Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene

Abstract

Ethanol was a major byproduct of 2,3-butanediol (2,3-BD) fermentation by Klebsiella oxytoca ME-UD-3. In order to achieve a high efficiency of 2,3-BD production, K. oxytoca mutants deficient in ethanol formation were successfully constructed by replace the aldA gene coding for aldehyde dehydrogenase with a tetracycline resistance cassette. The results suggested that inactivation of aldA led to a significantly improved 2,3-BD production. The carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained.

Use of Glycerol in Biotechnological Applications

“…Byproduct reduction approaches were made with K. oxytoca mutants defective in genes encoding lactate dehydrogenase and phosphotransacetylase, reducing lactate and acetate byproduct formation by 88% and 92%, respectively, but increasing 2,3-BDO production only by 7.8% (Ji et al, 2008). Also formation of ethanol, a major byproduct of 2,3-BDO production with K. oxytoca, could be eliminated by insertion mutagenesis of the aldehyde dehydrogenase gene and 2,3-BDO production from glucose in a fed-batch process was improved to yield 130 g l -1 2,3-BDO with a productivity of 1.63 g l -1 h -1 and a yield of 0.48 g g -1 (Ji et al, 2010). Many substrates have been used for the production of 2,3-BDO.…”

Developing Organisms for Consolidated Bioprocessing of Biomass to Ethanol

“…Compared to S. cerevisiae, all of the bacterial species discussed above are relatively sensitive to inhibitors associated with lignocellulosic hydrolysates (Bothast et al, 1999;Yamano et al, 1998;Ohta et al, 1991b). Engineering enhanced protein secretion allowed the successful secretion of endoglucanases in E. coli (Ji et al, 2009) and K. oxytoca ). E. coli and K. oxytoca strains capable of breaking down cellulose could also be modified to produce other commodity products such as lactic acid, succinic acid, acetic acid or 2, 3-butanediol (Ji et al, 2009).…”

“…Engineering enhanced protein secretion allowed the successful secretion of endoglucanases in E. coli (Ji et al, 2009) and K. oxytoca ). E. coli and K. oxytoca strains capable of breaking down cellulose could also be modified to produce other commodity products such as lactic acid, succinic acid, acetic acid or 2, 3-butanediol (Ji et al, 2009). The Geobacillus strain used by TMO Renewables Ltd. is capable of producing ethanol at appreciable titers from pretreated lignocellulosic feedstock and represents a very promising organism for CBP.…”

Biorefinery for Glycerol Rich Biodiesel Industry Waste

“…K. pneumoniae G31 was observed to transform glycerol to 2,3-BDO with a yield of 49.2 g/L. The process was greatly favoured by strong aeration and alkaline pH [34]. Saccharomyces cerevisiae is not a very suitable organism for using glycerol as a carbon source primarily because of its low utilization rates.…”