单倍体酿酒酵母的丙酮酸脱羧酶基因(pdc1)的敲除与鉴定

为了抑制单倍体酿酒酵母H14的副产物乙醇的合成,使得2,3-丁二醇产量的提升。利用基因工程手段构建载体pWCL-pdc1,获得两端含40 bp pdc1的同源重组片段-loxP-kanMX-loxP。利用Cre/loxP技术获得pdc1缺失菌株S. cerevisiae H14-01 (△pdc1)。并以野生型菌株S. cerevisiae H14为对照,进行摇瓶发酵试验。S. cerevisiae H14-01长势明显略低于原始菌株。在整个发酵期间,2,3-丁二醇的最高产量和转化率分别为0.373±0.016 g/L和0.005 g/g,分别较原始菌株提高了37.30%和4.66%,但原始菌株没有检测到乙偶姻和2,3-BD生成。另外S. cerevisiae H14-01的乙醇转化率降低了33.24%,但甘油产量提高了15.76%。说明了碳流流向乙醇被阻断之后,会增加2,3-丁二醇的产量,同时会使此部分碳流流向甘油。因此,并为进一步获得高产2,3-丁二醇的工程微生物群体奠定了基础。

Pyruvate Decarboxylase Gene (pdc1) of Haploid Saccharomyces Cerevisiae: Knockout and Identification

The aim is to inhibit the synthesis of ethanol, a by-product of haploid Saccharomyces cerevisiae H14, and increase the yield of 2,3-butanediol. The vector pWCL-pdc1 was constructed by genetic engineering to obtain the homologous recombinant fragment loxP-kanMX-loxP containing 40 bp pdc1 at both ends. The pdc1 deleted strain S. cerevisiae H14-01 (Δpdc1) was obtained by Cre/loxP technique. The shaking flask fermentation experiment was carried out with the wild type strain S. cerevisiae H14 as the control. The growth of S. cerevisiae H14-01 was significantly lower than that of the original strain. During the whole fermentation period, the maximum yield and conversion rate of 2,3-butanediol were 0.373 ±0.016 g/L and 0.005 g/g, respectively, which were 37.30% and 4.66% higher than that of the original strain, respectively. However, acetoin and 2,3-BD production was not detect in the original strain. In addition, the ethanol conversion of S. cerevisiae H14-01 decreased by 33.24%, but the glycerol production increased by 15.76%. It showed that after the carbon flow to ethanol was blocked, the production of 2, 3-butanediol could be increased, and part of the carbon flow will flow to glycerol. Therefore, the study lays a good foundation for further obtaining an engineering microbial population with high yield of 2,3-butanediol.