dhaBCE基因与yqhD基因串联表达载体的构建及其生物转化甘油

Construction of dhaBCE and yqhD Co-expression Vector and Its Biotransformation of Glycerol

将来自于肺炎克雷伯氏杆菌的甘油脱水酶基因插入到质粒pET28(a+)-yqhD的上游,并用SD序列隔开,串联构建重组质粒pET28(a+)dhaBCE-yqhD,转化到大肠杆菌E.coli Novablue中进行共表达。结果显示:含有pET28(a+)dhaBCE-yqhD的重组菌在28℃条件下,IPTG诱导16h后,甘油脱水酶和yqhD氧化还原酶的酶活力分别达到35 U/mg和82 U/mg,而对照组检测不到甘油脱水酶酶活;当甘油浓度为55g/L时,产物1,3-PD的产量可达39g/L;甘油浓度过量不利于产物合成,且产物1,3-丙二醇对合成反应具有一定的抑制作用。

dhaBCE from Klebsiella. pneumoniae was inserted into the upstream of yqhD gene in the plasmid pET28 （a＋）-yqhD. The recombinant plasmid pET28 （a ＋）dhaBCE-yqhD harboring the genes encoding dhaBCE and yqhD with a Shine-Dalgarno （SD） sequence was transformed into E. coli novablue to be co-expressed. The results showed that after 16 hours calculated from following the addition of inducer IPTG at inducing temperature 28℃, the enzyme activity of glycerol dehydratase and yqhD. oxidoreductase in recombined strain could reach 35 U/mg and 82 U/ mg, individually, higher than the wild type. The yield of 1,3 -PD arrived at 39g/L when 55g/L of substrate glycerol was added in the fermentation culture. Excessive glycerol could inhibit produce formation and inhibition of product on 1,3-propenediol biotransformation existed.