摘要
采用基因重组技术将黑曲酶糖化酶GAIcDNA与MF α1因子的启动子 信号序列及PGK基因的转录终止位点重组进大肠杆菌 酵母穿梭质粒构建酵母表达载体YepMGT2 0 ,用原生质体转化法引入酿酒酵母GRF1 8,在酵母MF α1启动子 信号序列及PGK终止位点的调控下 ,实现糖化酶的高表达 ,99%的酶活力分泌至胞外 ,构建的酿酒酵母GRF1 8(YEpMGT2 0 )在 1 0 %淀粉的培养基中培养 48h ,淀粉水解率达 96.1 % ;在 1 0 %淀粉的YPS培养基中发酵 96h可产生 5 .6% (v/v)的酒精 (在2 0 %淀粉培养基中酒精产量达 8.4% ) ,在无选择压力的SC培养基中培养 5d ,重组质粒的丢失率只有 1 .9%。
Glucoamylase GAI cDNA of Aspergillus niger, promoter and signal sequence of MF?α1 factor and terminator of PGK gene were cloned in a E.coli yeast shuttle plasmid resulting in expression vector YEpMGT20 which was then introduced into Saccharomyces cerevisaie GRF18 by protoplast transformation.Glucoamylase was efficiently expressed and 99% of enzyme activity secreted to the medium under the control of MF?α1 promoter signal sequence and PGK terminator.Starch degradation rate reached 96.1% when GRF18(YepMGT20) grew in medium containing 10% starch for 48 hours.5.6% ( v/v ) ethanol was produced in YPS medium containing 10% starch.Ethanol content was 8.4% ( v/v) in 20% starch.The missing rate of recombinant plasmid was 1.9% in SC medium for 5 days without selection stress.
出处
《工业微生物》
CAS
CSCD
2000年第4期15-18,共4页
Industrial Microbiology
基金
广东省自然科学基金 !( 940 6 2 8)
广州市重点研究项目资助!( 96 - 2 2 6 - 2 )
关键词
酵母工程菌
淀粉水解
酒精生产
构建
A.niger glucoamylase
gene expression
engineering yeast strain
starch degradation
ethanol production
