酿酒酵母产D-乳酸重组菌的构建与发酵  

Construction of metabolically engineered Saccharomyces cerevisiae for D-lactic acid production

作　　者：汪兆峰 石贵阳[1] 张梁[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室粮食发酵工艺与技术国家工程实验室,江苏无锡214122

出　　处：《生物加工过程》2015年第6期6-12,共7页Chinese Journal of Bioprocess Engineering

基　　金：国家高技术研究发展计划(863计划)(2011AA02A205;2012AA021201);教育部新世纪优秀人才支持计划(NCET-11-0665)

摘　　要：采用基因工程方法对酿酒酵母进行代谢改造,使酵母产生乳酸代谢途径。将来源于L.mesenteroides和E.coli的D-乳酸脱氢酶基因,分别插入带有G418抗性的酵母穿梭质粒p YX212-kan MX上,电转化酵母,得到2株生产D-乳酸的酿酒酵母重组菌S.cerevisiae WE1510和S.cerevisiae WB1186。进一步摇瓶发酵试验表明:重组菌S.cerevisiae WB1186在YEPD培养基、20 g/L糖、p H 5的条件下生长条件最好,并具有更好的产乳酸能力。经3 L发酵罐条件下验证,S.cerevisiae WB1186分批发酵96 h,最终乳酸积累量达到18.0 g/L;发酵条件为培养基YEPD,接种量10%,溶解氧(DO)30%,转速150 r/min,初始葡萄糖质量浓度10 g/L,控制pH 5.0,通气量3 L/min,OD600最大值转为厌氧发酵。A Saccharomyces cerevise strain was constructed by genetic engineering method, with lactic acid metabolic pathways. The gene dldh, encoding a lactate dehydrogenase from L. mesenteroides and E. coli, was cloned into a yeast shuttle vector PYX212-kanMX. The resultant plasmid pYX212-kanMX-DLDH was introduced into W303-1A by electroporation method and obtained two recombinant S. cerevisiae WE1510 and S. cerevisiae WBl186. Subsequently, a recombinant D-lactic acid producing yeast WBl186 was obtained,which had a better ability to produce D-lactic acid. It was up to 18.0 g/L of the lactic acid accumulation by batch fermentation in a 3-L fermenter for 96 h and the preliminary fermentation conditions were as follows, culture medium YEPD, inoculating rate 10%, DO 30%, stirring rate 150 r/min, concentration of glucose 10 g/L, pH 5.0, aeration rate 3 L/min, when OD60o up to maximum, the fermentation was turned to anaerobic.

关 键 词：D-乳酸 D-乳酸脱氢酶 LEUCONOSTOC mesenteroides 酿酒酵母 代谢改造 

分 类 号：TQ812[化学工程]