微生物学报

Foreign protein	Origin	T. reesei strain	Expression system	Yield	References
Chymosin	Bovine	Rut-C30	cbh1	cbh1	cbh1+20 aa	40 mg/L	[15]
Fab antibody fragments	Mouse	RUT-C30	cbh1	cbh1	cbh1	1 mg/L	[16]
Xylanase Ⅵ	Acrophialophora nainiana	RUT-C30	cbh1	cbh1	cbh1	172 mg/L	[17]
Cellobiose dehydrogenase	Corynascusthermophilus	QM9414	cdna1	cdh	trpC	29 mg/L	[18]
Mammalian proteins α-galactosidase A	Human	Rut-C30	cbh1	cbh1	cbh1	636 mg/L	[19]
Erythropoietin	Human	RutC-30 M3	cbh1	cbh1	cbh1	97 mg/L	[20]
β-lactoglobulin	Bovine	M1908	SES	pdc1	cbh1	1 g/L	[21]
Ovalbumin	Hen	M1908	SES	pdc1	cbh1	2 g/L	[21]
β-glucosidase	Aspergillus niger	QM53	cdna1	cbh1	cbh1	17.2 IU/mg	[22]
Te-cel7A	Talaromyces emersonii	QP4	cdna1	trpC	cbh1	0.93 U/L	[23]
Feruloyl esterase	Aspergillus niger	RUT-C30	cbh1	cbh1	cbh1	20.69 U/gds	[24]
Mannanase 5A	A. niger	TU-6	cbhl	cbh1	cbh1	1 204 U/mL	[25]
Alkaline endoglucanase	Humicola insolens	RUT-C30	xyn1/xyn2	xyn1/xyn2	xyn1/xyn2	15.7 U/mL	[26]
Interferon alpha-2b	Human	M504	cbh1	cbh1	cbh1	4.5 g/L	[27]
Mannanase 5A	Trichoderma harzianum	QM9414	pdc	pdc	ThMan5A	1.6 g/L	[28]
Dye-decolorizing peroxidase	Pleurotus sapidus	RUT-C30	cbh1	cbh1	rPsaDyP	55 000 U/L	[29]

Foreign protein	Origin	T. reesei strain	Expression system	Yield	References
Chymosin	Bovine	Rut-C30	cbh1	cbh1	cbh1+20 aa	40 mg/L	[15]
Fab antibody fragments	Mouse	RUT-C30	cbh1	cbh1	cbh1	1 mg/L	[16]
Xylanase Ⅵ	Acrophialophora nainiana	RUT-C30	cbh1	cbh1	cbh1	172 mg/L	[17]
Cellobiose dehydrogenase	Corynascusthermophilus	QM9414	cdna1	cdh	trpC	29 mg/L	[18]
Mammalian proteins α-galactosidase A	Human	Rut-C30	cbh1	cbh1	cbh1	636 mg/L	[19]
Erythropoietin	Human	RutC-30 M3	cbh1	cbh1	cbh1	97 mg/L	[20]
β-lactoglobulin	Bovine	M1908	SES	pdc1	cbh1	1 g/L	[21]
Ovalbumin	Hen	M1908	SES	pdc1	cbh1	2 g/L	[21]
β-glucosidase	Aspergillus niger	QM53	cdna1	cbh1	cbh1	17.2 IU/mg	[22]
Te-cel7A	Talaromyces emersonii	QP4	cdna1	trpC	cbh1	0.93 U/L	[23]
Feruloyl esterase	Aspergillus niger	RUT-C30	cbh1	cbh1	cbh1	20.69 U/gds	[24]
Mannanase 5A	A. niger	TU-6	cbhl	cbh1	cbh1	1 204 U/mL	[25]
Alkaline endoglucanase	Humicola insolens	RUT-C30	xyn1/xyn2	xyn1/xyn2	xyn1/xyn2	15.7 U/mL	[26]
Interferon alpha-2b	Human	M504	cbh1	cbh1	cbh1	4.5 g/L	[27]
Mannanase 5A	Trichoderma harzianum	QM9414	pdc	pdc	ThMan5A	1.6 g/L	[28]
Dye-decolorizing peroxidase	Pleurotus sapidus	RUT-C30	cbh1	cbh1	rPsaDyP	55 000 U/L	[29]

Promoter	Source gene	Type	Induction/regulation mode	Expression intensity (relative)	References
P _cbh1	Cellobiohydrolase CBH1/CEL7A	Inducible	Induced by cellulose, lactose, cellobiose, etc.	Very high	[43]
P _cbh2	Cellobiohydrolase CBH2/CEL6A	Inducible	Induced by cellulose, lactose	High	[44]
P _egl2	Endoglucanase EG2/CEL5A	Inducible	Induced by cellulose	Medium	[45]
P _xyn2	Xylanase XYN2	Inducible	Induced by xylan	High	[26]
P _pdc1	Pyruvate decarboxylase	Constitutive	Stable and continuous expression	Medium	[28]
P _gpd1	Glyceraldehyde-3-phosphate dehydrogenase	Constitutive	Stable and continuous expression	Medium	[46]
P _cdna1	cdna1 (unknown)	Constitutive	Stable and continuous expression	High	[18]
P _cbh1_PA	Modified from P _cbh1	Inducible	Induced by cellulose, lactose, cellobiose, etc.	Extremely high	[47]

Promoter	Source gene	Type	Induction/regulation mode	Expression intensity (relative)	References
P _cbh1	Cellobiohydrolase CBH1/CEL7A	Inducible	Induced by cellulose, lactose, cellobiose, etc.	Very high	[43]
P _cbh2	Cellobiohydrolase CBH2/CEL6A	Inducible	Induced by cellulose, lactose	High	[44]
P _egl2	Endoglucanase EG2/CEL5A	Inducible	Induced by cellulose	Medium	[45]
P _xyn2	Xylanase XYN2	Inducible	Induced by xylan	High	[26]
P _pdc1	Pyruvate decarboxylase	Constitutive	Stable and continuous expression	Medium	[28]
P _gpd1	Glyceraldehyde-3-phosphate dehydrogenase	Constitutive	Stable and continuous expression	Medium	[46]
P _cdna1	cdna1 (unknown)	Constitutive	Stable and continuous expression	High	[18]
P _cbh1_PA	Modified from P _cbh1	Inducible	Induced by cellulose, lactose, cellobiose, etc.	Extremely high	[47]

里氏木霉高效异源蛋白表达系统的构建与优化研究进展

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侯婧卓 ¹^,² , 李海燕 ¹ , 刘英 ² , 彭小伟 ²

微生物学报 | 综述 2026,66(8): 3793-3808

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微生物学报 |综述 2026 , 66 (8) : 3793 -3808

里氏木霉高效异源蛋白表达系统的构建与优化研究进展

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侯婧卓¹^,², 李海燕¹, 刘英², 彭小伟²

作者信息

^1.沈阳化工大学环境与安全工程学院，辽宁沈阳

^2.中国科学院过程工程研究所，生物药制备与递送全国重点实验室，北京

通讯作者:

李海燕, 彭小伟

作者简介:

作者贡献声明

侯婧卓：文献检索、图表绘制、初稿撰写及修改；李海燕：论文内容审阅与修订；刘英：论文润色修改；彭小伟：对论文学术规范性、逻辑严谨性及全面性的审阅与修订，学术指导，经费支持。

Research progress in the construction and optimization of an efficient heterologous protein expression system with Trichoderma reesei as the chassis

Jingzhuo HOU¹^,², Haiyan LI¹, Ying LIU², Xiaowei PENG²

Affiliations

^1.College of Environmental and Safety Engineering, Shenyang University of Chemical Technology, Shenyang, Liaoning, China

^2.State Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China

Corresponding Author:

E-mail: LI Haiyan, lihaiyan98@syuct.edu.cn;

PENG Xiaowei, xwpeng@ipe.ac.cn

出版时间: 2026-08-04 doi: 10.13343/j.cnki.wsxb.20260151

文章导航

摘要

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里氏木霉(Trichoderma reesei)是目前已知蛋白分泌能力最强的微生物，广泛应用于纤维素酶生产，并在异源蛋白生产及应用领域备受关注。近年来，该菌在表达元件筛选、菌种底盘改造、发酵工艺优化等方面取得了显著进展，但仍面临重要挑战，尚未实现异源蛋白表达的大规模应用。本文综述了里氏木霉高效表达异源蛋白的策略及近期研究进展，分析了其应用于异源蛋白生产的限制因素，并就提高异源蛋白产量提出了新的策略与建议。

关键词

里氏木霉 / 异源蛋白生产 / 高效表达策略 / 蛋白酶

Abstract

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Trichoderma reesei is recognized as the microorganism with the most potent protein secretion capability known to date. It is extensively utilized for cellulase production and has garnered significant attention in heterologous protein synthesis. Although notable progress has been achieved in the screening of expression elements, chassis engineering, and fermentation process optimization, critical challenges persist, and the large-scale industrial application of heterologous protein expression has not yet been fully realized. This paper reviews the strategies and recent research advances regarding the high-efficiency expression of heterologous proteins in T. reesei. Furthermore, this paper discusses the limiting factors for its industrial-scale application and proposes novel recommendations and strategies to enhance heterologous protein yields.

Key words

Trichoderma reesei / heterologous protein production / high-efficiency expression strategies / protease

引用本文

侯婧卓, 李海燕, 刘英, 彭小伟. 里氏木霉高效异源蛋白表达系统的构建与优化研究进展. 微生物学报, 2026 , 66 (8) : 3793 -3808 . DOI: 10.13343/j.cnki.wsxb.20260151

Jingzhuo HOU, Haiyan LI, Ying LIU, Xiaowei PENG. Research progress in the construction and optimization of an efficient heterologous protein expression system with Trichoderma reesei as the chassis[J]. Acta Microbiologica Sinica, 2026 , 66 (8) : 3793 -3808 . DOI: 10.13343/j.cnki.wsxb.20260151

正文

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里氏木霉(Trichoderma reesei)属于子囊菌门(Ascomycota)、木霉属(Trichoderma)，是一种嗜温丝状真菌^[1-2]。里氏木霉最早于第二次世界大战期间在所罗门群岛被分离，并由Mandels等^[3]于1957年首次鉴定并命名为QM6a。后因其表现出较强的纤维素酶分泌能力而受到广泛关注，据报道，里氏木霉工业菌株的纤维素酶分泌能力最高可达100 g/L^[4]。这种突出的分泌能力奠定了里氏木霉在生物经济中的重要地位，并使其在工业用酶、抗体等异源蛋白生产领域备受关注^[5]。此外，20世纪60年代，里氏木霉被美国食品和药物管理局(Food and Drug Administration, FDA)认证为生产食品添加剂的安全菌种(generally recognized as safe, GRAS)^[6]，目前已生产纤维素酶、木聚糖酶、淀粉酶、漆酶、果胶酶和植酸酶等多种工业酶，被广泛应用于食品添加剂、饲料、能源等领域^[7]。在我国，根据GB 2760—2024标准，国家食品安全风险评估中心(China National Center for Food Safety Risk Assessment, CFSA)已批准使用里氏木霉生产的食品用酶制剂(CFSA Standards, 2024)^[8]。

经过数十年的诱变与筛选育种，陆续涌现出一系列高产菌株，如QM9414^[9]、TU-6^[10]和RUT-C30^[11]，其纤维素酶产量可达原始菌株的10倍以上。作为真核生物，里氏木霉还拥有高效的蛋白分泌体系和完善的翻译后修饰机制，可实现与真核系统兼容的糖基化和二硫键形成，因此被视为表达异源蛋白的理想宿主，目前已有多种异源蛋白在里氏木霉中成功表达^[12]。里氏木霉生产异源蛋白的初期尝试主要集中在小牛凝乳酶^[13]。2007年，Mäntylä等^[14]将嗜热毛霉菌(Chaetomium thermophilum)的内切木聚糖酶在里氏木霉中重组表达，用于牛皮纸浆的生物漂白，结果表明里氏木霉可用于商业化酶的生产。近年来，随着基因编辑技术的迅猛发展，多种异源蛋白在里氏木霉中成功表达(表1)，包括β-葡萄糖苷酶、木聚糖酶、脂肪酶以及哺乳动物来源的肥胖抑制素等^[12,30]。

虽然里氏木霉具备极高的天然纤维素酶生产能力，已作为细胞工厂广泛用于重组蛋白的生产，但与内源蛋白相比，外源蛋白的生产水平通常较低(表1)。在利用里氏木霉表达异源蛋白的早期阶段，异源蛋白产量普遍低于1 g/L。因此，要充分发挥其作为异源蛋白生产理想宿主的全部潜力仍面临重大挑战，影响外源蛋白生产的因素包括宿主防御系统、外源基因在宿主中的转录、翻译、翻译后修饰及分泌途径等多个方面^[5]。

蛋白质的合成与分泌是一个复杂且受到严格调控的过程，为提升异源蛋白在里氏木霉中的合成和分泌水平，研究者采用了多种策略，例如过表达转录激活因子^[31-32]、破坏转录抑制因子^[33-34]、增加基因拷贝数^[35]、过表达分子伴侣或关键分泌组分^[36-37]、删除内源性蛋白酶^[38]以及优化培养条件^[39]。总体而言，提升蛋白质产量的策略主要可分为3类：(1)着重于表达元件的筛选与优化，从而提升转录和翻译水平；(2)对宿主底盘进行改造，通过编辑宿主背景蛋白、转录调控枢纽、胞外蛋白酶及分泌途径相关基因，进一步提高异源蛋白表达量并实现高效分泌，同时确保产物稳定存在；(3)优化发酵条件，通过对发酵体系及参数的优化进一步提升产量，加速产业化进程。本文将主要从上述3个方面综述当前里氏木霉在异源蛋白表达及产量提升方面的主要策略，并讨论各优化策略目前面临的挑战；此外，结合人工智能(artificial intelligence, AI)技术飞速发展的现状，对AI辅助设计策略进行总结并展望其未来发展，以期为里氏木霉异源蛋白的产业化发展提供参考。

1 表达元件筛选与优化

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1.1 启动子

启动子是转录阶段的重要元件，可被RNA聚合酶特异性识别并结合，决定基因在转录水平上的初始表达时间和强度^[40]。目前，里氏木霉外源基因表达常用的启动子根据基因表达调控机制的不同可分为组成型(constitutive)与诱导型(inducible)两大类^[5]。组成型启动子具有持续稳定的转录活性，其表达水平不受外界环境或转录因子调控的影响；与之相对，诱导型启动子依赖于特定的诱导物，可被纤维素、槐糖、乳糖等多种糖类诱导，并受分解代谢抑制调控^[41-42]。通过对里氏木霉启动子进行理性设计及改造，可实现目标基因的高效表达，常用启动子及其特点对比见表2。

具有高表达强度的启动子P _cbh1 是众多研究的重点。P _cbh1 可通过不同方式进行设计改造，通常通过添加转录激活因子的结合位点实现，如ACE2、HAP2/3/5和XYR1等^[48]。Zou等^[49]对P _cbh1 启动子进行改造，将转录抑制因子CRE1的结合位点替换为HAP2/3/5及转录激活因子ACE2的结合位点，结果显示，在诱导培养基中，改造后的P _cbh1_m2启动子驱动的绿色荧光蛋白表达量较野生型P _cbh1 提高了5.5倍；在抑制培养基中表达量提升了7.4倍，显示出显著的表达增强效果。Sun等^[47]改造启动子P _cbh1 以表达黑曲霉来源的甘露聚糖酶，将P _cbh1 上的3个CRE1 (5′-SYGGRG-3′)结合位点替换为1个ACE2 (5′-GGCTAATAA-3′)结合位点和2个HAP2/3/5 (5′-CCAAT-3′)结合位点，并将8个ACE1 (5′-A GGCA-3′)结合位点均替换为ACE2结合位点，设计得到启动子P _cbh1_PA，获得的产量为野生型P _cbh1 的3倍。

通过上述策略对启动子进行改造，通常可明显提升目标蛋白的转录水平，进而提高产量。然而，这类策略在工业放大过程中有时难以达到预期效果。转录水平大幅提高后，内质网负荷随之增加，错误折叠或未完全折叠的蛋白逐渐积累，持续诱发未折叠蛋白反应，进而影响细胞生长并增加产物降解风险^[22,50-51]。因此，对产业化而言，启动子工程的核心并非单纯追求更高强度，而是要在表达能力与宿主负荷之间找到最佳平衡点。

1.2 信号肽

信号肽是位于分泌蛋白或膜蛋白N端的一段短肽，在新生多肽链中作为识别元件，通过分泌途径引导蛋白质靶向并转运至细胞膜或内质网，信号肽可被信号肽酶切除，从而使蛋白质得以正确折叠并运往其功能位点^[52-53]。在酵母、丝状真菌和哺乳动物细胞中，改变信号肽能够显著影响目标蛋白质的表达水平^[54-55]。这表明，目标蛋白质的高效分泌需要信号肽与蛋白质转运机制之间具有高度匹配的兼容性，然而其内在分子机制迄今尚未得到充分阐明^[56]。

高表达内源性纤维素酶[如纤维二糖水解酶I (cellobiohydrolase I, CBHI)]的信号肽已被广泛用于引导异源蛋白进入里氏木霉的分泌途径^[23]。近期研究表明，信号肽的性能取决于具体环境，需根据不同的异源目标蛋白进行针对性优化。Ji等^[56]将里氏木霉、构巢曲霉(Aspergillus nidulans)和黑曲霉(Aspergillus niger) 3株丝状真菌的252条天然信号肽经系统发育聚类分为15大分支，从中抽取17条代表性序列构建单拷贝表达盒，并在同一基因组位点整合黑曲霉来源的内切β-1,4-甘露聚糖酶，结果显示，里氏木霉Cel74A信号肽表达酶活最高，是原始CBH1信号肽的3倍。然而，这一结论未必适用于其他异源蛋白。Rantasalo等^[57]评估了4种信号肽用于分泌酵母脂酶B (Candida antarctica lipase B, CALB)，其中异源α-淀粉酶(α-amylase-SS, AaSS)信号肽表现优于其他信号肽，其产生的胞外脂酶活性比CBHI/CBHII高出3.6倍；而由CBHI/CBHII驱动的表达导致信号肽切割不完全，产生带有残留肽段的截短型CALB变体，此类截短可能诱发错误折叠，触发内质网相关降解(endoplasmic reticulum associated degradation, ERAD)并降低产量。

综上可见，信号肽的性能会受到发酵条件的影响，这可能是由于发酵过程中菌丝形态、极性生长、囊泡运输和膜系统状态发生变化，进而出现胞内滞留、加工延迟或异常降解等情况。因此，信号肽的优化同样需要根据具体发酵条件找到与分泌系统的最佳组合。

1.3 融合表达

在里氏木霉中进行异源蛋白生产时，除适配启动子、调控转录因子外，确定一种合适的表达策略也是提高蛋白产量的重要方法。

构建融合表达载体是广泛采用的策略之一，该策略主要通过将内源性分泌蛋白与异源蛋白融合，实现异源蛋白的有效合成分泌。在里氏木霉中常采用分泌量最高的纤维素酶CBHI作为融合表达元件，将其融合至异源蛋白的N端以引导表达分泌^[16]。截至目前，通过构建CBHI-异源蛋白融合载体已成功表达了多种蛋白。例如，采用该策略成功将Fab抗体片段在里氏木霉中的产量提高了50倍以上^[58]；将肥胖抑制素(obestatin) (一种由23个氨基酸构成的人源小肽)的产量从约300 ng/mL提高到5.5 μg/mL^[30]。

尽管CBHI成功引导了多种外源蛋白在里氏木霉中的表达，并在产量上取得了一定提升，但由于其分子量过大(作为融合元件的序列长达481个氨基酸)，会显著增加表达盒长度，影响mRNA稳定性，且不利于简化遗传操作及后续蛋白分离纯化程序，后续研究中主要采用纤维素酶CBHII的N端纤维素结合结构域(cellulose binding domain, CBM)以及甘露聚糖酶I (Man5A或MANI)催化结构域，其中CBHII的CBM是目前已知用于促进异源蛋白表达分泌的最高效融合元件^[59]。虽然融合表达有助于目标产物的分泌，但后续切割、加工和纯化过程会增加下游加工的复杂度，从而提高产业化成本。

2 宿主底盘改造

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2.1 去除宿主背景蛋白

纤维素酶和半纤维素酶在里氏木霉分泌蛋白中占比超过90%，它们不仅与外源蛋白竞争翻译与分泌资源，也增加了下游纯化的难度与成本^[60]。Chai等^[61]利用规律成簇的间隔短回文重复序列系统及相关蛋白9 (clustered regularly interspaced short palindromic repeats associated 9, CRISPR-Cas9)技术，结合ura5标记回收系统，对里氏木霉11个主要纤维素酶基因(包括cbh2、egl1、xyn2、bgl1等)进行连续无痕基因敲除，构建了从HLΔ1到HLΔ11的一系列底盘菌株，改造后菌株的背景蛋白总量降低了52%；在此基础上，利用cbh1强启动子驱动表达细菌木聚糖酶(bacterial xylanase, XYL7)、真菌免疫调节蛋白(potential pharmaceutical protein, LZ8)和人血清白蛋白(human serum albumin, HSA) 3种异源蛋白，均实现了产量的显著提升。

除敲除背景蛋白基因外，还可利用组成型启动子表达，借用碳代谢阻遏(carbon catabolite repression, CCR)抑制背景蛋白的产生^[5]。Rantasalo等^[57]通过合成表达系统在葡萄糖培养基中培养，并删除主要纤维素酶基因(cbh1、cbh2、egl1、egl2)，实现了背景蛋白的大幅降低，最终使目标蛋白纯度达到约75%。

尽管上述方法可实现对背景蛋白的部分抑制，但目标蛋白的分泌并未达到预期水平。这是因为异源蛋白表达同时受到折叠、修饰、分泌调控及蛋白酶降解等多方面影响，导致异源蛋白无法完全取代纤维素酶利用其加工分泌途径，多项研究均证实了这一现象^[62]。

2.2 转录调控改造

转录调控在异源蛋白生产中起核心开关作用，里氏木霉基因组中注释了约700个转录因子，但截至目前仅对极少部分转录因子的功能进行了研究^[2]。这些转录因子大多为Zn₂Cys₆型转录调控因子，含有一个保守的C₆锌指结构DNA结合域及一个真菌特征性的转录因子结构域，尽管里氏木霉中该转录因子结构域的功能尚不清晰，但已有研究表明这一结构域对其调控功能至关重要，截短后会改变其对下游基因的调控模式^[63]。

Sun等^[22]通过敲除里氏木霉中p53样转录因子vib1，构建了纤维素酶基本沉默、胞外蛋白酶活性降低70%以上的Δvib1宿主；以cdna1启动子驱动、单拷贝整合黑曲霉β-葡萄糖苷酶(β-D-glucosidase, BGLA)后，Δvib1宿主在乳糖培养体系中酶活较QM53宿主提高17%，且SDS-PAGE结果显示背景蛋白显著减少；发酵上清在30 ℃存放4周后，酶活几乎无损失，而QM53体系损失超过60%；此外，Δvib1本底及其表达BGLA后的UPR/ERAD相关基因转录水平均显著低于QM53，表明其分泌压力明显减轻。上述结果表明，转录因子vib1的单次缺失即可同时削弱纤维素酶和蛋白酶背景，获得生长正常、分泌压力低、异源蛋白产量高且稳定性优的“洁净”表达平台，为工业低成本纯化敏感重组蛋白提供了优良策略。

通过改造XYR1、CRE1等核心转录因子可精准调控启动子活性，从而获得理想的表达谱。例如，突变型Xyr1变体(XYR1-A824V)的组成型表达可使xyn1和xyn2启动子实现高水平、不依赖诱导剂的表达，既省去了昂贵诱导剂的使用，又最大限度减少了背景蛋白的分泌^[64]。CRE1是介导碳代谢阻遏的核心转录抑制因子，在葡萄糖充足时其DNA结合域与靶基因启动子高效结合，从而抑制纤维素酶基因的表达，同时在碳代谢、氮吸收和真菌发育中起关键作用^[65-66]。已有多项研究通过改造启动子使目的蛋白在葡萄糖为碳源条件下表达，进而利用碳代谢阻遏抑制内源纤维素酶表达，获得高纯度目的蛋白^[46,67-68]。

尽管转录因子工程可通过重塑纤维素酶调控与分泌应激网络来提升里氏木霉的异源蛋白表达潜力，但受限于转录调控网络的复杂性和多样性、内源蛋白竞争及应激反馈抑制等因素，单一转录因子改造难以实现产能的显著突破。为解决这一困境，需将转录调控改造与分泌途径优化、动态调控及多组学建模相结合，从而实现异源蛋白的高效、稳定表达。

2.3 降低胞外蛋白酶对异源蛋白的降解

对里氏木霉QM6a的分泌蛋白组学分析表明，其培养上清液中可鉴定出多达39种分泌型蛋白酶^[69]。研究表明，这些酶可降解内源性和异源性蛋白，相关研究已证实蛋白酶活性与重组蛋白浓度降低存在关联，蛋白酶活性常被认为是异源重组蛋白表达过程中的主要挑战^[27,38]。

Landowski等^[27]采用逐步基因敲除策略，通过迭代删除包括slp7、amp2等在内的9个主要蛋白酶基因，成功将干扰素α-2b (interferon-alpha 2b, IFNα-2b)的摇瓶产量从0.2 g/L提高至2.4 g/L；在1 L发酵罐规模下补充添加大豆胰蛋白酶抑制剂后，产量进一步提升至4.5 g/L。为缓解蛋白酶对靶标异源蛋白的潜在降解风险，Chai等^[61]删除了蛋白酶激活因子Peal (tre123125)，成功将里氏木霉胞外蛋白酶活性降低了52%。

胞外蛋白酶降解是里氏木霉异源蛋白损失的主要原因，尤其在培养后期和高细胞密度条件下更为突出^[70]。因此，敲除主要蛋白酶基因、降低自溶水平或通过蛋白工程提升产物稳定性成为常见优化方向，但在放大培养过程中仍存在挑战。里氏木霉的蛋白酶体系复杂，多个蛋白酶之间存在功能冗余，单一敲除常难以获得稳定效果；多基因敲除虽可降低对异源蛋白的降解，但可能影响营养利用、细胞壁更新和长期培养稳态等。

2.4 调控菌丝形态

丝状真菌的蛋白质分泌在活跃生长的菌丝顶端最为旺盛，由此可推断，具有高分枝表型的丝状真菌由于菌丝顶端数量增加，可能更有利于蛋白质生产；同时，丝状真菌的高分枝表型有时伴随菌丝缩短，从而降低培养液黏度，这对高密度发酵有利^[71-72]。

在粗糙脉孢菌(Neurospora crassa)中，一种温度敏感的mcb突变体表现出较低的极性生长能力，单位长度菌丝的顶端数量更多，其胞外蛋白产量提高了3-5倍，羧甲基纤维素酶活性提高了20倍^[73]。删除编码Rho-GTPase RacA的racA基因可使黑曲霉菌丝尖端增加约20%，这种过度分枝的表型增加了菌丝尖端高尔基体后分泌囊泡的数量，最终使葡糖淀粉酶产量提高了4倍^[74]。上述结论为里氏木霉的菌丝形态改造提供了良好参考。

丝状真菌的极性和形态也可通过调节培养条件进行调控。在黑曲霉培养中添加氧化铝颗粒可形成自由分散的菌丝体，并使上清液中的果呋喃糖苷酶活性提高4倍^[75]；在微颗粒强化补料分批工艺中，产量提升了10倍^[76]。形态学改变(如菌丝分枝增加)可通过扩大蛋白质附着与扩散的表面积进一步增强分泌效率。例如，敲除里氏木霉gul1基因可提高分枝数量、降低发酵液黏度，提升整体分泌产量^[77]。此外，Gao等^[78]通过RNA干扰(RNA interference, RNAi)技术介导Trcot1基因沉默，成功在里氏木霉中诱导出高分枝菌丝表型，显著提高了单位菌丝生物量的纤维素酶分泌效率。丝状真菌的形态学特征在很大程度上决定了发酵液的流变学性质及溶氧、传质效率，是制约里氏木霉工业化放大的关键因素^[79]。尽管通过调控菌丝的极性和形态可改善发酵性能，但菌株改良仍需结合反应器类型、通气搅拌方式和目标蛋白特性进行系统性设计。里氏木霉作为异源蛋白表达宿主底盘的主要改造策略如图1所示。

3 发酵工艺优化

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3.1 培养基成分优化

里氏木霉的异源蛋白表达水平首先受碳、氮源种类及比例的制约。氮源是影响里氏木霉蛋白酶分泌的关键因素。Sun等^[80]系统研究了碳、氮源对胞外蛋白酶产量的调控作用，发现有机氮源(如蛋白胨、玉米浆、牛血清白蛋白)能强烈诱导蛋白酶分泌，这一发现为通过氮源优化抑制蛋白降解提供了理论基础。Haab等^[81]早期研究同样证实，有机氮源可显著诱导T. reesei QM9414胞外蛋白酶的形成。Šimkovič等^[82]进一步报道了有机氮源诱导绿色木霉(Trichoderma viride)分泌胞外蛋白酶的现象。这些研究表明，减少或替换有机氮源可作为降低蛋白酶活性的有效策略。Landowski等^[27]在干扰素α-2b生产研究中采用系统性培养基优化策略，通过减少酵母提取物和蛋白胨等有机氮源，配合蛋白酶基因敲除，实现了2.4 g/L的高产量，该研究强调培养基组成需与遗传改造策略协同优化，以最大限度降低蛋白降解。

碳源选择同样需与启动子类型相匹配。Li等^[46]利用pdc组成型启动子，在高浓度葡萄糖条件下实现了木聚糖酶Ⅱ的高效生产(9 266 IU/mL)，显著高于天然表达水平，证明葡萄糖作为碳源可有效驱动组成型表达并简化发酵工艺。然而，诱导型启动子(如P _cbh1 )虽具有高强度表达优势，但依赖纤维素类诱导物会导致大量天然纤维素酶共表达，进而增加下游纯化成本^[83]。因此，碳源选择需权衡表达强度与工艺经济性。

3.2 发酵参数调控

里氏木霉的发酵参数(如温度、pH、溶氧和营养供给方式)直接影响其生长代谢状态及蛋白分泌通路效率，从而决定异源蛋白的表达水平与产量。因而，系统优化发酵过程参数不仅有助于提升里氏木霉作为表达宿主的生产性能，也为实现异源蛋白的高效稳定制备提供了关键支撑。里氏木霉分泌蛋白的高峰通常出现在对数生长后期至稳定期初期，因此采用“批式-流加混合”模式可在不增加反应器复杂度的前提下，将比生长速率稳定控制在0.05-0.08 h^-1，从而延长蛋白合成期并减少底物抑制^[84]。在发酵过程中还可通过搅拌和通气增加氧气供应，以维持良好的细胞呼吸和代谢活性，从而提高蛋白表达和分泌效率^[85]。专利US9428780B2中，利用补料分批发酵(pH 3.0-6.0, 25-40 ℃)实现颗粒淀粉水解酶(granular starch hydrolyzing enzyme, GSHE)产量超过1 g/L^[86]。

温度阶段控制也被证实可进一步提高蛋白表达。将发酵前24 h温度设定为32 ℃以加速菌体生长，随后降至28 ℃以降低内源蛋白酶活性，可使异源蛋白降解率下降40%，终产量提高25%^[87]。另外，Jiang等^[68]发现，cdna1启动子具有热诱导特性，与30 ℃相比，在37 ℃下培养显著提高了β-甘露聚糖酶和聚半乳糖醛酸酶的产量，因此可通过基因工程与培养条件优化相结合提高重组蛋白产量。

3.3 工艺难题解决

里氏木霉在高通气、高搅拌条件下极易产生大量泡沫，导致逃液、染菌及蛋白损失。为解决这些问题，针对工艺瓶颈开展系统性优化与调控，有助于生产效率与产品质量的提升。管志欣等^[88]系统比较了化学消泡剂与工艺减泡策略，发现添加0.01%聚醚类消泡剂虽可瞬时破泡，但会显著抑制菌丝分枝及蛋白分泌，使纤维素酶终酶活下降22%；相反，采用初始低浓度糖含量结合6 g/L粗小麦麸皮的物理减泡方案，可将泡沫减少75%，蛋白产量提升30%，这是由于粗麸皮中的膳食纤维通过吸附表面活性物质并增加液膜机械强度，实现原位抑泡，同时对菌体无毒。

进一步研究发现，将补料速率控制在5-9 mL/h (1.5 L罐)并将溶氧维持在20%以上，可使发酵液表面张力稳定在52-55 mN/m，显著低于起泡临界值45 mN/m^[87]。对于5-50 L放大体系，采用底部微孔环形分布器替代传统单孔鼓泡，可将气泡平均直径由3.2 mm降至1.1 mm，泡沫层减少60%，同时氧传质系数提高35%^[84]。此外，通过在线泡沫电极联动消泡泵实现消泡剂脉冲式添加(每次50 μL)，可将消泡剂总用量减少70%，并维持泡沫层低于2 cm，确保蛋白回收率超过90%^[88]。

结合上述策略，将培养基成分优化、发酵参数调控、泡沫原位抑制三方面策略协同应用可实现里氏木霉异源蛋白表达水平的提高，为工业化规模高效制备异源蛋白提供了可参考的技术范式。

4 AI辅助设计与前沿技术进展

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随着生成式AI、蛋白质基础模型、自动化实验平台和数字孪生技术的快速发展，微生物细胞工厂的研发模式正在发生显著变化。过去，异源蛋白生产平台的开发主要依赖于目标蛋白逐个摸索、表达构件逐个替换、发酵条件逐步试错，在大量试验中寻找最优生产条件^[89-90]。近年来越来越多研究表明，AI已能够在目标蛋白可表达性预测、信号肽和调控元件筛选、蛋白稳定性改造、宿主设计和发酵过程控制等方面提供实质性帮助，并开始在细菌、酵母和部分丝状真菌中展现出提高研发效率的潜力^[91]。对里氏木霉而言，这一趋势意义尤其重大。作为工业上最具潜力的蛋白分泌宿主之一，里氏木霉具有天然高分泌能力，但其异源蛋白生产存在分泌途径复杂、糖基化差异、背景蛋白成分复杂等问题^[62]。因此，借助AI和数字化建设里氏木霉表达大数据平台，不仅可以实现技术升级，更可使科研工作从经验探索迈向可预测工程，目前已有学者利用AlphaFold2预测的XYR1结构对潜在靶点进行突变，实现了目标酶产量的显著提高^[92]。

4.1 微生物蛋白生产平台利用AI技术取得的实质性进步

近年AI在微生物蛋白生产领域的突破，首先体现在蛋白辅助设计方面。以ESM、ProtT5、ProteinMPNN、ProGen及其衍生模型为代表的蛋白语言模型，已不再局限于结构预测，而是开始被广泛用于蛋白可溶性、稳定性、突变效应和分泌潜力的预测^[93-94]。与早期的机器学习方法相比，蛋白语言模型能够利用大规模序列数据预测表达关键影响因素，进一步提高目标蛋白结构特征与宿主加工能力的匹配程度。

其次，AI在表达构件设计与组合优化中的应用更加广泛。近年来，大量研究开始从AI预测转向AI参与实验设计^[95-96]。例如，在酵母和细菌中，深度学习和主动学习已被用于启动子强度预测、核糖体结合位点(ribosome binding site, RBS)/非翻译区(untranslated region, UTR)设计、分泌信号肽筛选和多变量组合推荐^[97-98]。相比传统的单因素替换，这种方法可在大量排列组合中优先推荐高评分方案，显著提升优化效率^[99]。对于里氏木霉这样的丝状真菌宿主，由于其载体构建、菌株筛选和发酵验证周期远长于大肠杆菌，这样的设计流程可显著减少试错成本^[100]。

AI与自动化实验平台结合，正在推动设计-构建-测试-学习(design-build-test-learn, DBTL)闭环真正落地。过去，DBTL更多是一个概念框架；而近年在自动化液体工作站、微型生物反应器、在线检测和机器学习迭代算法的支持下，越来越多微生物工程研究开始形成可持续迭代的闭环系统^[101]。对于异源蛋白开发而言，这意味着研发过程可通过数据分析和模块化组装实现更高效的迭代开发。

最后，生物过程数字化和数字孪生(digital twins, DTs)正加速进入产业视野^[102]。尤其在生物制药、酵母发酵和连续培养系统中，机器学习辅助的模型预测控制研究明显增多^[103]。这表明AI的作用已不再局限于菌株构建前端，而开始贯穿发酵过程优化和放大控制。同时，物联网设备在生物加工中的集成实现了精确的数据收集，可实时调整和改进工艺效果^[104]。

4.2 AI辅助设计里氏木霉表达平台

里氏木霉虽然是蛋白分泌的优秀宿主，但异源蛋白能否在里氏木霉中实现高效表达，本质上取决于目标蛋白自身性质与宿主加工分泌机制之间的匹配程度，而非单纯由宿主的天然分泌能力决定。不同蛋白在转录负担、折叠需求、分泌途径适配性以及胞外抗降解能力等方面存在本质差异。与此同时，里氏木霉作为典型的丝状真菌，其异源蛋白生产过程还对培养条件和细胞生理状态表现出很强的敏感性，因此同一表达结构在不同培养体系或放大阶段常出现明显波动。

围绕里氏木霉异源蛋白表达体系的智能化开发，真正需要解决的并非单一实验条件的优化，而是如何将分散的案例经验汇总为可预测、可迭代、可迁移的知识体系，构建一个能够支撑预测、决策和持续学习的数字化体系，使里氏木霉表达平台从经验驱动走向理性设计^[105]。当前研究可围绕多个关键变量展开，例如启动子及信号肽的选择、胞外蛋白酶基因缺失和发酵参数等，利用模型根据已有数据预测可能的最优实验条件，再利用新获得的数据持续修正判断，逐轮逼近最优解^[106]。

总体而言，近几年AI辅助技术在微生物细胞工厂领域带来的进展已表明，数据驱动开发不再停留于概念验证阶段，而正在逐步成为宿主工程、蛋白设计与发酵工艺优化的重要支撑。对里氏木霉而言，当前特异性面向该宿主的智能化研究仍然有限，但该体系未来具有广阔的发展空间，有潜力发展为异源蛋白表达的重要底盘。

5 里氏木霉作为异源蛋白表达超级宿主的核心及展望

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里氏木霉作为已知分泌蛋白能力最强的微生物，在过去的开发利用中被寄予很高的期待，但目前异源蛋白产量远未达到预期目标，里氏木霉的超强蛋白合成及分泌系统并未在异源蛋白表达过程中充分发挥作用。未来研究将聚焦于提高异源蛋白表达效率及扩展表达蛋白种类2个方面，以下几个方面是重要的努力方向。

5.1 清除背景蛋白

删除内源性纤维素酶基因是构建里氏木霉低蛋白背景底盘菌株的关键策略。通过依次删除主要纤维素酶与半纤维素酶基因(cbh2、xyn2、egl1、egl2、cip1、cel74a、bgl1等)可大幅降低此类背景杂质，从而简化纯化流程并降低成本^[61]。当以葡萄糖为主要碳源时，采用组成型启动子或人工设计的合成启动子可通过碳代谢阻遏机制有效降低纤维素酶的本底表达^[65]。以上策略可消除天然酶合成的代谢负担，将资源重新导向异源蛋白生产，从而提高整体产量和系统效率。

5.2 充分利用诱导系统

目前普遍认为β-二糖是里氏木霉生产纤维素酶的有效诱导物，尤其是槐糖、纤维二糖和乳糖，其中槐糖是目前发现的最有效的可溶性诱导物^[7]。研究报道槐糖诱导木霉生产纤维素酶的效果是纤维二糖的2 500倍，是乳糖的200倍^[107]。本课题组Yang等^[108]从高温菌欧文湖热解纤维素菌(Caldicellulosiruptor owensensis)中筛选到一种多功能β-葡萄糖苷酶，催化葡萄糖合成槐糖的时空效率达到9.5 g/(L·h)，使利用槐糖诱导里氏木霉高效生产蛋白的产业化应用成为可能。然而，高强度诱导并非能达到预期效果，如何平衡转录强度与分泌能力仍是亟待解决的难题，主要瓶颈在于通过启动子的选择或工程化适配转录机制和分泌机制解决内质网过载的问题。

5.3 提高蛋白修饰能力

里氏木霉在生产药用蛋白方面展示出巨大的潜力与优势，其高效蛋白表达能力为此奠定了基础^[2]。与此同时，尽管糖基化修饰依旧是一个挑战，需要克服真菌高甘露糖型糖基化模式以避免免疫反应^[109]，但通过基因编辑可以进行有效调整。相较于传统的哺乳动物细胞，里氏木霉具有培养成本低、发酵速度快和产量高等优点。尽管要完全取代哺乳动物细胞仍需提升其重组蛋白的产出和改善糖基化路径，但通过创新的发酵工艺和合成生物学技术，该真菌平台有望在未来成为生物制药的关键工具。

6 总结与展望

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里氏木霉作为极具潜力的异源蛋白表达系统，其发展前景广阔，有望在生物制造与生物医药领域发挥更重要的作用。首先，基于其强大的蛋白合成分泌能力，通过进一步的代谢工程与分泌途径优化，有望成为工业蛋白生产的理想底盘微生物工厂。其次，其接近于哺乳动物的糖基化修饰能力，为复杂药用糖蛋白(如抗体、酶替代疗法药物)的规模化、低成本生产提供了独特优势，有望推动相关生物类似药或创新药的开发进程。此外，依托于清晰的可诱导表达调控机制，特别是对纤维素酶基因启动子及相应转录因子的深入解析，未来可构建高效、可控的表达系统，实现异源蛋白产量的显著提升。更重要的是，随着AI辅助技术在微生物细胞工厂领域的不断发展，异源蛋白的结构设计、表达优化、定向改造与发酵培养的持续检测优化拥有广阔的发展前景。总之，随着系统生物学、合成生物学与发酵技术的持续进步，里氏木霉将在异源蛋白生产，尤其是高附加值蛋白药物生产方面展现出更大的应用潜力与产业化价值。

基金

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国家自然科学基金(22478403)

参考文献

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文献

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2026年第66卷第8期

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doi: 10.13343/j.cnki.wsxb.20260151

接收时间：2026-02-24
首发时间：2026-08-21
出版时间：2026-08-04

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收稿日期：2026-02-24
录用日期：2026-04-02

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National Natural Science Foundation of China(22478403)

国家自然科学基金(22478403)

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^1.沈阳化工大学环境与安全工程学院，辽宁沈阳

^2.中国科学院过程工程研究所，生物药制备与递送全国重点实验室，北京

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Foreign protein	Origin	T. reesei strain	Expression system	Yield	References
Chymosin	Bovine	Rut-C30	cbh1	cbh1	cbh1+20 aa	40 mg/L	[15]
Fab antibody fragments	Mouse	RUT-C30	cbh1	cbh1	cbh1	1 mg/L	[16]
Xylanase Ⅵ	Acrophialophora nainiana	RUT-C30	cbh1	cbh1	cbh1	172 mg/L	[17]
Cellobiose dehydrogenase	Corynascusthermophilus	QM9414	cdna1	cdh	trpC	29 mg/L	[18]
Mammalian proteins α-galactosidase A	Human	Rut-C30	cbh1	cbh1	cbh1	636 mg/L	[19]
Erythropoietin	Human	RutC-30 M3	cbh1	cbh1	cbh1	97 mg/L	[20]
β-lactoglobulin	Bovine	M1908	SES	pdc1	cbh1	1 g/L	[21]
Ovalbumin	Hen	M1908	SES	pdc1	cbh1	2 g/L	[21]
β-glucosidase	Aspergillus niger	QM53	cdna1	cbh1	cbh1	17.2 IU/mg	[22]
Te-cel7A	Talaromyces emersonii	QP4	cdna1	trpC	cbh1	0.93 U/L	[23]
Feruloyl esterase	Aspergillus niger	RUT-C30	cbh1	cbh1	cbh1	20.69 U/gds	[24]
Mannanase 5A	A. niger	TU-6	cbhl	cbh1	cbh1	1 204 U/mL	[25]
Alkaline endoglucanase	Humicola insolens	RUT-C30	xyn1/xyn2	xyn1/xyn2	xyn1/xyn2	15.7 U/mL	[26]
Interferon alpha-2b	Human	M504	cbh1	cbh1	cbh1	4.5 g/L	[27]
Mannanase 5A	Trichoderma harzianum	QM9414	pdc	pdc	ThMan5A	1.6 g/L	[28]
Dye-decolorizing peroxidase	Pleurotus sapidus	RUT-C30	cbh1	cbh1	rPsaDyP	55 000 U/L	[29]

Foreign protein

Origin

T. reesei strain

Expression system

Yield

References

Chymosin

Bovine

Rut-C30

cbh1

cbh1+20 aa

40 mg/L

[15]

Fab antibody fragments

Mouse

RUT-C30

cbh1

1 mg/L

[16]

Xylanase Ⅵ

Acrophialophora

nainiana

RUT-C30

cbh1

172 mg/L

[17]

Cellobiose dehydrogenase

Corynascusthermophilus

QM9414

cdna1

cdh

trpC

29 mg/L

[18]

Mammalian proteins α-galactosidase A

Human

Rut-C30

cbh1

636 mg/L

[19]

Erythropoietin

Human

RutC-30 M3

cbh1

97 mg/L

[20]

β-lactoglobulin

Bovine

M1908

SES

pdc1

cbh1

1 g/L

[21]

Ovalbumin

Hen

M1908

SES

pdc1

cbh1

2 g/L

[21]

β-glucosidase

Aspergillus niger

QM53

cdna1

cbh1

17.2 IU/mg

[22]

Te-cel7A

Talaromyces emersonii

QP4

cdna1

trpC

cbh1

0.93 U/L

[23]

Feruloyl esterase

Aspergillus niger

RUT-C30

cbh1

20.69 U/gds

[24]

Mannanase 5A

A. niger

TU-6

cbhl

cbh1

1 204 U/mL

[25]

Alkaline endoglucanase

Humicola insolens

RUT-C30

xyn1/xyn2

15.7 U/mL

[26]

Interferon alpha-2b

Human

M504

cbh1

4.5 g/L

[27]

Mannanase 5A

Trichoderma harzianum

QM9414

pdc

ThMan5A

1.6 g/L

[28]

Dye-decolorizing peroxidase

Pleurotus sapidus

RUT-C30

cbh1

rPsaDyP

55 000 U/L

[29]

Promoter	Source gene	Type	Induction/regulation mode	Expression intensity (relative)	References
P _cbh1	Cellobiohydrolase CBH1/CEL7A	Inducible	Induced by cellulose, lactose, cellobiose, etc.	Very high	[43]
P _cbh2	Cellobiohydrolase CBH2/CEL6A	Inducible	Induced by cellulose, lactose	High	[44]
P _egl2	Endoglucanase EG2/CEL5A	Inducible	Induced by cellulose	Medium	[45]
P _xyn2	Xylanase XYN2	Inducible	Induced by xylan	High	[26]
P _pdc1	Pyruvate decarboxylase	Constitutive	Stable and continuous expression	Medium	[28]
P _gpd1	Glyceraldehyde-3-phosphate dehydrogenase	Constitutive	Stable and continuous expression	Medium	[46]
P _cdna1	cdna1 (unknown)	Constitutive	Stable and continuous expression	High	[18]
P _cbh1_PA	Modified from P _cbh1	Inducible	Induced by cellulose, lactose, cellobiose, etc.	Extremely high	[47]

Promoter

Source gene

Type

Induction/regulation mode

Expression intensity (relative)

References

P _cbh1

Cellobiohydrolase CBH1/CEL7A

Inducible

Induced by cellulose, lactose, cellobiose, etc.

Very high

[43]

P _cbh2

Cellobiohydrolase CBH2/CEL6A

Inducible

Induced by cellulose, lactose

High

[44]

P _egl2

Endoglucanase EG2/CEL5A

Inducible

Induced by cellulose

Medium

[45]

P _xyn2

Xylanase XYN2

Inducible

Induced by xylan

High

[26]

P _pdc1

Pyruvate decarboxylase

Constitutive

Stable and continuous expression

Medium

[28]

P _gpd1

Glyceraldehyde-3-phosphate dehydrogenase

Constitutive

Stable and continuous expression

Medium

[46]

P _cdna1

cdna1 (unknown)

Constitutive

Stable and continuous expression

High

[18]

P _cbh1_PA

Modified from P _cbh1

Inducible

Induced by cellulose, lactose, cellobiose, etc.

Extremely high

[47]