Inactivation of dhaD and dhaK abolishes by-product accumulation during 1,3-propanediol production in Klebsiella pneumoniae.
1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P(BAD) promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.
Horng
Yu-Tze
YT
Department of Laboratory, Medicine and Biotechnology, Tzu Chi University, College of Medicine, 701 Section 3, Zhongyang Road, Hualien 970, Taiwan, ROC.
Chang
Kai-Chih
KC
Chou
Ta-Chung
TC
Yu
Chung-Jen
CJ
Chien
Chih-Ching
CC
Wei
Yu-Hong
YH
Soo
Po-Chi
PC
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002072:Butylene Glycols / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D020868:Gene Silencing; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011409:Propylene Glycols / Q000378:metabolism*; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D002072:Butylene Glycols; D011409:Propylene Glycols; D019344:Lactic Acid; D000431:Ethanol; C026978:2,3-butylene glycol; C041787:1,3-propanediol; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase