辅酶A依赖的丙醛脱氢酶基因的克隆、表达及其对宿主菌生长的影响

Cloning and expression of CoA-dependent propionaldehyde dehydrogenase in Klebsiella pneumoniae and its influences on cell growth

辅酶A依赖的丙醛脱氢酶(Pdu P)是以甘油为底物催化合成聚3-羟基丙酸的关键酶。本研究从鼠伤寒沙门氏菌基因组中PCR扩增了pdu P基因,构建表达载体后转化肺炎克雷伯氏菌,得到重组菌K.p(p ET-pk-pdu P)。SDS-PAGE显示Pdu P实现了高效表达。生长曲线表明重组菌延迟进入平稳期,生物量较空质粒对照菌提高了24.68%,1,3-丙二醇和2,3-丁二醇的产量分别达到16.58 g/L和15.68 g/L,甘油转化率比空质粒对照菌和原代菌分别提高了40.62%和34.02%。上述结果表明,过表达pdu P重排了代谢流量,促进了菌体生长。

CoA-dependent propionaldehyde dehydrogenase( PduP) is a key enzyme in glycerol-based biosynthesis of poly-3-hydroxypropionic acid. In this study,the pdu P gene from Salmonella enterica serovar Typhimurium LT2 was amplified by the polymerase chain reaction( PCR) and cloned into Klebsiella pneumoniae,resulting in a recombinant strain named K. p( pET-pk-pduP). Sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE)analysis showed a high level of Pdu P expression. In contrast to the control strain K. p( pET-pk) which harbored an empty vector pET-pk,the strain K. p( pET-pk-pduP) entered a stationary phase later and the biomass increased by24. 68% after fermentation for 24 h. Moreover,this recombinant strain produced 1,3-propanediol at a concentration of 16. 58 g / L and 2,3-butanediol at a concentration of 15. 68 g / L. Compared with K. p( pET-pk) and wild type,the total glycerol conversion rate of K. p( pET-pk-pduP) increased by 40. 62% and 34. 02%,respectively. Overall these results indicate that the PduP overexpression reallocated the metabolic flux and thus stimulated cell growth.