Abstract: Glycerol can be transformed to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae with accompanying ethanol accumulation.Aldehyde dehydrogenase (ALDH) is one of the key enzymes for biosynthesis of ethanol, which competes for reducing equivalents NADH with 1,3-propanediol dehydrogenase which catalyzes biosynthesis of 1,3-PD.Based on metabolic analysis, one reasonable method to improve 1,3-PD yield is to inhibit the catalysis of ALDH so that ethanol production could be restrained. In this paper, a homologous recombination vector pUCAT was constructed, in which the ALDH gene of K.pneumoniae was disrupted by inserting tetracycline resistance gene (Tcr).The amplified DNA fragment of 5′ALDH-Tcr-3′ALDH from pUCAT was used to transform K.pneumoniae M5aL and the ALDH gene knockout recombinants were obtained.Comparing with those of the wild type K.pneumoniae M5aL, the ALDH activity of the recombinants were undetected, cell growth was inhibited obviously, ethanol yields were decreased by 43%—53%, 1,3-PD yields and molar conversions from glycerol to 1,3-PD were increased by 27%—42% and 19%—24% respectively.

摘要： 利用Klebsiella pneumoniae厌氧发酵甘油生产1,3-丙二醇时，一部分甘油通过氧化代谢途径大量合成副产物乙醇，降低了1,3-丙二醇的得率.醛脱氢酶ALDH是乙醇合成途径的关键酶之一，其催化作用不仅消耗了大量甘油，还将还原型辅酶NADH氧化为NAD⁺，降低了同为NADH依赖型的1,3-PD合成途径的效率.本文以醛脱氢酶ALDH为改造目标，以K.pneumoniae为宿主，通过同源重组技术在K.pneumoniae M5aL的ALDH基因中成功地插入了四环素抗性基因，经抗性筛选和基因水平鉴定，得到两株ALDH基因敲除的重组菌0623-1hb及0623-1hc.本文研究了这两株重组菌的生长代谢特性，结果表明两株重组菌的ALDH酶活基本检测不到，菌体生长受到明显抑制，乙醇合成浓度比出发菌株K.pneumoniae M5aL降低了43%～53%，1,3-PD合成浓度及摩尔得率分别提高了27%～42%和19%～24%.