氨甲酰磷酸不同合成途径在大肠杆菌中的比较

Comparison of synthesis of carbamoyl phosphate through different pathway in Escherichia coli

摘要:

【背景】氨甲酰磷酸是生物合成代谢中精氨酸与嘧啶的重要前体物质，在工业微生物生产精氨酸与嘧啶及其衍生物中发挥关键作用。【目的】在大肠杆菌Escherichia coli BW25113中比较氨甲酰磷酸不同合成途径的催化效率。【方法】在大肠杆菌Escherichia coli BW25113中过表达鸟氨酸氨甲酰基转移酶(OTC)的基础上，分别过表达大肠杆菌自身的氨基甲酸激酶(CK)和氨甲酰磷酸合酶(CPS Ⅱ)并表征其反应效果。通过优化底物供应(调整底物浓度与引入L-谷氨酰胺合成酶)对CK与CPS Ⅱ的催化反应进行优化。【结果】在大肠杆菌中过表达OTC，建立细胞水平氨甲酰磷酸检测体系。在此基础上比较不同来源的CK，发现大肠杆菌来源的CK效果最好，50 mmol/L NH4HCO3条件下全细胞催化9 h得到2.95±0.15 mmol/L L-瓜氨酸；过表达CPS Ⅱ时，50 mmol/L L-谷氨酰胺催化9 h得到3.16±0.29 mmol/L L-瓜氨酸。通过改变底物NH4HCO3浓度和引入外源L-谷氨酰胺合成酶(GS)等方式对CK与CPS Ⅱ的催化反应分别进行优化后，100 mmol/L NH4HCO3条件下，L-瓜氨酸浓度分别提高至4.67±0.55 mmol/L和6.12±0.38 mmol/L，且过表达GS后CPS Ⅱ途径可以利用NH3，不需要额外添加L-谷氨酰胺。【结论】引入L-谷氨酰胺合成酶后的CPS Ⅱ途径合成氨甲酰磷酸的能力优于CK途径，为精氨酸、嘧啶及其衍生物的合成提供了一种更加高效的策略。

Abstract:

[Background] Carbamoyl phosphate is an important precursor in metabolism of arginine and pyrimidine, and plays a key role in biosynthesis of arginine and pyrimidine and its derivatives in microorganisms. [Objective] The synthesis of carbamoyl phosphate through different pathways was investigated and compared in Escherichia coli BW25113. [Methods] Ornithine carbamoyltransferase (OTC) was overexpressed in E. coli BW25113 as a carbamoyl phosphate detecting system. Then carbamate kinase (CK) from different species or carbamoyl phosphate synthase II (CPS II) was overexpressed in E. coli BW25113 to enhance carbamoyl phosphate biosynthesis. These two carbamoyl phosphate biosynthetic pathways (CK pathway and CPS II pathway) were then optimized by adjusting substrates supply and introducing L-glutamine synthetase. [Results] A carbamoyl phosphate detection system was established in vivo by overexpressing OTC in E. coli. By overexpressing CKs from different species in E. coli with OTC, the carbamoyl phosphate biosynthetic pathway was enhanced, and the results showed that CK from E. coli was better than the other two, which produced 2.95±0.15 mmol/L L-citrulline in 9 h whole-cell bioconversion. By overexpressing CPS II in E. coli with OTC, the carbamoyl phosphate biosynthesis was improved, and 3.16±0.29 mmol/L L-citrulline was obtained in the whole-cell biocatalytic process. The CK and CPS II pathways were optimized by changing the concentration of substrate NH4HCO3 to 100 mmol/L and introducing exogenous L-glutamine synthetase (GS). As a result, 4.67±0.55 mmol/L and 6.12±0.38 mmol/L L-citrulline was achieved by strains with CK or CPS II pathway respectively. After the optimization of CPS II pathway with overexpressing GS, extra addition of L-glutamine was not required in the reaction system. [Conclusion] Biosynthesis of carbamoyl phosphate was improved by constructing and optimizing the CK synthetic pathway or CPS II synthetic pathway. CPS II pathway with introducing GS is better than that of the CK synthetic pathway in respect of carbamoyl phosphate synthesis, and provides a more efficient strategy for the synthesis of arginine, pyrimidine and its derivatives.