构建多酶复合体强化异戊二烯生物合成

doi:10.11949/0438-1157.20241454

化工学报 ›› 2025, Vol. 76 ›› Issue (7): 3436-3445.DOI: 10.11949/0438-1157.20241454

构建多酶复合体强化异戊二烯生物合成

孙慧¹(), 屈虹男², 孙甲琛³, 张根林¹(), 贾海洋²(), 李春¹^,²^,³^,⁴()

^1.石河子大学化学化工学院/化工绿色过程省部共建国家重点实验室培育基地，新疆石河子 832003
^2.北京理工大学化学与化工学院生物化工研究所/医药分子科学与制剂工程工业和信息化部重点实验室，北京 100081
^3.天津大学化工学院
^1.系统生物工程教育部重点实验室，天津 300072，清华大学化学工程系工业生物催化教育部重点实验室，北京 100084

收稿日期:2024-12-16 修回日期:2025-04-14 出版日期:2025-07-25 发布日期:2025-08-13
通讯作者: 张根林,贾海洋,李春
作者简介:孙慧（2000—），女，硕士研究生， s175908357@126.com
基金资助:
国家自然科学基金重点项目(22138006);国家自然科学基金面上项目(2247081930)

Construction of multi-enzyme complex to enhance isoprene biosynthesis

Hui SUN¹(), Hongnan QU², Jiachen SUN³, Genlin ZHANG¹(), Haiyang JIA²(), Chun LI¹^,²^,³^,⁴()

^1.State Key Laboratory Incubation Base for Green Processing of Chemical Engineering, School of Chemistry and Chemical Engineering, Shihezi University，Shihezi 832003, Xinjiang, China
^2.Key Laboratory of Medicinal Molecule Science and Pharmaceutical Engineering of Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China
^3.Key Laboratory of Systems Bioengineering, Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
^4.Key Laboratory of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Received:2024-12-16 Revised:2025-04-14 Online:2025-07-25 Published:2025-08-13
Contact: Genlin ZHANG, Haiyang JIA, Chun LI

摘要/Abstract

摘要：

针对大肠杆菌细胞工厂合成异戊二烯存在前体不足、有毒中间代谢物积累、产物流失等问题，利用合成生物学和代谢工程的方法，在大肠杆菌体内表达银白杨（Populus alba）来源的异戊二烯合酶，并通过多顺反子的形式过表达Dxs、Dxr和IspD的单个或多个基因，强化大肠杆菌内源的MEP途径。为缩短中间代谢产物在酶分子间的运输距离及时间，增强途径的底物隧道效应，运用蛋白支架策略将MEP途径中Dxs、Dxr和IspD酶进行共区域化，构建异戊二烯合成的多酶复合体，获得的工程菌株BL21（DE3）-ScaS，异戊二烯产量达到24 mg/L，较游离酶形式的工程菌株BL21（DE3）-FreeS的产量提高了35.7%。并通过工程菌株BL21（DE3）-ScaS质粒稳定性研究，发现发酵过程中补加抗生素能够显著提高不相容性重组质粒pET28a-DxsDxrIspD与pET21b-GSP-IspS的稳定性。同时，利用通量平衡分析（flux balance analysis，FBA）探究合成途径中关键酶的代谢通量分布情况，从理论层面证明了上述策略的有效性，为异戊二烯微生物细胞工厂的开发及应用提供了参考。

关键词: 异戊二烯, 异戊二烯合酶, 蛋白支架, 多酶复合体, 大肠杆菌

Abstract:

In view of the problems of insufficient precursors, accumulation of toxic intermediate metabolites and product loss in the synthesis of isoprene in Escherichia coli cell factories, we used synthetic biology and metabolic engineering methods to express isoprene synthase from Populus alba in Escherichia coli and overexpressed single or multiple genes of Dxs, Dxr and IspD in the form of polycistronic to strengthen the endogenous MEP pathway of Escherichia coli. In order to shorten the transport distance and time of intermediate metabolites between enzyme molecules and enhance the substrate channeling effect of the pathway, the protein scaffold strategy was applied to Dxs, Dxr and IspD enzyme colocalization in the MEP pathway to construct a multi-enzyme complex for isoprene synthesis, and obtained the engineered strain BL21(DE3)-ScaS produced isoprene yield of 24 mg/L, which was 35.7% higher than the engineered strain BL21(DE3)-FreeS in free enzyme form. And through the stability study of the engineered strain BL21(DE3)-ScaS plasmid, it was found that the supplementation of antibiotics during the fermentation process could significantly improve the stability of the incompatible recombinant plasmids pET28a-DxsDxrIspD and pET21b-GSP-IspS. At the same time, flux balance analysis (FBA) was used to explore the metabolic flux distribution of key enzymes in the synthetic pathway, the effectiveness of the above strategies is proved theoretically, which provides a reference for the development and application of isoprene microbial cell factories.

Key words: isoprene, isoprene synthase, protein scaffold, multi-enzyme complex, Escherichia coli

中图分类号:

Q 816

孙慧, 屈虹男, 孙甲琛, 张根林, 贾海洋, 李春. 构建多酶复合体强化异戊二烯生物合成[J]. 化工学报, 2025, 76(7): 3436-3445.

Hui SUN, Hongnan QU, Jiachen SUN, Genlin ZHANG, Haiyang JIA, Chun LI. Construction of multi-enzyme complex to enhance isoprene biosynthesis[J]. CIESC Journal, 2025, 76(7): 3436-3445.

图/表 10

参考文献 33

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质粒	描述	来源
pUC57-Isps	克隆载体连接Isps基因	公司合成
pET28a	T7 promoter, Kan^R, f1 ori	实验室储存
pET21b	T7 promoter, Amp^R, f1 ori	实验室储存
pET21b-IspS	pET21b连接IspS基因	本研究构建
pET21b-IspSq	pET21b连接IspSq基因	本研究构建
pET28a-Dxs	pET28a连接RBS-Dxs基因	本研究构建
pET28a-Dxr	pET28a连接RBS-Dxr基因	本研究构建
pET28a-DxsDxr	pET28a连接RBS-Dxs-RBS-Dxr基因	本研究构建
pET28a-DxsIspD	pET28a连接RBS-Dxs-RBS-IspD基因	本研究构建
pET28a-DxrIspD	pET28a连接RBS-Dxr-RBS-IspD基因	本研究构建
pET28a-DxsDxrIspD	pET28a连接RBS-Dxs-RBS-Dxr-RBS-IspD基因	本研究构建
pET28a-Dxr-GBD	pET28a连接Dxr-GBD基因	本研究构建
pET28a-IspD-SH3	pET28a连接IspD-SH3基因	本研究构建
pET28a-Dxs-PDZ	pET28a连接Dxs-PDZ基因	本研究构建
pET28a-Dxs-Linker-GBD lig	pET28a连接Dxs-Linker-GBD lig基因	本研究构建
pET28a-Dxr-Linker-SH3 lig	pET28a连接Dxr-Linker-SH3 lig基因	本研究构建
pET28a-IspD-Linker-PDZ lig	pET28a连接IspD-Linker-PDZ lig基因	本研究构建
pET28a-DxsDxrIspD-GSP lig	pET28a连接Dxs-Linker-GBD lig、Dxr-Linker-SH3 lig、IspD-Linker-PDZ lig基因	本研究构建
pET21b-GSP-IspS	pET21b连接GBD-SH3-PDZ基因和IspS基因	本研究构建
pET21b-GSP-IspSq	pET21b连接GBD-SH3-PDZ基因和IspSq基因	本研究构建

质粒	描述	来源
pUC57-Isps	克隆载体连接Isps基因	公司合成
pET28a	T7 promoter, Kan^R, f1 ori	实验室储存
pET21b	T7 promoter, Amp^R, f1 ori	实验室储存
pET21b-IspS	pET21b连接IspS基因	本研究构建
pET21b-IspSq	pET21b连接IspSq基因	本研究构建
pET28a-Dxs	pET28a连接RBS-Dxs基因	本研究构建
pET28a-Dxr	pET28a连接RBS-Dxr基因	本研究构建
pET28a-DxsDxr	pET28a连接RBS-Dxs-RBS-Dxr基因	本研究构建
pET28a-DxsIspD	pET28a连接RBS-Dxs-RBS-IspD基因	本研究构建
pET28a-DxrIspD	pET28a连接RBS-Dxr-RBS-IspD基因	本研究构建
pET28a-DxsDxrIspD	pET28a连接RBS-Dxs-RBS-Dxr-RBS-IspD基因	本研究构建
pET28a-Dxr-GBD	pET28a连接Dxr-GBD基因	本研究构建
pET28a-IspD-SH3	pET28a连接IspD-SH3基因	本研究构建
pET28a-Dxs-PDZ	pET28a连接Dxs-PDZ基因	本研究构建
pET28a-Dxs-Linker-GBD lig	pET28a连接Dxs-Linker-GBD lig基因	本研究构建
pET28a-Dxr-Linker-SH3 lig	pET28a连接Dxr-Linker-SH3 lig基因	本研究构建
pET28a-IspD-Linker-PDZ lig	pET28a连接IspD-Linker-PDZ lig基因	本研究构建
pET28a-DxsDxrIspD-GSP lig	pET28a连接Dxs-Linker-GBD lig、Dxr-Linker-SH3 lig、IspD-Linker-PDZ lig基因	本研究构建
pET21b-GSP-IspS	pET21b连接GBD-SH3-PDZ基因和IspS基因	本研究构建
pET21b-GSP-IspSq	pET21b连接GBD-SH3-PDZ基因和IspSq基因	本研究构建

菌株	描述	来源
E.coli Top10	野生型菌株用于分子克隆	公司购买
E.coli C600	以基因组为模板克隆IspD基因	实验室储存
Bacillus subtilis SL-14	以基因组为模板克隆Dxr、Dxs基因	实验室储存
BL21(DE3)	野生型表达宿主	实验室储存
BL21(DE3)-IspS	BL21(DE3)带有重组质粒pET-21b-IspS	本研究构建
BL21(DE3)-IspSq	BL21(DE3)带有重组质粒pET-21b-IspSq	本研究构建
BL21(DE3)-Dxs-IspS	BL21(DE3)-IspS带有重组质粒pET28a-Dxs	本研究构建
BL21(DE3)-Dxr-IspS	BL21(DE3)-IspS带有重组质粒pET28a-Dxr	本研究构建
BL21(DE3)-DxsDxr-IspS	BL21(DE3)-IspS带有重组质粒pET28a-DxsDxr	本研究构建
BL21(DE3)-DxsIspD-IspS	BL21(DE3)-IspS带有重组质粒pET28a-DxsIspD	本研究构建
BL21(DE3)-DxrIspD-IspS	BL21(DE3)-IspS带有重组质粒pET28a-DxrIspD	本研究构建
BL21(DE3)-FreeS	BL21(DE3)-IspS带有重组质粒pET28a-DxsDxrIspD	本研究构建
BL21(DE3)-Freeq	BL21(DE3)-IspSq带有重组质粒pET28a-DxsDxrIspD	本研究构建
BL21(DE3)-ScaS	BL21(DE3)带有重组质粒pET28a-DxsDxrIspD-GSP lig和pET21b-GSP-IspS	本研究构建
BL21(DE3)-Scaq	BL21(DE3)带有重组质粒pET28a-DxsDxrIspD-GSP lig和pET21b-GSP-IspSq	本研究构建

菌株	描述	来源
E.coli Top10	野生型菌株用于分子克隆	公司购买
E.coli C600	以基因组为模板克隆IspD基因	实验室储存
Bacillus subtilis SL-14	以基因组为模板克隆Dxr、Dxs基因	实验室储存
BL21(DE3)	野生型表达宿主	实验室储存
BL21(DE3)-IspS	BL21(DE3)带有重组质粒pET-21b-IspS	本研究构建
BL21(DE3)-IspSq	BL21(DE3)带有重组质粒pET-21b-IspSq	本研究构建
BL21(DE3)-Dxs-IspS	BL21(DE3)-IspS带有重组质粒pET28a-Dxs	本研究构建
BL21(DE3)-Dxr-IspS	BL21(DE3)-IspS带有重组质粒pET28a-Dxr	本研究构建
BL21(DE3)-DxsDxr-IspS	BL21(DE3)-IspS带有重组质粒pET28a-DxsDxr	本研究构建
BL21(DE3)-DxsIspD-IspS	BL21(DE3)-IspS带有重组质粒pET28a-DxsIspD	本研究构建
BL21(DE3)-DxrIspD-IspS	BL21(DE3)-IspS带有重组质粒pET28a-DxrIspD	本研究构建
BL21(DE3)-FreeS	BL21(DE3)-IspS带有重组质粒pET28a-DxsDxrIspD	本研究构建
BL21(DE3)-Freeq	BL21(DE3)-IspSq带有重组质粒pET28a-DxsDxrIspD	本研究构建
BL21(DE3)-ScaS	BL21(DE3)带有重组质粒pET28a-DxsDxrIspD-GSP lig和pET21b-GSP-IspS	本研究构建
BL21(DE3)-Scaq	BL21(DE3)带有重组质粒pET28a-DxsDxrIspD-GSP lig和pET21b-GSP-IspSq	本研究构建

构建多酶复合体强化异戊二烯生物合成

Construction of multi-enzyme complex to enhance isoprene biosynthesis

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