微生物细胞工厂介导的多酶级联反应赋能生物质高值化应用

doi:10.19586/j.2095-2341.2026.0039

生物技术进展 ›› 2026, Vol. 16 ›› Issue (3): 496-509.DOI: 10.19586/j.2095-2341.2026.0039

微生物细胞工厂介导的多酶级联反应赋能生物质高值化应用

孙雨晴(), 彤丽格(), 房文霞()

广西科学院生物科学与技术研究所，非粮生物质能技术全国重点实验室，南宁 530007

收稿日期:2026-02-14 接受日期:2026-04-03 出版日期:2026-05-25 发布日期:2026-07-14
通讯作者: 彤丽格,房文霞
作者简介:孙雨晴 E-mail：yqsun20@163.com
基金资助:
国家自然科学基金项目(32560226);广西青苗人才计划项目(2024QMJH-1913)

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

摘要/Abstract

摘要：

面对全球化石资源枯竭与环境问题加剧的双重挑战，可再生生物质的高值化应用已成为实现绿色可持续发展的关键路径。微生物细胞工厂凭借其高效的代谢网络整合、辅因子自主再生及模块化组装等优势，已成为介导多酶级联反应的核心载体，为突破单一酶催化效率低、产物单一及过程复杂等瓶颈提供了创新解决方案。系统综述了微生物细胞工厂介导的多酶级联反应的核心构建策略，阐述了大肠杆菌、酿酒酵母、黑曲霉等典型宿主细胞的改造与适配机制。重点介绍了其在体内级联与混合级联体系中的应用特性。同时，总结了该技术在复杂生物质高值转化的最新研究进展，深入分析了当前面临的限速酶识别与改造、酶系反应条件兼容性、宿主代谢流竞争及规模化应用成本等关键挑战，以期为微生物细胞工厂介导的多酶级联反应在生物质高值转化领域的工业化应用提供参考。

关键词: 多酶级联反应, 微生物细胞工厂, 生物质, 高值转化

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

中图分类号:

Q815

孙雨晴, 彤丽格, 房文霞. 微生物细胞工厂介导的多酶级联反应赋能生物质高值化应用[J]. 生物技术进展, 2026, 16(3): 496-509.

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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链接本文: https://swjsjz.magtechjournal.com/CN/10.19586/j.2095-2341.2026.0039

https://swjsjz.magtechjournal.com/CN/Y2026/V16/I3/496

图/表 6

参考文献 98

[1]	LELIEVELD J, HAINES A, BURNETT R, et al.. Air pollution deaths attributable to fossil fuels: observational and modelling study[J/OL]. BMJ, 2023, 383: e077784[2026-02-01]. .
[2]	NARGOTRA P, SHARMA V, LEE Y C, et al.. Microbial lignocellulolytic enzymes for the effective valorization of lignocellulosic biomass: a review[J/OL]. Catalysts, 2023, 13(1): 83[2026-02-01]. .
[3]	CHENG L, WANG L, WEI L, et al.. Combined mild chemical pretreatments for complete cadmium release and cellulosic ethanol co-production distinctive in wheat mutant straw[J]. Green Chem., 2019, 21(13): 3693-3700.
[4]	SHARMA A, BALDA S, CAPALASH N, et al.. Engineering multifunctional enzymes for agro-biomass utilization[J/OL]. Bioresour. Technol., 2022, 347: 126706[2026-02-01]. .
[5]	黄文倩,王迎夏,田维圣,等.多酶催化级联反应在天然产物酶法合成中的应用研究进展[J].中国中药杂志,2023,48(2):336-348.
	HUANG W Q, WANG Y X, TIAN W S, et al.. Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products[J]. China J. Chin. Mater. Med., 2023, 48(2): 336-348.
[6]	CAO Y, YANG P, ZHAO R, et al.. Recent progress in enzyme immobilization to metal-organic frameworks to enhance the CO₂ conversion efficiency[J/OL]. Molecules, 2025, 30(2): 251[2026-02-01]. .
[7]	LIU M, WANG Y, JIANG H, et al.. Synthetic multienzyme assemblies for natural product biosynthesis[J/OL]. ChemBioChem, 2023, 24(6): e202200518[2026-02-01]. .
[8]	SU W, QU G, WU H, et al.. Biocatalytic synthesis of 7-aminodesacetoxycephalosporanic acid with engineered E. coli dual cells and immobilized enzymes[J]. ACS Sustainable Chem. Eng., 2025, 13(27): 10316-10325.
[9]	HAN R, TU W, LIU S, et al.. Novel multienzyme cascade for efficient synthesis of d-allulose from inexpensive sucrose[J/OL]. Food Biosci., 2023, 56: 103303[2026-02-01]. .
[10]	SUN Z, ZHAO Q, HAAG R, et al.. Responsive emulsions for sequential multienzyme cascades[J]. Angew. Chem. Int. Ed., 2021, 60(15): 8410-8414.
[11]	ZHU Y, LIU Y, AI M, et al.. Surface display of carbonic anhydrase on Escherichia coli for CO₂ capture and mineralization[J]. Synth. Syst. Biotechnol., 2021, 7(1): 460-473.
[12]	CAO Y, QIAO W, ZHANG C, et al.. Programmable enzyme catalysis based on multiscale confinements[J]. Nat. Synth., 2025, 4(11): 1338-1348.
[13]	LEDESMA-FERNANDEZ A, VELASCO-LOZANO S, SANTIAGO-ARCOS J, et al.. Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis[J/OL]. Nat. Commun., 2023, 14: 2587[2026-02-01]. .
[14]	LOBEL B T, BAIOCCO D, AL-SHARABI M, et al.. Current challenges in microcapsule designs and microencapsulation processes: a review[J]. ACS Appl. Mater. Interfaces, 2024, 16(31): 40326-40355.
[15]	GHOSH B. Artificial cell design: reconstructing biology for life science applications[J]. Emerg. Top. Life Sci., 2022, 6(6): 619-627.
[16]	ZHAN K, BAO A, ZHANG S, et al.. Nanoconfined enzymatic cascade strategy for highly efficient synthesis of D-allulose[J/OL]. Chem. Eng. J., 2025, 525: 170472[2026-02-01]. .
[17]	GONG H, MENG Y, SUN Y, et al.. Design of a programmable and recyclable protein scaffolding material with geometrically precise enzyme patterning for improved cascade catalysis[J/OL]. Adv. Funct. Mater., 2026, 36(20): e14761[2026-02-01]. .
[18]	WANG Z, SUNDARA SEKAR B, LI Z. Recent advances in artificial enzyme cascades for the production of value-added chemicals[J/OL]. Bioresour. Technol., 2021, 323: 124551[2026-02-01]. .
[19]	李举谋,石焜,张志钧,等.多酶级联反应的构建及其在双官能团功能化学品合成中的应用[J].生物工程学报,2023,39(6):2158-2189.
	LI J M, SHI K, ZHANG Z J, et al.. Construction of multi-enzyme cascade reactions and its application in the synthesis of bifunctional chemicals[J]. Chin. J. Biotechnol., 2023, 39(6): 2158-2189.
[20]	VAAJE-KOLSTAD G, WESTERENG B, HORN S J, et al.. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[J]. Science, 330(6001): 219-222.
[21]	ZHAO H, SU H, SUN J, et al.. Bioconversion of α-chitin by a lytic polysaccharide monooxygenase OsLPMO10A coupled with chitinases and the synergistic mechanism analysis[J]. J. Agric. Food Chem., 2024, 72(13): 7256-7265.
[22]	余婷.蛋白支架组装纤维素酶[D].上海:华东理工大学,2015.
[23]	GUO X, AN Y, LU F, et al.. Optimization of synergistic degradation of steam exploded corn straw by lytic polysaccharide monooxygenase R17L and cellulase[J/OL]. Ind. Crops Prod., 2022, 182: 114924[2026-02-01]. .
[24]	FAN Y, YAN Y, NWOKONKWO O, et al.. Tuning nitrate reduction reaction selectivity via selective adsorption in electrified membranes[J]. Nat. Chem. Eng., 2025, 2(6): 379-390.
[25]	刘丽霞,魏国光,刘占英.纤维素降解酶改造研究进展[J].中国生物工程杂志,2025,45(8):130-138.
	LIU L X, WEI G G, LIU Z Y. Research progress on cellulose degrading enzyme[J]. China Biotechnol., 2025, 45(8): 130-138.
[26]	SONG W, WANG J H, WU J, et al.. Asymmetric assembly of high-value α-functionalized organic acids using a biocatalytic chiral-group-resetting process[J/OL]. Nat. Commun., 2018, 9(1): 3818[2026-02-01]. .
[27]	SUN J, LOH K C. Navigating the interface between lignocellulose pretreatment and bioconversion in lignin-inclusive fermentation: a review[J/OL]. Bioresour. Technol., 2026, 439: 133325[2026-02-01]. .
[28]	ZHANG D P, JING X R, WU L J, et al.. Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory[J/OL]. Microb. Cell Fact., 2021, 20(1): 11[2026-02-01]. .
[29]	WANG Y, LUO X, ZHOU M, et al.. An efficient whole-cell platform for Rebaudioside M biotransformation: cascade design, expression regulation, process engineering[J]. Green Chem., 2026, 28(4): 1972-1985.
[30]	TANG X, ARSALAN A, ZHANG G, et al.. Coexpression of D-allulose 3-epimerase and L-rhamnose isomerase in Bacillus subtilis through a dual promoter enables high-level biosynthesis of D-allose from D-fructose in one pot[J]. J. Agric. Food Chem., 2025, 73(3): 2056-2067.
[31]	SAUER B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae [J]. Mol. Cell. Biol., 1987, 7(6): 2087-2096.
[32]	KIM H, KIM M, KIM S, et al.. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes[J]. Lab. Anim. Res., 2018, 34(4): 147-159.
[33]	ROSSI M, SALOMON A, CHAUMONTEL N, et al.. Warning regarding hematological toxicity of tamoxifen activated CreERT2 in young Rosa26CreERT2 mice[J/OL]. Sci. Rep., 2023, 13: 5976[2026-02-01]. .
[34]	SONG F, ZHANG H, WANG K, et al.. In vivo continuous evolution via phenotypic sorting to alleviate metabolic bottlenecks in β-alanine production[J]. Trends Biotechnol., 2026, 44(2): 430-449.
[35]	LU X, LI J, HUANG C, et al.. Development of new multi-glycosylation routes to facilitate the biosynthesis of sweetener mogrosides from bitter immature Siraitia grosvenorii using engineered Escherichia coli [J]. J. Agric. Food Chem., 2024, 72(32): 18078-18088.
[36]	WAN L, ZHU Y, CHEN G, et al.. Efficient production of 2′-fucosyllactose from L-fucose via self-assembling multienzyme complexes in engineered Escherichia coli [J]. ACS Synth. Biol., 2021, 10(10): 2488-2498.
[37]	SUN M, CHEN M, YU H, et al.. Triple-signal hierarchical dynamic regulation synergizes with multi-level metabolic engineering to enhance lutein biosynthesis in S. cerevisiae [J/OL]. Chem. Eng. J., 2025, 526: 171417[2026-02-01]. .
[38]	RIBEAUCOURT D, HÖFLER G T, YEMLOUL M, et al.. Tunable production of (R)-or (S)-citronellal from geraniol via a bienzymatic cascade using a copper radical alcohol oxidase and old yellow enzyme[J]. ACS Catal., 2022, 12(2): 1111-1116.
[39]	HAN S, XUE Y, YAN Q, et al.. Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-β-D-glucosamine from powdery chitin[J/OL]. Bioresour. Technol., 2024, 393: 130024[2026-02-01]. .
[40]	LI Y, LI C, MUHAMMAD A S, et al.. Enhanced expression of xylanase in Aspergillus niger enabling a two-step enzymatic pathway for extracting β-glucan from oat bran[J/OL]. Bioresour. Technol., 2023, 377: 128962[2026-02-01]. .
[41]	ZHU L, SHEN B, SONG Z, et al.. Permeabilized TreS-expressing Bacillus subtilis cells decorated with glucose isomerase and a shell of ZIF-8 as a reusable biocatalyst for the coproduction of trehalose and fructose[J]. J. Agric. Food Chem., 2020, 68(15): 4464-4472.
[42]	LIU G, GONG H, TANG H, et al.. Enhanced lignocellulose degradation in Bacillus subtilis RLI2019 through CRISPR/Cas9-mediated chromosomal integration of ternary cellulase genes[J/OL]. Int. J. Biol. Macromol., 2025, 306: 141727[2026-02-01]. .
[43]	HABIBI N, SAMIAN M R, HASHIM S Z M, et al.. EcoliOverExpressionDB: a database of recombinant protein overexpression in E. coli [J]. Protein Expr. Purif., 2014, 95: 92-95.
[44]	WEN Q, XU X, HE Q, et al.. Dual-pathway engineering enables robust vanillin bioproduction in Pseudomonas putida [J/OL]. Chem. Eng. J., 2025, 526: 171181[2026-02-01]. .
[45]	ZHU F Y, GU Y Y, CAI X, et al.. Metabolic engineering of microbial chassis cells for D-pantothenate production: a review[J]. ACS Synth. Biol., 2025, 14(10): 3831-3848.
[46]	MOLINA-VÁZQUEZ E R, CASPETA L, GOSSET G, et al.. Tailoring Escherichia coli BL21 (DE3) for preferential xylose utilization via metabolic and regulatory engineering[J/OL]. Appl. Microbiol. Biotechnol., 2025, 109(1): 54[2026-02-01]. .
[47]	ZHANG X, XIN J, LIU M, et al.. Cost-effective whole-cell biosynthesis of ursodeoxycholic acid using engineered Escherichia coli with a multienzyme cascade[J/OL]. Front. Microbiol., 2025, 16: 1538237[2026-02-01]. .
[48]	KRETZER L G, KNYCHALA M M, SILVA L C, et al.. Screening and engineering yeast transporters to improve cellobiose fermentation by recombinant Saccharomyces cerevisiae [J/OL]. Fermentation, 2024, 10(9): 490[2026-02-01]. .
[49]	CAI X, HU C H, WU A Y, et al.. Integrated protein engineering and expression balancing enable efficient D-allulose production[J]. J. Agric. Food Chem., 2025, 73(40): 25562-25572.
[50]	赵婧邑,郭燕,冯婷婷,等.多酶级联系统制备蒜糖醇的细胞工厂构建及工艺优化[J].食品科学技术学报,2022,40(3):88-97.
	ZHAO J Y, GUO Y, FENG T T, et al.. Construction and process optimization of cell factory for allitol synthesis with multienzymatic cascade system[J]. J. Food Sci. Technol., 2022, 40(3): 88-97.
[51]	LIU H, WU L, ZHAO S, et al.. Enzyme symphony in bio-inspired multi-enzyme cascades for enhanced biosynthesis[J/OL]. Biotechnol. Adv., 2026, 87: 108789[2026-02-01]. .
[52]	GAO X, TANG G, YAN J, et al.. Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars[J]. Chin. J. Catal., 2025, 72: 95-105.
[53]	CAO C, BAI Y, ZHANG M, et al.. Synthesis of 2′-fucosyllactose using multi-enzyme cascade with cofactor regeneration[J/OL]. Sci. Rep., 2026, 16: 2457[2026-02-01]. .
[54]	LI L, JIANG H, XU T, et al.. Engineering bienzymatic cascade for efficient biosynthesis of citronellal and citronellol[J]. Biotechnol. Bioeng., 2025, 122(11): 3205-3215.
[55]	LIU J, LI Z. Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell-free system[J]. Biotechnol. Bioeng., 2019, 116(3): 536-542.
[56]	QING L, GAO J, DU L, et al.. Chitinase and deacetylase-based chitin-degrading bacteria: one-pot cascade bioconversion of chitin to chitooligosaccharides[J]. J. Agric. Food Chem., 2024, 72(43): 23937-23946.
[57]	WANG Y, YANG X, CHEN P, et al.. Homologous overexpression of genes in Cordyceps militaris improves the production of polysaccharides[J/OL]. Food Res. Int., 2021, 147: 110452[2026-02-01]. .
[58]	JUNG S W, YEOM J, PARK J S, et al.. Recent advances in tuning the expression and regulation of genes for constructing microbial cell factories[J/OL]. Biotechnol. Adv., 2021, 50: 107767[2026-02-01]. .
[59]	POURESMAEIL M, AZIZI-DARGAHLOU S. Factors involved in heterologous expression of proteins in E. coli host[J/OL]. Arch. Microbiol., 2023, 205(5): 212[2026-02-01]. .
[60]	JANG S K, KRÄUSSLICH H G, NICKLIN M J, et al.. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation[J]. J. Virol., 1988, 62(8): 2636-2643.
[61]	WEINGARTEN-GABBAY S, ELIAS-KIRMA S, NIR R, et al.. Systematic discovery of cap-independent translation sequences in human and viral genomes[J/OL]. Science, 2016, 351(6270): aad4939[2026-02-01]. .
[62]	SUBRAMANIAN V, SCHUSTER L A, MOORE K T, et al.. A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei [J/OL]. Biotechnol. Biofuels, 2017, 10(1): 34[2026-02-01]. .
[63]	LI D, LIU J, SUN L, et al.. Developing polycistronic expression tool in Yarrowia lipolytica [J]. Synth. Syst. Biotechnol., 2025, 10(1): 127-132.
[64]	SUBRAMANIAN V, FARMER S J, HEILAND K L, et al.. A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei [J/OL]. J. Ind. Microbiol. Biotechnol., 2023, 49(6): kuac027[2026-02-01]. .
[65]	UNKLES S E, VALIANTE V, MATTERN D J, et al.. Synthetic biology tools for bioprospecting of natural products in eukaryotes[J]. Chem. Biol., 2014, 21(4): 502-508.
[66]	ZHANG R, CUI Y, WANG H, et al.. In silico characterization of Rhodotorula toruloides ELO-like elongases and production of very-long-chain fatty acids by expressing Rtelo2, RtKCR, RtHCD, and RtECR through IRES-mediated bicistrons[J/OL]. World J. Microbiol. Biotechnol., 2024, 40(12): 395[2026-02-01]. .
[67]	WANG B, WU Y, LYU X, et al.. Synergistic regulation of chassis cell growth and screening of promoters, signal peptides and fusion protein linkers for enhanced recombinant protein expression in Bacillus subtilis [J/OL]. Int. J. Biol. Macromol., 2024, 280: 136037[2026-02-01]. .
[68]	PATEL D K, VETTIATIL D, PATEL D H, et al.. Linkers: a synergistic way for the synthesis of chimeric proteins[J/OL]. Protein Expr. Purif., 2022, 191: 106012.
[69]	CHEN X, ZARO J L, SHEN W C. Fusion protein linkers: property, design and functionality[J]. Adv. Drug Deliv. Rev., 2013, 65(10): 1357-1369.
[70]	HAN Z, SONG B, YANG J, et al.. Curcumin-encapsulated fusion protein-based nanocarrier demonstrated highly efficient epidermal growth factor receptor-targeted treatment of colorectal cancer[J]. J. Agric. Food Chem., 2022, 70(49): 15464-15473.
[71]	WU X, YUN Z, SU N, et al.. Characterization of maltose-binding protein-fused heparinases with enhanced thermostability by application of rigid and flexible linkers[J]. Biotechnol. Appl. Biochem., 2025, 72(1): 5-16.
[72]	AMET N, LEE H F, SHEN W C. Insertion of the designed helical linker led to increased expression of tf-based fusion proteins[J]. Pharm. Res., 2009, 26(3): 523-528.
[73]	CHEN X, LEE H F, ZARO J L, et al.. Effects of receptor binding on plasma half-life of bifunctional transferrin fusion proteins[J]. Mol. Pharm. 2011, 8(2): 457-465.
[74]	BERGERON L M, GOMEZ L, WHITEHEAD T A, et al.. Self-renaturing enzymes: design of an enzyme-chaperone Chimera as a new approach to enzyme stabilization[J]. Biotechnol. Bioeng., 2009, 102(5): 1316-1322.
[75]	SABOURIN M, TUZON C T, FISHER T S, et al.. A flexible protein linker improves the function of epitope-tagged proteins in Saccharomyces cerevisiae [J]. Yeast, 2007, 24(1): 39-45.
[76]	ZHANG T, ZHANG F, WANG N, et al.. Generation of SST-P2A-mCherry reporter human embryonic stem cell line using the CRISPR/Cas9 system (WAe001-A-2C)[J/OL]. Stem Cell Res., 2024, 77: 103397[2026-02-01]. .
[77]	HADPECH S, JINATHEP W, SAOIN S, et al.. Impairment of a membrane-targeting protein translated from a downstream gene of a "self-cleaving" T2A peptide conjunction[J]. Protein Expr. Purif., 2018, 150: 17-25.
[78]	PI W C, WANG J, SHIMADA M, et al.. E2A-PBX1 functions as a coactivator for RUNX1 in acute lymphoblastic leukemia[J]. Blood, 2020, 136(1): 11-23.
[79]	EFIMOVA V S, ISAEVA L V, OREKHOV P S, et al.. Using a viral 2A peptide-based strategy to reconstruct the bovine P450scc steroidogenic system in S. cerevisiae: bovine P450scc system expression using 2A peptides[J]. J. Biotechnol., 2021, 325: 186-195.
[80]	FRANCE S P, HEPWORTH L J, TURNER N J, et al.. Constructing biocatalytic cascades: in vitro and in vivo approaches to de novo multi-enzyme pathways[J]. ACS Catal., 2017, 7(1): 710-724.
[81]	GAD S, AYAKAR S. Protein scaffolds: a tool for multi-enzyme assembly[J/OL]. Biotechnol. Rep., 2021, 32: e00670[2026-02-01]. .
[82]	ALI M Y, GAO J, ZHANG Z, et al.. Directional co-immobilization of artificial multimeric-enzyme complexes as a robust biocatalyst for biosynthesis curcumin glucosides and regeneration of UDP-glucose[J/OL]. Int. J. Biol. Macromol., 2024, 278: 135035[2026-02-01]. .
[83]	HAN J, JO Y, SUN H, et al.. The increased efficiency of porphyran hydrolysis by constructing a multifunctional enzyme complex from marine microorganisms[J/OL]. Enzyme Microb. Technol., 2023, 165: 110207[2026-02-01]. .
[84]	WANG L, SUN Y, LV D, et al.. Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production[J]. Appl. Microbiol. Biotechnol., 2020, 104(24): 10493-10502.
[85]	ZHONG X, MA Y, ZHANG X, et al.. More efficient enzymatic cascade reactions by spatially confining enzymes via the SpyTag/SpyCatcher technology[J/OL]. Mol. Catal., 2022, 521: 112188[2026-02-01]. .
[86]	WANG J, LU Y, CHENG P, et al.. Construction of bi-enzyme self-assembly clusters based on SpyCatcher/SpyTag for the efficient biosynthesis of (R)-ethyl 2-hydroxy-4-phenylbutyrate[J/OL]. Biomolecules, 2023, 13(1): 91[2026-02-01]. .
[87]	LI M, ZHANG J, ZHANG K, et al.. Improving the thermostability and catalytic activity of RgDAAO by a combinatorial strategy using sequence consensus design and SpyTag/SpyCatcher self-cyclization[J]. J. Agric. Food Chem., 2025, 73(38): 24211-24221.
[88]	WANG B P, YIN X, HUANG M Y, et al.. A self-assembling γPFD-SpyCatcher hydrogel scaffold for the coimmobilization of SpyTag-enzymes to facilitate the catalysis of regulated enzymes[J]. J. Agric. Food Chem., 2024, 72(36): 19940-19947.
[89]	VAN DEN BERG VAN SAPAROEA H B, HOUBEN D, DE JONGE M I, et al.. Display of recombinant proteins on bacterial outer membrane vesicles by using protein ligation[J/OL]. Appl. Environ. Microbiol., 2018, 84(8): e02567[2026-02-01]. .
[90]	DUARTE M, ALVES V D, CORREIA M, et al.. Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies[J]. Int. J. Biol. Macromol., 2023, 224: 55-67.
[91]	WANG Z, DAI Y, AZI F, et al.. Constructing protein-scaffolded multienzyme assembly enhances the coupling efficiency of the P450 system for efficient daidzein biosynthesis from (2S)-naringenin[J]. J. Agric. Food Chem., 2024, 72(11): 5849-5859.
[92]	PAN Y, LIU Y, PHAN T L, et al.. Biomanufacturing of inositol from corn stover with biological pretreatment by an in vitro synthetic biology platform[J]. ACS Sustainable Chem. Eng., 2025, 13(1): 436-446.
[93]	YU X, LI J, SUN Y, et al.. Co-immobilized multi-enzyme biocatalytic system on reversible and soluble carrier for saccharification of corn straw cellulose[J/OL]. Bioresour. Technol., 2024, 395: 130325[2026-02-01]. .
[94]	REN Y, WANG Y, XIA Y, et al.. Elimination of calnexin overcomes the secretion bottleneck of Trichoderma reesei β-glucosidase Cel3B which boosts corncob saccharification[J/OL]. Bioresour. Technol., 2026, 444: 133944[2026-02-01]. .
[95]	DING S Y, RINCON M T, LAMED R, et al.. Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens [J]. J. Bacteriol., 2001, 183(6): 1945-1953.
[96]	FIERER J O, TOVAR-HERRERA O E, WEINSTEIN J Y, et al.. Affinity-induced covalent protein-protein ligation via the SpyCatcher-SpyTag interaction[J]. Green Carbon, 2023, 1(1): 33-42.
[97]	张巧,李永成. Rhizopus stolonifer几丁质脱乙酰酶的异源表达及其与几丁质酶的协同作用研究[J].食品与发酵工业,2024,50(2):177-183.
	ZHANG Q, LI Y C. Heterologous expression of a chitin deacetylase from Rhizopus stolonifer and its synergistic hydrolysis with a chitinase[J]. Food Ferment. Ind., 2024, 50(2): 177-183.
[98]	DU C, ZHOU Y, LIU L, et al.. Bacterial surface-assembled chitinosome for dismantling chitin into N-acetyl glucosamine[J]. ACS Sustainable Chem. Eng., 2023, 11(30): 11239-11247.

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]

微生物细胞工厂介导的多酶级联反应赋能生物质高值化应用

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

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