基于启动子定向进化的葡萄糖-6-磷酸异构酶高效分泌表达体系的构建

游文慧; 付刚; 张大伟

文章摘要

基于启动子定向进化的葡萄糖-6-磷酸异构酶高效分泌表达体系的构建

Development of an Efficient Secretory Expression System for Glucose-6-Phosphate Isomerase Based on Promoter Directed Evolution

DOI：doi:10.3969/j.issn.1005-7021.2026.02.002

中文关键词: 枯草芽孢杆菌（Bacillus subtilis） MATE表达系统启动子突变葡萄糖-6-磷酸异构酶培养基优化

英文关键词: Bacillus subtilis MATE expression system promoter mutation glucose-6-phosphate isomerase medium optimization

基金项目:中国科学院战略性先导科技专项（XDC0120203）；天津市科技计划项目（24ZYCGYS00690）

作者	单位
游文慧	1.大连工业大学生物工程学院,辽宁大连 116034；2.中国科学院天津工业生物技术研究所,天津 300308
付刚	2.中国科学院天津工业生物技术研究所,天津 300308；3.中国科学院大学,北京 10004；4.低碳合成工程生物学全国重点实验室中国科学院工业生物技术研究所,天津 300308；5.国家合成生物技术创新中心,天津 300308
张大伟	1.大连工业大学生物工程学院,辽宁大连 116034；2.中国科学院天津工业生物技术研究所,天津 3003083；3.中国科学院大学,北京 10004；4.低碳合成工程生物学全国重点实验室中国科学院工业生物技术研究所,天津 300308；5.国家合成生物技术创新中心,天津 300308；6.天津科技大学生物工程学院,天津 300457

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中文摘要:

为提升诱导型启动子P_malA的表达强度并实现目标蛋白的高效胞外表达，基于前期所构建的麦芽糖诱导型表达系统(MATE表达系统)，通过易错PCR构建启动子突变文库，并以绿色荧光蛋白（Green fluorescent protein, GFP）为报告蛋白筛选高表达突变体，获得突变位点位于-35区域的增强型启动子M3。以M3为调控元件构建目标酶葡萄糖-6-磷酸异构酶（Pgi）表达载体，融合Sec型信号肽AmyS实现其胞外分泌表达。通过添加不同碳源进行诱导表达优化，筛选出适配性最优条件后，在3 L发酵罐中进行高密度发酵，Pgi产量达7.26 g/L。功能验证表明，本体系表达的Pgi催化活性与大肠埃希菌（Escherichia coli）对照表达体系相当，说明该系统在提升表达强度的同时保持了蛋白功能的稳定性。本研究构建了一个优化后的高效MATE表达体系，为代谢工程中关键酶的高效表达提供了可行路径，并为枯草芽孢杆菌（Bacillus subtilis）在工业酶制剂开发中的应用提供了支撑。

英文摘要:

To enhance the transcriptional strength of the inducible promoter P_malA and achieve efficient extracellular expression of target proteins, in this study, error-prone PCR was employed to construct a promoter mutant library based on a previously established maltose-inducible expression system (MATE expression system). Using green fluorescent protein(GFP) as a reporter protein, a high-expression mutant designated as M3, harboring a single-base substitution in the-35 region, was identified. The mutant promoter M3 was then utilized as a regulatory element to construct a recombinant expression vector for glucose-6-phosphate isomerase (phospho-glucose isomerase，Pgi), which was fused at its N-terminus to the Sec-type signal peptide AmyS to facilitate secretion. To further optimize inducible expression, various carbon sources were evaluated, and the optimal condition was applied in a 3-L bioreactor for high-density fermentation, yielding a maximum Pgi titer of 7.26 g/L. Functional assays confirmed that the catalytic activity of Pgi expressed in this system was comparable to that of the control produced in Escherichia coli, indicating that enhanced expression did not compromise protein functionality. This study establishes an optimized and efficient MATE-based expression system, offering a feasible strategy for high-level expression of key enzymes in metabolic engineering and supporting the application of Bacillus subtilis in the development of industrial enzyme preparations.

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