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

Overexpression and functional characterization of ACSMU and PhaCMU from Massilia sp. UMI-21

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acs_MU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-acs_MU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, ¹H-NMR)分析结果表明，使用ACS_Pt-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】acs_MU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ACS_Pt-PCT_CP-PhaC_Re合成体系，ACS_MU替代ACS_Pt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。

Abstract:

[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 acs_MU 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-acs_MU 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 (¹H-NMR) results showed that products from the all three PHB synthesis pathways, ACS_Pt-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 acs_MU and phaC_MU can be overexpressed in the E. coli expression system to yield active soluble proteins. Compared with the ACS_Pt-PCT_CP-PhaC_Re pathway, substitution of ACS_Pt 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 a crucial role in PHB synthesis under identical precursor concentrations.