budC敲除及ldhA过表达对Klebsiella pneumoniae合成D-乳酸的影响

为了提高K. pneumoniae中D-乳酸的合成效率，本文以BUD和LDH为改造目标，扩增丁二醇脱氢酶基因budC，并在其中插入四环素抗性基因tet，构建了基因敲除载体pTBT，转化K. pneumoniae，利用同源重组技术，敲除K. pneumoniae染色体上的budC基因，得到重组菌K. pneumonia B-；在此基础上，构建了表达载体pKP-ldhA，转化K. pneumoniae B-，过量表达乳酸脱氢酶基因ldhA，得到重组菌K. pneumoniae B-L+。摇瓶发酵结果显示，重组菌K. pneumoniae B-L+的丁二醇合成浓度比原始菌降低了90%以上，D-乳酸合成浓度比K. pneumoniae B-和原始菌分别提高了77.1%和41.4%，发酵罐实验D-乳酸产量68.4g/L，转化率0.78，生产强度1.22g/(L·h)。结果表明，敲除budC及过表达ldhA有利于改善克雷伯肺炎杆菌中D-乳酸的合成。

Effects of budC gene knockout and ldhA overexpression on D-lactic acid production by Klebsiella pneumoniae

In the metabolic pathway of Klebsiella pneumoniae,2,3-butanediol is a byproduct and its accumulation decreases the yield of main product,D-lactic acid.Butanediol dehydrogenase(BUD) is one of the key enzymes for butanediol biosynthesis,which competes with lactate dehydrogenase(LDH) for reducing equivalents of nicotinamide adenine dinucleotide(NADH).In order to reduce the butanediol accumulation and improve the D-lactic acid production,in this study,a homologous recombination vector pTBT was constructed and transformed into K.pneu-moniae,resulting in K.pneumoniae B-,in which the budC gene encoding butanediol dehydrogenase was disrupted by inserting a tetracycline resistant gene(tet).Simultaneously,the expression vector pKP-ldhA harboring the ldhA gene was constructed and transformed into K.pneumoniae B-to overexpress lactate dehydrogenase.The resulting recombinant strain K.pneumoniae B-L + exhibited a nearly abolished butanediol formation(decreased by 90%)but a significantly improved NADH availability and D-lactic acid production.In flask culture,the D-lactic acid concentrations were 77.1% and 41.4%,respectively,higher than those of the parent strains,K.pneumoniae B-and K.pneumoniae.In fed-batch fermentation with glycerol,the D-lactic acid yield,conversion and productivity reached 68.4 g/L,0.78,and 1.22 g/(L.h),respectively.