代谢工程改造野生耐酸酵母生产L-乳酸

Metabolic engineering of wild acid-resistant yeast for L-lactic acid production

以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5(乳酸调节)的培养基中生长且不利用乳酸的酵母(初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因(ldhA)插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C.magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C.magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最适pH为3.5,并在pH 2.5时正常发酵产乳酸。本研究成果为首次从自然界筛选到一株耐高浓乳酸的耐酸酵母菌,并以此为宿主菌构建了具有生产L-乳酸能力的耐酸重组酵母,为以耐酸酵母菌高产L-乳酸进行了有益的探索。

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content,one wild acid-resistant yeast strain isolated from natural samples,was found to be able to grow well in YEPD medium（20 g/L glucose,20 g/L tryptone,10 g/L yeast extract,adjusted pH 2.5 with lactic acid） without consuming lactic acid.Based on further molecular biological tests,the strain was identified as Candida magnolia.Then,the gene ldhA,encoding a lactate dehydrogenase from Rhizopus oryzae,was cloned into a yeast shuttle vector containing G418 resistance gene.The resultant plasmid pYX212-kanMX-ldhA was introduced into C.magnolia by electroporation method.Subsequently,a recombinant L-lactic acid producing yeast C.magnolia-2 was obtained.The optimum pH of the recombinant yeast is 3.5 for lactic acid production.Moreover,the recombinant strain could grow well and produce lactic acid at pH 2.5.This recombinant yeast strain could be useful for producing L-lactic acid.