摘要
【目的】在酿酒酵母体内设计代谢通路,使酿酒酵母能利用纤维素水解产物纤维二糖生产乙醇。【方法】首先,用大肠杆菌DH5α总DNA为模板克隆编码大肠杆菌乳糖透过酶的LacY基因。为过表达LacY基因,以质粒YEplac181作为载体,将酿酒酵母PGK1p强启动子加到LacY基因之前,CYC1t终止子加到LacY基因之后,构建质粒YEplac181-PGK1p-LacY-CYC1t。之后,将纤维二糖转运蛋白LacY表达质粒和β-葡萄糖苷酶(β-glucosidase,BGL)表达质粒pRS316-PGK1p-gh1-1-CYC1t依次转入野生型酿酒酵母W303-1A中,使野生型酿酒酵母W303-1A异源表达可转运纤维二糖的LacY蛋白和β-葡萄糖苷酶GH1-1,构建可利用纤维二糖的酿酒酵母工程菌W303-1A GL。最后,通过发酵测定酿酒酵母工程菌W303-1A GL的纤维二糖利用情况和乙醇产量,并对纤维二糖代谢通路中纤维二糖酶活力进行测定。【结果】本研究构建了纤维二糖转运蛋白LacY和β-葡萄糖苷酶GH1-1协同表达的酿酒酵母工程菌W303-1AGL。W303-1AGL可以有效利用纤维二糖发酵生产乙醇,W303-1A GL发酵24 h时乙醇产量达到3.25 g/L,得率为0.325 g乙醇/g纤维二糖,利用葡萄糖产乙醇理论得率为0.511 g乙醇/g纤维二糖,达到葡萄糖产乙醇理论得率的64%,细胞密度最高在第54 h达到OD600=10.84,胞内β-葡萄糖苷酶的酶活在72 h最高,可达到0.51 U/mg。【结论】本研究成功构建了能有效利用纤维二糖的重组酿酒酵母工程菌W303-1A GL,为提高纤维素乙醇生产效率、降低纤维素乙醇生产成本提供了新思路。
[Objective]A metabolic pathway was designed in Saccharomyces cerevisiae so that it could produce ethanol from cellobiose,a hydrolysate of cellulose.[Methods]First,with the total DNA of Escherichia coli DH5αas template,the LacY gene encoding lactose permease was cloned.With plasmid YEplac181 as the vector,the strong promoter PGK1p of S.cerevisiae was added in front of the LacY,and the CYC1t terminator was added behind LacY to construct the plasmid YEplac181-PGK1p-LacY-CYC1t.Then,the cellobiose transporter(LacY)expression plasmid andβ-glucosidase expression plasmid pRS316-PGK1p-gh1-1-CYC1t were successively transferred into wild-type S.cerevisiae W303-1A.The engineered S.cerevisiae W303-1A GL was constructed by heterologous expression of cellobiose transporter LacY andβ-glucosidase GH1-1 in W303-1A.Finally,the cellobiose utilization and ethanol yield of W303-1A GL were determined based on fermentation,and the cellobiose activity in the cellobiose metabolic pathway was detected.[Results]W303-1A GL which expressed LacY and GH1-1 was developed,which can use cellobiose to produce ethanol.The ethanol yield of W303-1A GL was up to 3.25 g/L at 24 h,and the production rate was 0.325 g ethanol/g cellobiose,64%of the theoretical production rate from glucose(0.511 g ethanol/g cellobiose).The highest cell density was OD600=10.84 at 54 h,and the activity of intracellularβ-glucosidase peaked(0.51 U/mg)at 72 h.[Conclusion]W303-1A GL which can effectively utilize cellobiose is developed.This study is expected to serve as a reference for improving the production of cellulosic ethanol at lower cost.
作者
鲁尚昆
苏意德
邵文举
张爱利
LU Shangkun;SU Yide;SHAO Wenju;ZHANG Aili(School of Chemical Engineering and Technology,Hebei University of Technology,Tianjin 300130,China)
出处
《微生物学报》
CAS
CSCD
北大核心
2023年第1期170-180,共11页
Acta Microbiologica Sinica
基金
国家自然科学基金(21978065,21206028)
教育部“春晖计划”合作科研项目(Z2017012)。
