不同信号肽对酿酒酵母蔗糖转化酶Suc2分泌表达水平的影响  

Effect of Different Signal Peptide on Secretary Level of Invertase Suc2 in Saccharomyces cerevisiae

作　　者：王天琦 王碧莹 付彤 陈晓艺[1] 李宪臻[1] 杨帆[1] WANG Tian-qi;WANG Bi-ying;FU Tong;CHEN Xiao-yi;LI Xian-zhen;YANG Fan(Schl.of Bio-Engin,Dalian Polytechnic Uni,Dalian 116034)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《微生物学杂志》2023年第5期9-20,共12页Journal of Microbiology

基　　金：国家自然科学基金青年基金项目(31801469);辽宁省科学研究项目(J2020041);。

摘　　要：目前,绝大多数酿酒酵母(Saccharomyces cerevisiae)菌株利用菊糖生产乙醇的能力有限,而蔗糖转化酶Suc2是酿酒酵母水解菊糖的关键酶,其分泌水平直接影响酿酒酵母转化菊糖为乙醇的性能。为提高酿酒酵母中蔗糖转化酶Suc2的分泌表达水平,利用生物信息学的分析方法选择出11种不同的分泌信号肽,包括酿酒酵母内源性、其他菌株来源以及已报道序列优化改造的信号肽,将它们融合至Suc2并构建了相应的酿酒酵母BY4741重组菌。其中,酿酒酵母内源分泌信号肽AGA2能使蔗糖转化酶Suc2更有效的分泌,含有信号肽AGA2的重组菌BY-AG的蔗糖酶酶活和菊糖酶酶活相对于含有天然信号肽的原始菌BY-S分别提高42%和26%,其利用菊糖产乙醇能力较原始菌提高了32%,乙醇产量达到78.11 g/L。在使用毕赤酵母(Pichia pastoris)分泌信号肽MSB2时,蔗糖转化酶Suc2的分泌水平也有提高,含有信号肽MSB2的重组菌BY-MS较原始菌BY-S的蔗糖酶酶活和菊糖酶酶活分别提高了80%和74%,同时,利用菊糖产乙醇能力也提高了56%,产量达到86.31 g/L。最后,对重组菌BY-MS摇瓶发酵过程中的生物量、蔗糖酶酶活、残糖总量和乙醇产量进行了监测,结果表明,重组菌BY-MS的发酵性能较原始菌BY-S有显著提高。本研究为提高蔗糖转化酶Suc2的分泌水平、构建高效菊糖基乙醇生产菌株提供参考。At present,most Saccharomyces cerevisiae strains lack the ability to convert inulin into bioethanol,and sucrose invertase Suc2 is the key enzyme of S.cerevisiae to hydrolyze inulin.The secretion level of Suc2 directly affects the inulin-assimilating ability of S.cerevisiae.Therefore,to improve the secretion level of Suc2,bioinformatic analysis methods was adopted to select 11 different signal peptides secrete from S.cerevisiae strains,includes endogeneity,and other strain sources,and previously reported optimized signal peptides.These signal peptides were respectively fused to Suc2 and constructed corresponding recombinant S.cerevisiae strains BY4741.Among them endogenous signal peptide AGA2 of S.cerevisiae could more effectively secrete Suc2.And activities of sucrase and inulinase of AGA2 that contained signal peptide of recombinant BY-AG increased by 42%and 26%respectively as compared with the original strain BY-S.And its ethanol production ability using inulin increased by 32%as compared with original strain and the ethanol production reached up to 78.11 g/L.When using signal peptide MSB2 secreted from Pichia pastoris,the secretion level of Suc2 was also increased.The invertase activity and inulinase activity of recombinant strain BY-MS that contained signal peptide MSB2 increased by 80%and 74%respectively as compared with original strain BY-S.At the same time,the ethanol production of BY-MS reached up to 86.31 g/L increased by 56%.Finally,the biomass,invertase activity,total residual sugar and ethanol production in shake flask fermentation process of BY-MS were monitored.The results showed that the fermentation performance of recombinant strain BY-MS was significantly enhanced as compared with original strain BY-S.This study provided theoretical basis for increasing the secretion level of sucrose invertase Suc2 and constructing production strains with highly efficient inulin-based ethanol producing activity.

关 键 词：酿酒酵母 蔗糖转化酶Suc2 菊糖 分泌水平 分泌信号肽 生物乙醇 

分 类 号：Q939.97[生物学—微生物学]