基于表达元件和宿主优化促地衣芽胞杆菌高效表达L-天冬酰胺酶  

Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host

作　　者：杨新愿 饶忆 张梦茜 王佳琪 刘文渊 蔡冬波 陈守文[1] YANG Xinyuan;RAO Yi;ZHANG Mengxi;WANG Jiaqi;LIU Wenyuan;CAI Dongbo;CHEN Shouwen(State Key Laboratory of Biocatalysis and Enzyme Engineering,Environmental Microbial Technology Center of Hubei Province,College of Life Sciences,Hubei University,Wuhan 430062,Hubei,China)

机构地区：[1]湖北大学生命科学学院、湖北省环境微生物工程技术研究中心、省部共建生物催化与酶工程国家重点实验室,湖北武汉430062

出　　处：《生物工程学报》2023年第3期1096-1106,共11页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2021YFC2100200)。

摘　　要：L-天冬酰胺酶(L-asparaginase, L-ASN)广泛用于恶性肿瘤治疗及低丙烯酰胺食品生产,然而其较低的表达水平限制了应用推广。异源蛋白表达是提高目标酶表达水平的有效策略,芽胞杆菌广泛用于酶蛋白的高效生产,本研究拟通过表达元件及宿主优化提高芽胞杆菌(Bacillus)中L-天冬酰胺酶产量。首先,筛选了5种信号肽(SPSacC、SPAmyL、SPAprE、SPYwbN、SPWapA)用于L-天冬酰胺酶的分泌表达,其中SPSacC介导下L-天冬酰胺酶分泌效果最好,酶活达到157.61 U/mL。随后,选取了4种芽胞杆菌强启动子(P43、PykzA-P43、PUbay、Pbac A),其中串联启动子PykzA-P43介导的L-天冬酰胺酶表达量最高,较对照菌株提高了52.94%。最后,筛选了3种芽胞杆菌表达宿主:地衣芽胞杆菌(Bacillus licheniformis)Δ0F3和BL10、枯草芽胞杆菌(B. subtilis) WB800,其中,地衣芽胞杆菌BL10作为宿主时,L-天冬酰胺酶酶活最高,达到了438.3 U/mL,较对照菌株提高了81.83%,为目前报道的L-天冬酰胺酶摇瓶酶活最高水平。综上所述,本研究成功构建了一株高产L-天冬酰胺酶的地衣芽胞杆菌工程菌株BL10/PykzA-P43-SPsacC-ansZ,为L-天冬酰胺酶工业化生产奠定了基础。L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production,however,the low expression level hampers its application.Heterologous expression is an effective strategy to increase the expression level of target enzymes,and Bacillus is generally used as the host for efficient production of enzymes.In this study,the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host.Firstly,five signal peptides (SPSacC,SPAmyL,SPAprE,SPYwbNand SPWapA) were screened,among which SPSacCshowed the best performance,reaching an activity of 157.61 U/mL.Subsequently,four strong promoters (P43,PykzA-P43,PUbayand PbacA) from Bacillus were screened,and tandem promoter PykzA-P43showed the highest yield of L-asparaginase,which was 52.94%higher than that of control strain.Finally,three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10,B. subtilis WB800) were investigated,and the maximumL-asparaginase activity,438.3 U/mL,was reached by B.licheniformis BL10,which was an 81.83%increase compared with that of the control.This is also the highest level of L-asparaginase in shake flask reported to date.Taken together,this study constructed a B.licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase,which laid the foundation for industrial production of L-asparaginase.

关 键 词：L-天冬酰胺酶 地衣芽胞杆菌 信号肽 启动子 表达宿主 

分 类 号：Q936[生物学—微生物学]