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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