一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

Construction of recombinant Saccharomyces cerevisiae producing 1,3-propanediol by one step method

摘要:

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。

Abstract:

1，3-Propanediol is one of the most important industrial chemicals for its highly desired properties and its wide applications as a key component of an emerging polymer business. Biological production of 1，3-propanediol has been a novel and competitive way. In our previous job，the gene dahB encoding for glycerol dehydratase from Klebsiella and the gene yqhD encoding for 1，3-propanediol oxidoreductase isoenzyme from E. coli were cloned respectively. The two genes were then tandemly ligated and expressed successfully in E. coli. The recombinant E. coli strain could produce 1，3-propanediol from D-glycerol. In the current research，the expression vectors including pGAPZB-yqhD，pGAPZB-dhaB and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB were furtherly constructed on the basis of our previous job. Then the vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB was introduced into Saccharomyces cerevisiae W303-1A using LiAc transformation method successfully. D-glucose is used as substrate to produce 1，3-propanediol with the recombinant strain after fermentation for 72h. SDS-PAGE analysis showed recombinant products at about 61kD、43kD、21kD、15kD，consistent with the molecular weight from the report. The specific enzymatic activity of the glycerol dehydratase and 1，3-propanediol oxidoreductase isoenzyme of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD -pGAP-dhaB were 24U/mg protein and 15U/mg protein，respectively，while those of the control were undetectable. In contrast to the wild strain without 1，3-propanediol output，1，3-propanediol concentration of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB reaches about 1.5g/L. The above results showed that the engineered S. cerevisiae strain which can convert the D-glucose as substrate to produce 1，3-propanediol by one-step fermentation was constructed successfully. This accomplishment bodes well for future construction of recombinant yeast strain which could overproduce 1，3-propanediol with the lower cost feedstock D-glucose by introducing the two genes yqhD and dhaB into the yeast strain overproducing glycerol with D-glucose (e.g. Candida glycerinogenes WL2002-5，which is capable of producing glycerol more than 120g/L with D-glucose as substrate and has been used for the commercial production of glycerol).