摘要
本文对高生物量富硒酵母的菌种选育和培养条件初步优化进行了研究；并对γ-谷氨酰半胱氨酸合成酶基因GSH-I进行了克隆，在酿酒酵母(Saccharomyces cerevisiae)中表达，有效提高了酿酒酵母的谷胱甘肽(GSH)生成量。
从300株工业生产用酵母中，筛选出对亚硒酸钠抗性较高的菌株。再从中选出生物量较高的二倍体菌株S．cerevisiae ZY-67和细胞硒含量较高的二倍体菌株Saccharomyces kluyveri ZY-198，对其进行生孢子培养和单倍体分离。用亚硝基胍(NTG)对生物量较高的单倍体ZY-67-18(α)和细胞硒含量较高的单倍体ZY-198-21(α)进行诱变，从突变株中选出生物量较高的ZY-67-18-34(α，leu~-)和硒含量较高的ZY-198-21-6(α，trp~-)作为融合亲株。通过原生质体融合，选育具有双亲的优良性状，且遗传性状稳定的融合子ZFF-28，其硒总含量分别是原始亲株ZY-67和ZY-198的2.8倍和2.0倍。
通过单因素实验和正交试验设计L_(16)(4~3×2~6)，确定了融合子ZFF-28的优化培养条件。在优化培养条件下，菌株ZFF-28的生物量可达8.2g／L，硒含量达2050μg／g，硒总含量达到了16810μg／L，是培养条件优化前的1．3倍，且细胞硒含量的91％为有机硒。
以GSH含量相对高的1~#酿酒酵母的DNA为模板，通过PCR扩增出4.2kb的目的基因GSH-I片段。将目的基因片段插入穿梭质粒YEp352的多克隆位点，构建重组质粒pGF-2。pGF-2转化单倍体酿酒酵母YS58，转化子的GSH含量是受体菌的1.7倍，且转化子的生物量没有因为质粒pGF-2的转入而受到影响。
将0.43kb的CUPI启动子片段，0.9kb的金属硫蛋白基因MTI片段，GSH-I基因片段，BamHI／SalI双酶切的YEp352片段，用T_4连接酶连接，构建重组质粒pGMF。用重组质粒pGMF转化青岛啤酒酵母S．cerevisiae YSF-31，以铜抗性(Cu~(2+))为选择标记筛选转化子。在非选择条件下培养36h，转化子YSF-31-1(pGMF)的GSH含量是受体菌YSF-31的1.5倍，同时，转化子的生物量没有受到影响。且在非选择条件下连续转接培养144h，85％的转化子含有质粒。
This paper not only focused on the breeding of a high-biomass, selenium-enriched yeast strain and optimization of cultivation conditions, but also focused on increasing glutathione (GSH) formation by functional expression of the γ-glutamylcysteine synthetase gene GSH-I in Saccharomyces cerevisiae.
Strains exhibiting comparatively high Se resistance were selected out of 300 industrial strains. Among them, S.cerevisiae ZY-67 with high biomass and S. kluyveri ZY-198 with high Se content were chosen as initial parental strains. Haploids ZY-67-18 (a) with high biomass and ZY-198-21 (a) with high Se content were mutagenized by NTG. The auxotrophic mutant ZY-67-18-34 (a, leu") with high biomass and ZY-198-21-6 (a, trp-) with high Se content were chosen as parental strains for fusion. Fusant ZFF-28 was selected with higher biomass and Se content than the parental strains. Its biomass and Se content are 2.8 times and 2.0 times of the initial parental strains ZY-67 and ZY-198. Fusant ZFF-28 is stable in genetics.
The cultivation conditions were optimized through single factor tests and a L16 (43 26) orthogonal design. Under the optimized cultivation conditions, the biomass (dry weight) reached 8.2 g/L and the Se content of the cells reached 2050 g/g. Organic Se content of the cells is 91%.
A 4.2kb DNA amplified by PCR was inserted into the SmaI site of YEp352 to construct plasmid pGF-2. The pGF-2 was introduced into S.cerevisiae YS58. The GSH content of the recombinant strain was 1.5 fold of that in the host.
A recombinant plasmid pGMF, containing GSH-I gene from pGF-2, was constructed with a copper resistance gene MTI as the selection marker and was first introduced into S.cerevisiae YSF-31 from Tsingtao Beer Brewery. The GSH content of the recombinant strain was 1.5 fold of that in the host strain. In addition, the biomass of the transformant was not affected by introduction of the plasmid pGMF. After growing the transformants under non-selective conditions (in YEPD) for 144h, 85% of cells still contained plasmid.
引文
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