerol, a useful byproduct in the biodiesel industry, was employed to convert into bioethanol, using engineered E. aerogenes SUMI014. Under optimal conditions of fermentation (34 °C, pH 7.5, 78 h), bioethanol production by the mutant strain was 34.54 g/L, 1.5 times greater than that produced by its wild type (13.09 g/L). Subsequent overexpression of alcohol dehydrogenase (adhE) gene in the mutant strain; increased the production of bioethanol up to 38.32 g/L. By the combination of gene deletion and overexpression, the bioethanol yield was 0.48 g/g when employing 80 g/L glycerol. Hence, a significant enhancement in ethanol production was observed. These results may provide valuable guidelines for further engineering bioethanol producers.

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