Microbial Cell Factory Mediated Multi-enzyme Cascades Reactions Empower High-value Utilization of Biomass

doi:10.19586/j.2095-2341.2026.0039

Current Biotechnology ›› 2026, Vol. 16 ›› Issue (3): 496-509.DOI: 10.19586/j.2095-2341.2026.0039

• Special Forum on Microbial Cell Factory • Previous Articles Next Articles

Microbial Cell Factory Mediated Multi-enzyme Cascades Reactions Empower High-value Utilization of Biomass

Yuqing SUN(), Lige TONG(), Wenxia FANG()

State Key Laboratory of Non-food Biomass Energy Technology，Institute of Biological Science and Technology，Guangxi Academy of Sciences，Nanning 530007，China

Received:2026-02-14 Accepted:2026-04-03 Online:2026-05-25 Published:2026-07-14
Contact: Lige TONG,Wenxia FANG

Abstract

Abstract:

Facing the dual challenges of global fossil resource depletion and worsening environmental problems， the high-value utilization of renewable biomass has become a critical pathway to achieve green and sustainable development. Microbial cell factories， with unique advantages in efficient metabolic network integration， autonomous cofactor regeneration and modular assembly， have emerged as core platforms for driving multi-enzyme cascade reactions. They provide innovative solutions to address bottlenecks including low catalytic efficiency of single enzymes， limited product diversity and complicated processing procedures. This review systematically summarized the core construction strategies of multi-enzyme cascade reactions mediated by microbial cell factories， focusing on the modification and adaptation mechanisms of typical host cells represented by Escherichia coli， Saccharomyces cerevisiae and Aspergillus niger， as well as the application features of in vivo cascade and hybrid cascade systems. Furthermore， we highlighted recent advances in the high-value conversion of complex biomass via this technology， and analyzed key challenges in depth， such as screening and modification of rate-limiting enzymes， compatibility of enzymatic reaction conditions， metabolic flux competition in host cells， and cost constraints restricting large-scale industrialization. This review aims to offer theoretical references and future perspectives for the industrial promotion and application of microbial cell factory-based multi-enzyme cascade reactions in biomass biorefinery.

Key words: multi-enzyme cascade reaction, microbial cell factories, biomass, high-value conversion

CLC Number:

Q815

Yuqing SUN, Lige TONG, Wenxia FANG. Microbial Cell Factory Mediated Multi-enzyme Cascades Reactions Empower High-value Utilization of Biomass[J]. Current Biotechnology, 2026, 16(3): 496-509.

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Figures/Tables 6

References 98

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宿主	底物摄取能力	能量与辅因子代谢	内源代谢网络复杂度	遗传操作便利性	应用
大肠杆菌	对疏水性底物的摄取存在膜通透性限制；高浓度底物可能产生抑制或毒性，影响细胞活性与催化效率	NAD(P)H再生能力强；可通过工程改造优化辅因子偏好性	代谢网络清晰，竞争途径多	遗传工具成熟（基因编辑、重组系统）；转化效率高；多质粒系统稳定	甜味罗汉果苷^[35]、2'-岩藻糖基乳糖^[36]
酿酒酵母	能通过工程化提升前体供应；对疏水性底物的摄取与存储能力可通过脂滴工程增强	具有线粒体呼吸链；ATP与NADH再生效率高	代谢网络较复杂；存在碳流竞争	拥有丰富的遗传工具（启动子库、融合标签、基因编辑等）；支持多层次、多信号的代谢工程策略	叶黄素^[37]、香茅醛^[38]
黑曲霉	碳源利用能力极强	需氧发酵，需控制溶氧；代谢网络完整，支持高密度培养和酶的高效合成	代谢网络复杂但可塑性强；内源酶系丰富：自带多种水解酶，可简化外源酶系构建	遗传工具成熟，启动子、信号肽可工程化；适合构建高效分泌表达系统	N-乙酰-β-D-氨基葡萄糖^[39]、β-葡聚糖^[40]
枯草芽孢杆菌	对于大分子底物，需依赖分泌的酶进行降解；透性化处理可增强胞内酶与底物的接触	辅因子代谢灵活，适应不同氧条件	代谢网络较简单，竞争途径少；适合次级代谢产物合成	可实现基因敲除、整合、多拷贝表达；质粒转化、基因组编辑效率高；易构建无抗生素标记的工程菌	海藻糖^[41]、纤维素降解^[42]

宿主	底物摄取能力	能量与辅因子代谢	内源代谢网络复杂度	遗传操作便利性	应用
大肠杆菌	对疏水性底物的摄取存在膜通透性限制；高浓度底物可能产生抑制或毒性，影响细胞活性与催化效率	NAD(P)H再生能力强；可通过工程改造优化辅因子偏好性	代谢网络清晰，竞争途径多	遗传工具成熟（基因编辑、重组系统）；转化效率高；多质粒系统稳定	甜味罗汉果苷^[35]、2'-岩藻糖基乳糖^[36]
酿酒酵母	能通过工程化提升前体供应；对疏水性底物的摄取与存储能力可通过脂滴工程增强	具有线粒体呼吸链；ATP与NADH再生效率高	代谢网络较复杂；存在碳流竞争	拥有丰富的遗传工具（启动子库、融合标签、基因编辑等）；支持多层次、多信号的代谢工程策略	叶黄素^[37]、香茅醛^[38]
黑曲霉	碳源利用能力极强	需氧发酵，需控制溶氧；代谢网络完整，支持高密度培养和酶的高效合成	代谢网络复杂但可塑性强；内源酶系丰富：自带多种水解酶，可简化外源酶系构建	遗传工具成熟，启动子、信号肽可工程化；适合构建高效分泌表达系统	N-乙酰-β-D-氨基葡萄糖^[39]、β-葡聚糖^[40]
枯草芽孢杆菌	对于大分子底物，需依赖分泌的酶进行降解；透性化处理可增强胞内酶与底物的接触	辅因子代谢灵活，适应不同氧条件	代谢网络较简单，竞争途径少；适合次级代谢产物合成	可实现基因敲除、整合、多拷贝表达；质粒转化、基因组编辑效率高；易构建无抗生素标记的工程菌	海藻糖^[41]、纤维素降解^[42]

类型	Linker序列	连接酶	表达宿主	产生结果	文献
刚性	(EAAAK)₅	麦芽糖结合蛋白、3种肝素酶	大肠杆菌	提升热稳定性和活性	[71]
	(EAAAK)	α-淀粉酶、牛α-乳清蛋白	枯草芽孢杆菌	提升分泌量和产量	[67]
	LEA(EAAAK)₄ALEA(EAAAK)₄ALE	转铁蛋白	HEK293细胞	提高表达水平，改善体外生物活性	[72]
	LEA(EAAAK)ALEAA(EAAAK)ALE(H4)₂	转铁蛋白、生长激素	HEK293	提高体内稳定性	[73]
柔性	(GGGGS) _n (n=1, 2, 4)	自复性酶-分子伴侣	大肠杆菌	提升活性和稳定性	[74]
	(GGGGS)₂	靶向肽、疏水蛋白	毕赤酵母	稳定表达，提高产量	[70]
	G₈	Myc-Est2p	酿酒酵母	改善蛋白功能，维持端粒长度	[75]
2A肽	P2A(GSG)ATNFSLLKQAGDVEENPGP	生长抑素和荧光蛋白	H1人胚胎干细胞（hESCs）	保持干细胞特性与多能性	[76]
	T2A(GSG)EGRGSLLTCGDVEENPGP	N-豆蔻酰化蛋白和荧光蛋白	HEK293T	亚细胞定位	[77]
	E2A(GSG)QCTNYALLKLAGDVESNPGP	E2A的激活结构域+ PBX1的同源域	HEK293T	协同激活转录	[78]
	F2A(GSG)VKQTLNFDLLKLAGDVESNPGP	P450scc-F2A-AdR-F2A-Adx	酿酒酵母	共表达多蛋白	[79]
IRES	(EMCV) IRES	Rtelo2-IRES-RtKCR;RtHCD-IRES-RtECR	产油酵母	简化多基因共表达的工程化流程；提高产量	[66]

类型	Linker序列	连接酶	表达宿主	产生结果	文献
刚性	(EAAAK)₅	麦芽糖结合蛋白、3种肝素酶	大肠杆菌	提升热稳定性和活性	[71]
	(EAAAK)	α-淀粉酶、牛α-乳清蛋白	枯草芽孢杆菌	提升分泌量和产量	[67]
	LEA(EAAAK)₄ALEA(EAAAK)₄ALE	转铁蛋白	HEK293细胞	提高表达水平，改善体外生物活性	[72]
	LEA(EAAAK)ALEAA(EAAAK)ALE(H4)₂	转铁蛋白、生长激素	HEK293	提高体内稳定性	[73]
柔性	(GGGGS) _n (n=1, 2, 4)	自复性酶-分子伴侣	大肠杆菌	提升活性和稳定性	[74]
	(GGGGS)₂	靶向肽、疏水蛋白	毕赤酵母	稳定表达，提高产量	[70]
	G₈	Myc-Est2p	酿酒酵母	改善蛋白功能，维持端粒长度	[75]
2A肽	P2A(GSG)ATNFSLLKQAGDVEENPGP	生长抑素和荧光蛋白	H1人胚胎干细胞（hESCs）	保持干细胞特性与多能性	[76]
	T2A(GSG)EGRGSLLTCGDVEENPGP	N-豆蔻酰化蛋白和荧光蛋白	HEK293T	亚细胞定位	[77]
	E2A(GSG)QCTNYALLKLAGDVESNPGP	E2A的激活结构域+ PBX1的同源域	HEK293T	协同激活转录	[78]
	F2A(GSG)VKQTLNFDLLKLAGDVESNPGP	P450scc-F2A-AdR-F2A-Adx	酿酒酵母	共表达多蛋白	[79]
IRES	(EMCV) IRES	Rtelo2-IRES-RtKCR;RtHCD-IRES-RtECR	产油酵母	简化多基因共表达的工程化流程；提高产量	[66]

支架蛋白	位置	功能	应用	文献
SpyTag/SpyCatcher	体内	提高酶的稳定性和催化活性	分子内自环化	[87]
	体内	提高催化效率	合成手性醇	[86]
	体外	具有重复使用性	实现姜黄素糖苷的高效生物合成及UDP-葡萄糖的原位再生	[82]
		增加油脂合成	催化天然甘油三酯向烃类生物燃料的转化	[85]
		固定化酶	形成γPFD-SpyCatcher自组装水凝胶支架	[88]
Snoop-SnoopCatcher	体外	实现长度可调的精确交联，赋予几何可编程性	降解聚对苯二甲酸乙二醇酯（polyethylene terephthalate，PET）	[17]
Snoop-SnoopCatcher	体内	连接不同蛋白	构建模块化外膜囊泡抗原展示平台	[89]
Cohesin-dockerin	体外	提高底物广谱性和水解活性	实现了对红藻来源的卟啉多糖的高效降解	[83]
Cohesin-dockerin	体外	增强结合亲和力	黄色瘤胃球菌（Ruminococcus flavefaciens）通过单结合模式的Coh‑Doc相互作用组装纤维素体	[90]
SH3-PDZ-GBD	体内	提高偶联效率和活性	合成大豆苷元；优化靛蓝素合成途径的酶空间组织将SH3、PDZ、GBD这3个结构域作为完整组合支架进行应用的文献相对较少，更多聚焦于对其各组成结构域进行独立开发	[91]
SH3-PDZ-GBD	体内	缓解中间产物抑制		[84]

Microbial Cell Factory Mediated Multi-enzyme Cascades Reactions Empower High-value Utilization of Biomass

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