来源于马赛菌(Massilia sp.)UMI-21的ACS_(MU)和PhaC_(MU)体外表达及功能研究  

Overexpression and functional characterization of ACS_(MU)and PhaC_(MU)from Massilia sp.UMI-21

作　　者：王明 李雪 韩雪容 WANG Ming;LI Xue;HAN Xuerong(Jilin Province Key Laboratory of Fungal Phenomics,Jilin Agricultural University,Changchun 130118,Jilin,China;School of Life Science and Technology,Changchun University of Science and Technology,Changchun 130022,Jilin,China)

机构地区：[1]吉林农业大学、吉林省菌物表型组学重点实验室,吉林长春130118 [2]长春理工大学生命科学技术学院,吉林长春130022

出　　处：《微生物学报》2024年第4期1162-1174,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31971252)。

摘　　要：【目的】构建马赛菌(Massilia sp.)UMI-21来源乙酰辅酶A合成酶ACS_(MU)和聚羟基脂肪酸酯(polyhydroxyalkanoate,PHA)合酶PhaC_(MU)的体外重组表达体系并过表达2种酶,利用体外合成体系确定2种酶在Massilia sp.UMI21聚3-羟基丁酸(polyhydroxybutyrate,PHB)合成途径中的主要功能。【方法】利用无缝克隆技术将来源于Massilia sp.UMI-21的乙酰辅酶A合成酶基因acsMU和PHA合酶基因phaC_(MU)扩增后与pQE-80L质粒连接,转导大肠杆菌(Escherichia coli)BL21(DE3)构建2个基因的重组表达体系;利用6×His标签纯化蛋白ACS_(MU)和PhaC_(MU),并采用5,5′-二硫双(2-硝基苯甲酸)[5,5′-dithiobis-(2-nitrobenzoic acid),DTNB]法测定其活性;使用体外单相合成系统(one-phase reaction system,OPRS),以(R)-3HB为底物,验证ACS_(MU)和PhaC_(MU)这2种酶在合成PHB途径中的功能。【结果】成功构建了ACS_(MU)和PhaC_(MU)蛋白重组表达菌株BL21-pQE-80L-acsMU和BL21-pQE-80L-phaC_(MU),提纯得到过表达蛋白ACS_(MU)和PhaC_(MU)产率分别为24.8 mg/L和25.6 mg/L;ACS_(MU)酶比活力为(0.148±0.011)U/mg。PhaC_(MU)酶对(R)-3HBCoA的比活力为(0.102±0.011)U/mg;核磁共振氢谱(nuclear magnetic resonance hydrogen spectroscopy,^(1)H-NMR)分析结果表明,使用ACSPt-PCT_(CP)-PhaC_(Re)、ACS_(MU)-PCT_(CP)-PhaC_(Re)和ACS_(MU)-PCT_(CP)-PhaC_(MU)这3条OPRS途径均能合成PHB,产量分别为0.62、0.76和0.64 g/L。【结论】acsMU和phaC_(MU)基因可利用大肠杆菌表达体系过表达并可获得具有活性的可溶性蛋白;对比ACSPt-PCT_(CP)-PhaC_(Re)合成体系,ACS_(MU)替代ACSPt合成PHB产量增加22.58%,在聚合酶相同的情况下,PHB的合成产量依赖乙酰辅酶A合成酶(acetyl-CoA synthase,ACS)合成乙酰辅酶A的稳定性。使用PhaC_(MU)代替PhaC_(Re),对比ACS_(MU)-PCT_(CP)-PhaC_(Re)组合,合成PHB产量减少了15.79%。在聚合前体浓度相同的情况下,PHB合成量依赖聚合酶的活性。[Objective]To obtain the proteins of acetyl-CoA synthase(ACS_(MU))and PHA synthase(PhaC_(MU))from Massilia sp.UMI-21 by structuring an in vitro recombinant expression system,and to elucidate their roles in the biosynthesis of polyhydroxybutyrate(PHB)using the one-phase reaction system(OPRS).[Methods]Seamless cloning was employed to ligate the acetyl-CoA synthase gene acsMU and the PHA synthase gene phaC_(MU)amplified from Massilia sp.UMI-21 to the pQE-80L plasmid to construct the recombinant plasmids.The recombinant plasmids were transformed into Escherichia coli BL21(DE3),and the recombinant strains were obtained.ACS_(MU)and PhaC_(MU)were purified using a 6×His tag,and their activities were determined by the 5,5′-dithiobis-(2-nitrobenzoic acid)(DTNB)method.With 3HB as a substrate,the one-phase reaction system(OPRS)was employed to validate the functions of ACS_(MU)and PhaC_(MU)in the biosynthesis of PHB.[Results]The recombinant strains BL21-pQE-80L-acsMU and BL21-pQE-80L-phaC_(MU)were successfully engineered,with the ACS_(MU)and PhaC_(MU)yields of 24.8 mg/L and 25.6 mg/L,respectively.The specific activity of ACS_(MU)was(0.148±0.011)U/mg,and that of PhaC_(MU)for(R)-3HBCoA was(0.102±0.011)U/mg.Nuclear magnetic resonance hydrogen spectroscopy(^(1)H-NMR)results showed that products from the all three PHB synthesis pathways,ACSPt-PCT_(CP)-PhaC_(Re),ACS_(MU)-PCT_(CP)-PhaC_(Re),and ACS_(MU)-PCT_(CP)-PhaC_(MU),in OPRS were PHB.The yields of PHB via the three pathways were 0.62,0.76,and 0.64 g/L,respectively.[Conclusion]The genes acsMU and phaC_(MU)can be overexpressed in the E.coli expression system to yield active soluble proteins.Compared with the ACSPt-PCT_(CP)-PhaC_(Re)pathway,substitution of ACSPt with ACS_(MU)increased the PHB yield by 22.58%.The yield of PHB was contingent upon the stability of acetyl-CoA synthase(ACS),which provided acetyl-CoA for reaction under identical PhaC.Replacing PhaC_(Re)with PhaC_(MU)decreased the PHB yield by 15.79%compared with ACS_(MU)-PCT_(CP)-PhaC_(Re).The polymerase PhaC plays

关 键 词：马赛菌UMI-21 乙酰辅酶A合成酶(ACS) 聚羟基脂肪酸酯(PHA)合酶 聚3-羟基丁酸(PHB)合成途径 

分 类 号：Q936[生物学—微生物学] Q78