复合诱变和原生质体融合选育S-腺苷甲硫氨酸酿酒酵母高产菌株

Breeding of Saccharomyces cerevisiae giving an enhanced yield of S-adenosyl-L-methionine by composite mutagenesis and protoplast fusion

对生产S-腺苷甲硫氨酸(SAM)的酿酒酵母(S.cerevisiae)菌株R1进行复合诱变和原生质体融合。采用酶解法获得出发菌株S.cerevisiae R1的单倍体菌株hR10,对其进行UV和DES复合诱变,筛选出了4个单倍体正突变株DR1-3、NM14、NM39和NM45。制备了单倍体正突变株的原生质体,采用热灭活和UV灭活法灭活双亲原生质体,在聚乙二醇(PEG)的诱导下进行原生质体融合,筛选得到融合子F35,其生产SAM的产量为22.5 mg/L,与出发菌株R1(11.1mg/L)相比,提高了103%,并且能够稳定遗传。采用正交试验优化了F35发酵生产SAM的培养基和发酵时间,优化了的培养基配方为麦芽糖100 g/L,蛋白胨10 g/L,NH4H2PO4 4 g/L,NH4Cl 5 g/L,MgSO4 0.1 g/L,KH2 PO4 6 g/L,发酵时间为72 h。

Composite mutagenesis and protoplast fusion have been employed in an attempt to improve the production of S-adenosyl-L-methionine（SAM） by S.cerevisiae R1.The haploidy of R1,hR10,was prepared by enzymatic hydrolysis and treated with UV and diethylstilbesterol（DES）.Four haploid mutants named DR1-3,NM14,NM39,and NM45 were selected and their protoplasts were grouped.One group was inactivated by heat,and another group was inactivated by UV irradiation.Then polyethylene glycol（PEG） was used to mediate protoplast fusion and one fusant F35 with improved production of SAM was obtained by screening.The production of SAM by F35 reached 22.5 mg/L,an increase of 103% compared with the value for the starting strain R1.Subculture experiments indicated that the hereditary character of high production was stable.The results of orthogonal tests showed that a medium containing 100g/L of maltose,10g/L of peptone,4g/L of NH4H2PO4,5g/L of NH4Cl,0.1g/L of MgSO4,and 6g/L of KH2PO4 with a fermentation time of 72h was suitable for production of SAM by F35.