药学学报

Application	Chassis	Enzyme	Substrate	Product	Mutation	Result	Ref.
Improve the catalytic activity of enzymes	E. coli	PdxA, PdxJ	4-Phosphohydroxy-L-threonine, 3-phosphohydroxy-1-aminoacetone	Pyridoxine	PdxA2 (H136N), PdxJ1 (E104T/I218L/G194C)	Using two mutants combined with metabolic engineering to construct an engineering strain, the production of pyridoxine was increased to 1.4 g·L^-1	[34]
	S. cerevisiae	CnVS	Farnesyl pyrophosphate	Valencene	M560L	The production of (+)‐nootkatone in the strain constructed by utilizing mutants and combining with metabolic engineering increased to 804.96 mg·L^-1	[35]
	E. coli	SmpepD	β-Ala, L-His	L-Carnosine	T168S/G148D	A strain was constructed using this mutant and transporter engineering, resulting in an increase in the production yield of L-carnosine to 133.2 mmol·L^-1	[36]
	S. cerevisiae	ERG7	2, 3-Oxidosqualene	Lanosterol	F699T, I705K	Replacing wild-type ERG7 in yeast with mutants with reduced activity reduces lanosterol production and indirectly increases triterpenoid production	[37]
Changing the regioselectivity of enzymes towards substrates	E. coli	P450BM3	m-Alkylphenols	m-Alkylbenzene-1, 4-diols	R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V, R47L/Y51F/F87V/L181Q/L188P/I401P, R47I/F87V/L188P	Coexpression of mutants and cofactors in E. coli and biocatalysis for efficient synthesis of m-alkylbenzene-1, 4-diol	[38]
	—	AmGT8	Cycloastragenol, UDP-Glu	Cycloastragenol-3/6-O-glycoside, cycloastragenol-3-(2′-O-glycoside)-O-glycoside	A394F, T131V, P192E	By combining different mutants and utilizing different glycosyl donors, it synthesized the main saponin component, astragaloside Ⅲ and Ⅳ	[39]
	—	AmAT7-3	Astragaloside Ⅳ, UDP-Xyl	Isoastragaloside Ⅱ, cyclocephaloside Ⅱ, C3′, C4′-O acetylated astragaloside Ⅳ	A310G, A310W	Specific C3′-O and C4′-O acetylation of astragaloside Ⅳ was successfully achieved	[40]
Changing the substrate range of enzymes	E. coli	HpaBC	Tyrosol	Hydroxytyrosol	—	Mutant HpaBC can simultaneously catalyze tyrosol/tyramine hydroxylase, significantly improving the efficiency of hydroxytyramine biosynthesis	[43]
	E. coli	DEBS	(2S, 3R)-3-Hydroxy-2-methylpentanoic acid-SNAC thioester, methylmalonyl-CoA	(4S, 5R)-3-Oxo-2, 4-dimethyl-5-hydroxy-heptanoic acid-D-lactone	—	A novel polyketide compound was successfully synthesized	[44]
	E. coli	MpOMT	(2S)-Naringenin, (2S)-eriodictyol	(2S)-Eriodictyol, (2S)-hesperetin	S142V	The mutant S142V can mainly catalyzes (2S) - estrictyl, and its yield increased to 27.5 mg·L^-1 in E. coli	[45]
Changing the product spectrum of enzymes	—	β-1, 3-Xylanase	β-1, 3-Xylan	Xylose, β-1, 3-xylobiose, β-1, 3-xylotriose	—	Unnatral β-1, 3-xylanase AncXyl10 hydrolyzes xylose and only produces β-1, 3-xylobiose and β-1, 3-xylotriose without producing xylose	[48]
	E. coli	AlbC	aa-tRNAs	Diketopiperazine	F186L	F186L mutant can catalyze the synthesis of new diketopiperazine molecules that cannot be produced by wild enzymes	[50]
	—	OAC TKS	Malonyl, pentyltetra-β-ketide-CoA	Pentyltetra-β-ketide-CoA, olivetolic acid	OAC (F24I) TKS (L190G)	OAC and TKS mutants can catalyze the formation of cannabinoid cores with linear fatty acyl segments of up to 11 Cs	[52]
Improving the adaptability of enzymes to pH	C. glutamicum	GAD	L-Glutamate	GABA	E89Q and C-terminal deletion	Changed the pH preference of glutamic acid decarboxylase, resulting in a GABA production of 38 g·L^-1 in C. glutamicum	[54]
	—	PhDac	GlcNAc	GlcN	G74D/H152E/W232A/Q29E/K106E/N176ER221E/L271E	Mutant M20 maintains high catalytic efficiency under low pH conditions	[56]
	—	BsGDH	L-Glutamate	α-Ketoglutaric acid	N16D, K218D	Lowering the optimal catalytic pH range of BsGDH to its stable pH range	[57]
Improving the adaptability of enzymes to temperature	—	LmSP	Sucrose, glycerol	αGG	V23L/S424R	The half-life of the mutant at 50 ℃ is twice that of the wild-type, and the sucrose conversion rate is 76.3%	[60]
	E. coli	CHST11	Chondroitin, PAPS	CSA	E114D/A159N/P170E/I67D/L134V/I206T/K218R/S286A/S297D/Y298S/Y304S/A305G/T316A	The T_m and half-life of the mutant were increased by 6.9 ℃ and 3.5 h, respectively, and a whole cell CSA catalytic system with a conversion rate of 89.5% was established	[64]
	—	PcEST	Lovastatin	Monacolin J	D106A	Mutant D106A has better solubility and thermal stability	[65]
Relieve inhibition of substrate or product concentration	Y. lipolytica	CarRP	Geranylgeranyl pyrophosphate, lycopene	Phytoene, β-carotene	Y27R	The mutant Y27R is no longer inhibited by substrates and maintains enzyme activity	[67]
	E. coli	ADC	L-Aspartic acid	β-Alanine	R12V	When the concentration of L-aspartic acid reached 100 g·L^-1, the mutant yield remained at 0.45 g·g^-1, and substrate inhibition was greatly alleviated	[68]
	S. cerevisiae	Aro3	Erythrose 4-phosphate, phosphoenol pyruvate	DAHP	D154N	The D154N mutant can effectively alleviate feedback inhibition of phenylalanine, and the production of salidroside reaches 2.4 g·L^-1	[69]
Improve the soluble expression of enzymes	E. coli	LovD	Monacolin J	Simvastatin	C40A, C60N	Mutants C40A and C60N significantly increased protein solubility, resulting in a 27% and 26% increase in catalytic activity	[72]
	—	reTP	Inulin	Fructose, glucose, inulooligosaccharides	Y128H/A316T/E344K/T504M	The expression level of soluble enzymes in the mutant is five times higher than that of wild enzymes	[73]
	—	GroEL/S	—	—	GroES (Y71H, V26A/Y71R, V26A/Y71H/V83M), GroEL (A145V/D490G, A163V/D490G, D188G/M488V)	Mutants of GroEL/S can better promote the folding and soluble expression of recombinant proteins	[74]

Application	Chassis	Enzyme	Substrate	Product	Mutation	Result	Ref.
Improve the catalytic activity of enzymes	E. coli	PdxA, PdxJ	4-Phosphohydroxy-L-threonine, 3-phosphohydroxy-1-aminoacetone	Pyridoxine	PdxA2 (H136N), PdxJ1 (E104T/I218L/G194C)	Using two mutants combined with metabolic engineering to construct an engineering strain, the production of pyridoxine was increased to 1.4 g·L^-1	[34]
	S. cerevisiae	CnVS	Farnesyl pyrophosphate	Valencene	M560L	The production of (+)‐nootkatone in the strain constructed by utilizing mutants and combining with metabolic engineering increased to 804.96 mg·L^-1	[35]
	E. coli	SmpepD	β-Ala, L-His	L-Carnosine	T168S/G148D	A strain was constructed using this mutant and transporter engineering, resulting in an increase in the production yield of L-carnosine to 133.2 mmol·L^-1	[36]
	S. cerevisiae	ERG7	2, 3-Oxidosqualene	Lanosterol	F699T, I705K	Replacing wild-type ERG7 in yeast with mutants with reduced activity reduces lanosterol production and indirectly increases triterpenoid production	[37]
Changing the regioselectivity of enzymes towards substrates	E. coli	P450BM3	m-Alkylphenols	m-Alkylbenzene-1, 4-diols	R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V, R47L/Y51F/F87V/L181Q/L188P/I401P, R47I/F87V/L188P	Coexpression of mutants and cofactors in E. coli and biocatalysis for efficient synthesis of m-alkylbenzene-1, 4-diol	[38]
	—	AmGT8	Cycloastragenol, UDP-Glu	Cycloastragenol-3/6-O-glycoside, cycloastragenol-3-(2′-O-glycoside)-O-glycoside	A394F, T131V, P192E	By combining different mutants and utilizing different glycosyl donors, it synthesized the main saponin component, astragaloside Ⅲ and Ⅳ	[39]
	—	AmAT7-3	Astragaloside Ⅳ, UDP-Xyl	Isoastragaloside Ⅱ, cyclocephaloside Ⅱ, C3′, C4′-O acetylated astragaloside Ⅳ	A310G, A310W	Specific C3′-O and C4′-O acetylation of astragaloside Ⅳ was successfully achieved	[40]
Changing the substrate range of enzymes	E. coli	HpaBC	Tyrosol	Hydroxytyrosol	—	Mutant HpaBC can simultaneously catalyze tyrosol/tyramine hydroxylase, significantly improving the efficiency of hydroxytyramine biosynthesis	[43]
	E. coli	DEBS	(2S, 3R)-3-Hydroxy-2-methylpentanoic acid-SNAC thioester, methylmalonyl-CoA	(4S, 5R)-3-Oxo-2, 4-dimethyl-5-hydroxy-heptanoic acid-D-lactone	—	A novel polyketide compound was successfully synthesized	[44]
	E. coli	MpOMT	(2S)-Naringenin, (2S)-eriodictyol	(2S)-Eriodictyol, (2S)-hesperetin	S142V	The mutant S142V can mainly catalyzes (2S) - estrictyl, and its yield increased to 27.5 mg·L^-1 in E. coli	[45]
Changing the product spectrum of enzymes	—	β-1, 3-Xylanase	β-1, 3-Xylan	Xylose, β-1, 3-xylobiose, β-1, 3-xylotriose	—	Unnatral β-1, 3-xylanase AncXyl10 hydrolyzes xylose and only produces β-1, 3-xylobiose and β-1, 3-xylotriose without producing xylose	[48]
	E. coli	AlbC	aa-tRNAs	Diketopiperazine	F186L	F186L mutant can catalyze the synthesis of new diketopiperazine molecules that cannot be produced by wild enzymes	[50]
	—	OAC TKS	Malonyl, pentyltetra-β-ketide-CoA	Pentyltetra-β-ketide-CoA, olivetolic acid	OAC (F24I) TKS (L190G)	OAC and TKS mutants can catalyze the formation of cannabinoid cores with linear fatty acyl segments of up to 11 Cs	[52]
Improving the adaptability of enzymes to pH	C. glutamicum	GAD	L-Glutamate	GABA	E89Q and C-terminal deletion	Changed the pH preference of glutamic acid decarboxylase, resulting in a GABA production of 38 g·L^-1 in C. glutamicum	[54]
	—	PhDac	GlcNAc	GlcN	G74D/H152E/W232A/Q29E/K106E/N176ER221E/L271E	Mutant M20 maintains high catalytic efficiency under low pH conditions	[56]
	—	BsGDH	L-Glutamate	α-Ketoglutaric acid	N16D, K218D	Lowering the optimal catalytic pH range of BsGDH to its stable pH range	[57]
Improving the adaptability of enzymes to temperature	—	LmSP	Sucrose, glycerol	αGG	V23L/S424R	The half-life of the mutant at 50 ℃ is twice that of the wild-type, and the sucrose conversion rate is 76.3%	[60]
	E. coli	CHST11	Chondroitin, PAPS	CSA	E114D/A159N/P170E/I67D/L134V/I206T/K218R/S286A/S297D/Y298S/Y304S/A305G/T316A	The T_m and half-life of the mutant were increased by 6.9 ℃ and 3.5 h, respectively, and a whole cell CSA catalytic system with a conversion rate of 89.5% was established	[64]
	—	PcEST	Lovastatin	Monacolin J	D106A	Mutant D106A has better solubility and thermal stability	[65]
Relieve inhibition of substrate or product concentration	Y. lipolytica	CarRP	Geranylgeranyl pyrophosphate, lycopene	Phytoene, β-carotene	Y27R	The mutant Y27R is no longer inhibited by substrates and maintains enzyme activity	[67]
	E. coli	ADC	L-Aspartic acid	β-Alanine	R12V	When the concentration of L-aspartic acid reached 100 g·L^-1, the mutant yield remained at 0.45 g·g^-1, and substrate inhibition was greatly alleviated	[68]
	S. cerevisiae	Aro3	Erythrose 4-phosphate, phosphoenol pyruvate	DAHP	D154N	The D154N mutant can effectively alleviate feedback inhibition of phenylalanine, and the production of salidroside reaches 2.4 g·L^-1	[69]
Improve the soluble expression of enzymes	E. coli	LovD	Monacolin J	Simvastatin	C40A, C60N	Mutants C40A and C60N significantly increased protein solubility, resulting in a 27% and 26% increase in catalytic activity	[72]
	—	reTP	Inulin	Fructose, glucose, inulooligosaccharides	Y128H/A316T/E344K/T504M	The expression level of soluble enzymes in the mutant is five times higher than that of wild enzymes	[73]
	—	GroEL/S	—	—	GroES (Y71H, V26A/Y71R, V26A/Y71H/V83M), GroEL (A145V/D490G, A163V/D490G, D188G/M488V)	Mutants of GroEL/S can better promote the folding and soluble expression of recombinant proteins	[74]

蛋白质工程技术及其在药用天然产物合成生物学中的应用研究进展

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孙晓燕 , 陈晶晶 , 陈天娇 , 巩婷 , 杨金玲 ^* , 朱平 ^*

药学学报 | 综述 2024,59(6): 1601-1615

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药学学报 | 综述 2024, 59(6): 1601-1615

蛋白质工程技术及其在药用天然产物合成生物学中的应用研究进展

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孙晓燕, 陈晶晶, 陈天娇, 巩婷, 杨金玲^*, 朱平^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 国家卫生健康委员会天然药物生物合成重点实验室, 中国医学科学院酶与天然药物生物催化重点实验室, 北京 100050

通讯作者:

^*杨金玲, Tel: 86-10-63165199, E-mail: yangjl@imm.ac.cn;

朱平, Tel: 86-10-63165199, E-mail: zhuping@imm.ac.cn

Research progress on protein engineering technology and its application in the synthesis biology of medicinal natural products

Xiao-yan SUN, Jing-jing CHEN, Tian-jiao CHEN, Ting GONG, Jin-ling YANG^*, Ping ZHU^*

Affiliations

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, NHC Key Laboratory of Biosynthesis of Natural Products, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2024-0105

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摘要

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天然产物是药物发现的重要源泉。通过传统提取分离和化学合成方法获得天然产物时, 存在操作复杂、成本高、效率低和易造成环境污染等问题。利用合成生物学方法构建微生物细胞工厂以生产药用天然产物具有高效、低成本和绿色环保的优势。然而, 在微生物细胞工厂中, 由于酶的局限性, 限制了产物范围和产量提高。蛋白质工程被认为是克服这些问题最有效的方法之一。本文介绍了蛋白质工程技术的常用方法, 综述了蛋白质工程技术在药用天然产物合成生物学领域改善酶的性能及作用环境、促进合成生物学工具发展等方面的具体应用, 还分析了蛋白质工程目前存在的瓶颈和面临的挑战, 并对其应用前景进行了展望, 可为蛋白质工程技术发展和应用提供借鉴。

关键词

药用天然产物 / 生物合成 / 酶 / 蛋白质工程 / 合成生物学

Abstract

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Natural products are important sources of drug discovery. However, the traditional methods of extraction and isolation, as well as chemical synthesis for obtaining natural products are associated with issues such as operational complexity, high costs, low efficiency, and environmental pollution. Constructing microbial cell factories through synthetic biology methods to produce medicinal natural products has the advantages of high efficiency, low cost, and environmental protection. Nevertheless, the scope and yield improvement of the products are limited by the limitations of enzymes in microbial cell factories. Protein engineering is considered one of the most effective approaches to overcome these limitations. This article introduces commonly used methods of protein engineering technology and summarizes its specific applications in improving enzyme performance, modifying the enzymatic environment, and promoting the development of synthetic biology tools in the field of pharmaceutical natural product synthesis. Furthermore, it analyzes the current bottlenecks and challenges in protein engineering and looks forward to its future application prospects, offering insights for the development and practical use of protein engineering technology.

Key words

medicinal natural product / biosynthesis / enzyme / protein engineering / synthetic biology

引用本文

孙晓燕, 陈晶晶, 陈天娇, 巩婷, 杨金玲, 朱平. 蛋白质工程技术及其在药用天然产物合成生物学中的应用研究进展. 药学学报, 2024 , 59 (6) : 1601 -1615 . DOI: 10.16438/j.0513-4870.2024-0105

Xiao-yan SUN, Jing-jing CHEN, Tian-jiao CHEN, Ting GONG, Jin-ling YANG, Ping ZHU. Research progress on protein engineering technology and its application in the synthesis biology of medicinal natural products[J]. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1601 -1615 . DOI: 10.16438/j.0513-4870.2024-0105

正文

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天然产物是生物体在进化过程中为适应环境、抵御外侵、竞争拮抗、传递信息等而产生的代谢产物^{[1, 2]}。因此天然产物本身就被赋予了特定的生物活性。在药物研发领域, 天然产物及其衍生物发挥了重要作用^{[3, 4]}。然而, 由于生长速度缓慢或易受环境因素干扰, 很多原始宿主并不适合用于生产药用天然产物。大多数药用天然产物在原始宿主中的含量非常低, 提取和分离过程也比较困难。虽然化学合成方法是获取药用天然产物的另一重要途径, 但有时成本太高, 容易造成环境污染, 并且一些结构复杂的化合物也很难通过化学合成方法获得。微生物繁殖速度快, 生长周期短, 发酵工艺成熟, 且产物易于提取分离。因此使用遗传背景清晰的大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)、枯草芽孢杆菌(Bacillus subtilis) 等模式微生物作为生产药用天然产物的底盘细胞^[5], 不仅可以提高生产效率, 降低生产成本, 而且可以确保生产过程对环境友好。随着药用天然产物生物合成途径的解析, 通过合成生物学方法构建微生物细胞工厂用以生产药用天然产物已成为研究热点^[6-8]。

利用微生物底盘细胞进行药用天然产物的生物合成也存在一些限制因素。一般来说, 可以通过增加酶的表达水平等经典方法来提高微生物细胞工厂的特定代谢通量^[9], 从而提高目的产物产量。但利用天然酶构建生物合成途径时, 大多数酶都表现出酶活性有限、底物范围窄、稳定性差, 甚至在异源宿主中失去功能等局限性, 这些缺点会极大地限制它们在实际生产中的应用。

现有研究表明, 酶的特性可以通过蛋白质工程来进行改造^[10]。蛋白质工程是指在基因层面上对DNA进行设计重组, 从而实现对现有蛋白质的改造或制造一种全新的蛋白质, 以满足人类对生产和生活需求的过程^{[11, 12]}。除了提高酶的稳定性和活性外, 蛋白质工程还是改造酶的特异性和选择性的唯一途径^[13]。早在21世纪初, 蛋白质工程就已被应用于生产天然药物^[14]。蛋白质工程在合成生物学中扮演着重要的角色, 为设计、构建和优化生物合成系统提供了有力的工具, 能够帮助高效地生产药用天然产物或合成新的活性非天然产物。本文介绍了蛋白质工程技术的常用策略, 并总结了其在药用天然产物合成生物学领域的最新应用, 以期为利用合成生物学技术生产药用天然产物提供借鉴。

1 蛋白质工程的常用方法

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蛋白质工程通常包括3个步骤: ①通过随机突变构建突变体库或理性设计构建功能相关位点的突变体, 甚至通过从头设计创造全新的蛋白质; ②表达这些突变体蛋白或新蛋白质; ③进行评估和筛选, 以获得符合要求的蛋白质^[15]。由于步骤②所涉及的异源表达已经成为一种常规的分子生物学技术, 因此本文只综述了步骤①所涉及的常用突变方法和步骤③所涉及的常用筛选方法。

1.1 随机突变

对于结构-功能信息不足的目标蛋白, 可以采用随机突变的方法进行蛋白质工程改造。最早的诱变技术通过使用化学或物理诱变剂在目标序列中引入随机突变^[16], 但由于这些方法引入的突变类型仍具有一定的偏好性并且需要进行进一步的操作以获得双链突变DNA, 因此早已被其他方法取代。

目前, 易错PCR (error prone PCR, epPCR)^[17]是随机突变最常使用的技术之一。在epPCR中, 通常使用低保真度聚合酶(如Taq聚合酶) 和改变反应条件(如增加Mn²⁺、Mg²⁺离子的浓度及使用不同浓度的脱氧核糖核苷酸) 以增加错误频率(图 1A)。这种技术的缺点是容易产生移码突变, 使大部分的突变体功能丧失。Laure等^[18]利用epPCR方法建立了来源于黑曲霉菌(Aspergillus niger) 的β-葡萄糖苷酶BGL1的突变体库, 其中95%的突变体失去了活性, 但有12个突变体对底物硝基苯-β-D-葡萄糖苷表现出比野生型更高的活性, 且突变体Y305C降低了高浓度底物对水解反应的抑制作用。

另一种应用较为广泛的随机突变技术是DNA改组(DNA shuffling)^[19]。该技术通过DNase Ⅰ对两个以上序列和功能相近但来自不同物种的同源基因进行酶切, 得到10~50个碱基对的随机双链寡核苷酸片段, 通过DNA聚合酶在无引物PCR中实现部分同源性导致的重组错配, 错配的片段互为模板进行延伸, 与此同时, 不同来源的基因间也可发生重组, 形成融合基因(图 1B)。DNA改组模拟自然重组, 通过剪接基因片段, 可以组合一些优势性状, 因此, 它能够实现比epPCR更高的突变率及更好的效果。4-羟基苯基丙酮酸双加氧酶(4-hydroxyphenylpyruvate dioxygenase, HPPD) 是抗除草剂的重要靶点。Guo课题组^[20]从水稻(Oryza sativa L.) 的HPPD和与其具有高度同源性的玉米(Zea mays L.) 的HPPD的DNA改组文库中筛选得到了一种突变体MFRR-2, 突变体MFRR-2转基因水稻对甲基磺草酮的抗性是野生型HPPD水稻的1.5倍, 成功利用DNA改组定向进化提高了水稻的抗除草剂能力。

1.2 理性设计

蛋白质的理性设计是蛋白质工程经典方法之一。与随机突变方法相比, 理性设计聚焦于蛋白质序列中的特定位置, 这些位置必须基于先验知识确定, 如结构信息、序列比对、生化数据、功能、机制或基于计算机的预测^[21] (图 2A)。与随机突变方法相比, 理性设计的优点是可以显著减少突变体数量, 从而极大地降低盲目性和工作量, 并提高有益突变的概率^[22]。在没有高通量筛选方法的情况下, 这一优点尤为突出。β-内酰胺酶GNCA可以降解多种β-内酰胺类抗生素(如青霉素、头孢噻肟), 但催化效率较低, 而β-内酰胺酶TEM-1仅对青霉素类有降解活性, 且其活性比GNCA高约两个数量级。为了提高GNCA对青霉素的选择性和催化效率, Modi等^[23]通过理性设计的方法, 以TEM-1的功能残基为参考设计GNCA, 得到的具有21个位点突变的GNCA突变体不仅对青霉素的降解活性提高了3倍, 还对头孢噻肟的降解活性降低了1万倍, 选择性显著提高。

1.3 从头设计

从头设计是一种基于对天然蛋白质的序列、三维结构、反应机制或过渡状态等的了解, 从头设计和开发蛋白质的方法, 最终可以在体外合成蛋白质或合成其基因用于异源表达^[24] (图 2B)。最初, 从头设计的酶与目标性能往往不能完全匹配, 但随着计算方法的发展和人工智能(artificial intelligence, AI) 的应用, 在从头合成新蛋白质方面已取得了令人瞩目的进展。Baker团队^[25]利用Rosetta软件进行从头设计, 创造了一种新的蛋白质Neo-2/15, 它是白细胞介素2和15的类似物, 具有抗癌和治疗自身免疫性疾病的潜力。加州伯克利的一家公司采用类似ChatGPT的蛋白质工程深度学习语言模型ProGen, 首次实现了人工智能预测的蛋白质的合成^[26]。这些利用AI算法快速模拟、预测和优化的蛋白质结构与已知的蛋白质完全不同, 最低相似度甚至只有31.4%, 但它们与天然蛋白质同样具有活性, 并且最终设计出的蛋白质, 是通常需要数百年才能进化出来的高活性蛋白质。近日, Ingraham团队^[27]开发了一种名为Chroma的生成式AI模型, 该模型建立在扩散模型和图神经网络的框架上, 通过利用超过30亿年进化的蛋白质样本, 已从头生成了310个自然界中不存在的蛋白质。实验证明, 这些蛋白质不仅可以表达并折叠成一定的空间结构, 还具有良好的生物活性。这种能够利用AI技术从头开始创造功能性蛋白质的能力, 标志着正进入蛋白质从头设计的新时代。

1.4 定向进化及高通量筛选

传统的定向进化包括突变文库的生成和目标突变体的筛选^[28]。定向进化是指通过利用自然进化的原理, 快速选择出具有特定功能的蛋白质突变体^[29]。对目标蛋白反复诱变和筛选, 使用每轮中的最佳突变体作为下一轮突变的起点, 直至达到目标功能, 这种筛选技术依赖于在空间上分离每种突变体, 然后单独评估它们的活性。这种方法有效但繁琐, 尤其是在筛选较大的文库时耗时又费力。为了解决这一问题, 开发了更快、成本效益更高、自动化更强的高通量筛选方法^{[30, 31]}, 如平板筛选法、展示法、荧光筛选法、质谱法、红外检测法等, 极大提高了筛选效率以及准确性。Yang课题组^[32]利用细胞膜表面的半乳糖转运体对底物及糖基化产物之间通透性的差异, 建立了首个基于荧光激活细胞分选技术在单细胞层面检测岩藻糖基转移酶突变体的筛选体系, 筛选速度达到10⁷ h^-1以上, 一举打破了这类酶的筛选瓶颈, 成功获得了目前报道催化效率最高的岩藻糖基转移酶突变体。因此, 开发更快速、灵敏、准确的高通量筛选技术, 是提高蛋白质工程改造效率的关键。

2 蛋白质工程技术在药用天然产物合成生物学中的应用

收起

2.1 改善酶的性能

2.1.1 提高酶的催化活性

天然产物的生物合成过程离不开酶的催化作用(图 3A), 但酶的催化效率较低会极大地限制产物产量。蛋白质工程技术通过改变酶的结构, 可以对酶的性能进行改善。因此, 为了提高目标产物的产量, 可以通过蛋白质工程来提高酶的催化活性^[33] (图 3B)。

吡哆醇(图 4A) 生物合成过程中的限速酶PdxA和PdxJ的催化效率较低, 受到代谢途径的严格调控。Liu等^[34]采用基于结构的酶再设计方法对这两个限速酶进行改造, 通过这种方式有效解决了酶催化效率低的问题。利用这两个酶活性升高的突变体构建了一株大肠杆菌工程菌, 通过结合多模块迭代优化策略, 使吡哆醇产量达1.4 g·L^-1, 首次突破了克级水平。在圆柚酮(图 4B) 的工业化生产中, 异源酶在微生物底盘中表达活性不佳, 难以实现圆柚酮的生产。Gao课题组^[35]基于酶的催化环境和机制对限速倍半萜合酶CnVS进行理性设计, 得到活性提高60%的突变体M560L。将蛋白质工程与代谢途径优化和多拷贝整合方法等技术手段相结合, 成功构建了一株能够高效合成圆柚酮的酿酒酵母工程菌株。在5 L生物反应器中, 该菌株圆柚酮最高产量达到了804.96 mg·L^-1。此外, Rao课题组^[36]通过理性设计改造了L-肌肽(图 4C) 生物合成途径中的二肽酶, 使其产量增加了41.6%。随后, 他们应用转运体工程来进一步提高L-肌肽的产量, 在5 L生物反应器中, L-肌肽的摩尔转化率达到了49.3%, 产量达到了133.2 mmol·L^-1。

总的来说, 通过蛋白质工程对药用天然产物生物合成途径中的限速酶进行改造以提高其活性是提高药用天然产物产量的有效方法。除此之外, 还可以通过蛋白质工程降低支路代谢途径的酶活性来间接提高目标产物产量。例如, 在酿酒酵母中2, 3-氧化角鲨烯是三萜类化合物的前体, 但其在内源性羊毛甾醇合酶的催化下转化为羊毛甾醇, 从而影响三萜类化合物的生产。Huo课题组^[37]通过改造酿酒酵母底盘细胞中羊毛甾醇合酶的关键氨基酸残基以降低其活性, 将更多的碳通量导向三萜, 从而显著提高了酿酒工程菌中三萜类化合物的产量。

2.1.2 改变酶对底物的区域选择性

在酶的众多选择性中(如区域选择性、立体选择性), 最常见也最重要的便是区域选择性。由于酶对特定底物的区域选择性差, 可能导致目标化合物的转化效率低。通过蛋白质工程改变酶对底物的区域选择性, 可以有效地合成目标化合物甚至是非天然的新化合物, 并且能够避免不必要的副产物的生成, 促进资源的高效利用(图 3C)。

苯酚等芳香族化合物的区域选择性羟基化可以产生许多有价值的化学品, 但通常缺乏有效的催化剂。间烷基酚的对羟基化能够产生药物中间体间烷基苯-1, 4-二醇, 但没有天然的酶可以催化这一反应。单加氧酶P450BM3的羟基化催化具有广泛的底物范围。Li团队^[38]通过理性设计和定向进化获得的P450BM3具有高度区域选择性, 可以作为催化剂用于间烷基苯酚的对(p)-羟基化, 通过生物羟基化反应以高产率合成了药物中间体间烷基苯-1, 4-二醇(图 4D)。Qiao团队^[39]从药用植物黄芪(Astragalus membranaceus) 中鉴定了一种多功能糖基转移酶AmGT8, 可催化三萜3-/6-/2′-O-糖基化反应。为了提高其区域选择性, 研究人员利用半理性设计方法构建了一个小型突变体库, 发现突变体A394F、A394D和T131V分别特异性催化6-O、3-O和2′-O糖基化, 而突变体P192E提高了催化3-O木糖基化活性。通过组合使用AmGT8的突变体A394F、T131V和P192E并利用不同糖基供体, 成功合成了黄芪根中主要皂苷类成分——黄芪皂苷Ⅲ和Ⅳ (图 4E、F)。此外, 研究人员还通过基于结构的蛋白质工程对黄芪的区域特异性皂苷乙酰转移酶AmAT7-3进行了改良^[40]。黄芪甲苷是黄芪的主要药理活性成分, 但由于黄芪的化学成分复杂, 需要大量纯化才能获得特定的黄芪甲苷。因此, 有必要提高酶的区域选择性使其能够有效地合成目标化合物。AmAT7-3能催化黄芪皂苷Ⅳ的C3′/C4′-O-乙酰化以及其他7种重要的药用皂苷的乙酰化反应。研究人员通过蛋白质工程获得的突变体A310G和A310W能特异地乙酰化黄芪皂苷Ⅳ的C3′-O和C4′-O (图 4G、H), 这极大降低了生产成本, 为将来工业化生产特定的黄芪甲苷奠定了基础。

2.1.3 改变酶的底物范围

通过蛋白质工程扩大酶的底物范围, 使其能够接受更广泛的底物, 这有助于拓展天然产物的生物合成途径, 提高合成产物的多样性。并且, 由于过量表达酶会给细胞带来代谢负担, 会对生物合成产生负面影响^[41], 因此, 在多途径网络中通过蛋白质工程获得具有杂泛性的酶即扩大酶的底物范围(图 3D), 是减少过表达酶数量的有效途径。蛋白质工程可以有效提高酶对底物的活性, 但是设计并快速鉴别能够扩大底物范围的突变体是目前面临的重大挑战^[42]。

定向趋异进化是将生物合成途径中的不同底物引入相应通路酶的有力工具。Tang团队^[43]采用定向趋异进化策略, 通过蛋白质工程将单加氧酶HpaBC改造为可以同时催化酪醇和酪胺变为羟基酪胺(图 4I) 的酪醇/酪胺羟化酶, 以尽量减少宿主细胞中过表达的外源蛋白的数量。利用这个具有杂泛性的酶, 使酪醇和酪胺向羟基酪胺的两种生物合成途径同时进行, 显著提高了羟基酪胺的生物合成效率。而对于聚酮合酶(polyketide synthase, PKS) 而言, 将整个AT结构域替换为具有不同底物特异性的同源物是扩大底物范围和引入非天然底物的常见方法之一。Keasling团队^[44]通过KAL-AT-PAL1交换策略对罕见的AT结构域进行了研究, 重组的新型PKS对异丁基丙二酰-CoA、异戊基丙二酰-CoA、2-甲基丁基丙二酰-CoA和苯基丙二酰-CoA等罕见底物具有活性, 并成功合成了新型聚酮化合物, 可用于生产非天然聚酮化合物。

除了扩大底物范围, 蛋白质工程还可以通过提高酶对单一底物的特异性来实现目标化合物的高效合成(图 3E)。Shan团队^[45]在先前的研究中确定了将柚皮素(图 4J) 转化为橙皮素(图 4L) 的途径, 首先羟基化酶F3′H将柚皮素氧化为圣草酚(图 4K), 然后甲基转移酶F4′OMT将圣草酚转化为橙皮素。从胡椒薄荷(Mentha piperita) 中获得的甲基转移酶MpOMT具有比F4′OMT更高的活性, 然而, MpOMT更倾向于催化柚皮素而不是圣草酚。因此, 研究团队对MpOMT进行了半理性设计, 得到了突变体S142V, 该突变体催化圣草酚生成橙皮素效率高且副产物生成少, 和野生酶具有不同的底物偏好性。利用这个突变体并在大肠杆菌中重新构建了橙皮素的生物合成途径, 实现了橙皮素的高效合成。经过优化代谢路径和发酵条件, 橙皮素最终产量可达27.5 mg·L^-1。通过提高酶对底物的特异性, 可以有效且有选择性地转化目标底物, 从而减少副产物的生成, 提高产物的纯度和产率, 有助于加速新药研发的进程。

2.1.4 改变酶的产物谱

除了要保证酶对底物的特异性之外, 在药用天然产物的生物合成过程中, 分离制备成本高、目标化合物产率低也是一个必须要面对的现实问题。当一种酶催化单一底物生成多种产物时, 通过蛋白质工程提高酶对特定产物的生成率(图 3F), 可以减少原料的浪费, 降低生产成本, 最终实现资源的有效利用, 并使目标化合物更易分离纯化^[46]。

β-1, 3-低聚木糖(β-1, 3-木二糖和β-1, 3-木三糖) 已被报道具有多种生物活性, 如抗凝血活性、抗氧化活性和抗肿瘤活性^[47], 这两种低聚木糖主要通过木聚糖水解产生, 然而, 现有酶的水解产物中含有大量的单糖。Zhang团队^[48]应用蛋白质工程, 通过优化祖先序列重建^[49]和数据挖掘, 筛选水解产物中不含单糖的酶, 构建了一个自然界中不存在的新型β-1, 3-木聚糖酶AncXyl10, 其水解木聚糖只产生β-1, 3-木二糖(60.38%) 和β-1, 3-木三糖(39.62%), 而不产生单糖, 有利于制备高纯度的低聚糖。

另一个限制酶的应用范围的因素是酶的产物谱非常有限, 如何扩大酶的产物谱从而提高产物多样性是更前沿、更具挑战性的研究方向。环二肽合酶利用氨酰-tRNA底物可以合成二酮哌嗪骨架, 含有这类骨架的天然产物可以通过肠屏障和血脑屏障, 是重要的药物先导化合物。然而, 基于蛋白质工程改造环二肽合酶的成功案例非常有限, 部分原因在于缺乏高通量的产物分析方法。为了生产新的用于治疗和工业应用的二酮哌嗪衍生物, Zhang等^[50]应用无标记质谱筛选技术对环二肽合酶AlbC进行定向进化改造, 快速得到了F186L突变体, 其能催化合成野生酶无法产生的新二酮哌嗪分子, 扩大了该酶的产物谱, 丰富了二酮哌嗪类天然产物的多样性。这不仅加速了酶定向进化的过程, 也使得研究人员能够更好地理解酶的催化机制, 同时新产物可能具备更好的药物性能, 为开发更有效的药物先导化合物提供了新的途径。

大麻素是大麻中的有效活性成分, 其中Δ⁹-四氢大麻酚(图 4M) 和大麻二酚(图 4N) 是最重要的大麻素, 具有镇痛、止吐、抗炎和抗癫痫的生物活性^[51]。在大麻素的生物合成途径中, 首先在四酮合酶TKS和橄榄酸环化酶OAC的作用下完成橄榄酸的合成, 再转移上异戊烯基单元, 最终通过环化得到大麻素。大麻素的间苯二酚烷基链是一个重要的药效团, 其链长可以以长度依赖的方式增加与大麻素受体的亲和力。因此, 对间苯二酚烷基链进行结构修饰, 有可能得到更多具有应用前景的活性大麻素类化合物。Morita课题组^[52]通过定向进化的方式对OAC和TKS进行蛋白质工程改造, 扩大了其产物谱, 使其催化具有高达11个C的线性脂肪酰基片段的大麻素核心的形成, 进而完成橄榄酸衍生物的生成。这为药物研发领域提供了一种新策略, 即通过蛋白质工程改造酶的催化性能, 实现对药效团的定向合成, 从而扩大产物谱以获得更多具有活性的化合物(图 3G), 为新药研发提供更多选择和可能性。

2.1.5 提高酶对pH的适应性

利用不同来源的酶在宿主细胞中重建生物合成途径时, 除了关注酶的活性、选择性等自身特性外, 还应该关注酶-宿主的相容性, 即宿主细胞的环境是否是酶的最适反应条件^[53]。当在谷氨酸棒杆菌(Corynebacterium glutamicum) 中生产γ-氨基丁酸(gamma-aminobutyrate, GABA, 图 4O) 时, 来源于大肠杆菌的谷氨酸脱羧酶的最适反应pH为4.5, 而谷氨酸棒状杆菌的细胞生长最适pH为7。为了解决这一问题, Choi等^[54]利用蛋白质工程技术, 通过去除谷氨酸脱羧酶的C端区域和引入E89Q的点突变来改变谷氨酸脱羧酶的pH偏好, 该谷氨酸脱羧酶突变体可在谷氨酸棒杆菌中高效表达, 经补料分批发酵, GABA产量为38 g·L^-1。

除了关注酶-宿主的相容性, 还需要关注保持酶活性和保持产物稳定的最适pH之间的区别。氨基葡萄糖(glucosamine, GlcN) (图 4P) 是一种天然的水溶性单糖, 能修复和保护软骨组织, 刺激软骨细胞生长, 促进软骨形成^[55]。通过生物催化, 利用N-乙酰氨基葡萄糖(N-acetylglucosamine, GlcNAc) 作为底物制备GlcN, 这一方法因其原料丰富且环境友好而受到广泛关注。来源于掘越氏火球菌(Pyrococcus horikoshii) 的几丁二糖脱乙酰酶PhDac对GlcNAc具有脱乙酰活性, 其最适温度为75 ℃, 最适pH值为7.5, 但GlcN在pH低于4.8时才能保持稳定, 且高温会加速GlcN的降解。因此, Huang等^[56]首先基于定点饱和诱变得到了催化效率提高2.21倍的突变体M14, 又通过表面电荷修饰来降低M14的最适pH, 最终获得的突变体M20最适pH降至6.0, 在低pH条件下保持较高的催化效率, 最终在pH 6.0、温度40 ℃条件下, GlcNAc转化为GlcN的转化率为94.7%。该结果提高了PhDac的催化效率, 促进了生物合成GlcN在工业生产中的应用。

谷氨酸脱氢酶(glutamate dehydrogenase, GDH) 催化L-谷氨酸氧化脱氨生成α-酮戊二酸(图 4Q), 枯草芽孢杆菌来源的谷氨酸脱氢酶BsGDH具有氧化和还原双向酶活性, 最适催化温度分别为60 ℃和65 ℃, 最适催化pH分别为8.0和7.5, 但在弱碱环境下(pH 7.0~8.0) 该酶稳定性较差。为提高BsGDH在催化合成α-酮戊二酸应用中的经济性, Wu等^[57]基于蛋白表面电荷工程, 对该酶进行分子改造, 以期将其催化最适pH调至其稳定性较高的中性环境。研究人员首先通过理性设计选择7个突变候选位点, 结果显示突变体N16D和K218D催化谷氨酸合成α-酮戊二酸的最适pH由8.0下调至7.0, 并且在pH 7.0条件下酶活性比野生型分别提高2.9倍和5.4倍, 成功将BsGDH最适催化pH范围下调至其稳定性良好的pH范围, 为通过生物催化工业化生产α-酮戊二酸奠定了基础。由此可见, 通过蛋白质工程改变酶自身的反应条件如pH从而提高其催化活性也是通过生物合成方法高效生产药用天然产物的重要途径。

2.1.6 提高酶对温度的适应性

在工业生产中往往需要高温来提高底物溶解度, 但高温易导致酶迅速变性失活, 因此需要获得热稳定性增强的酶^[30]。甘油葡萄糖苷(2-O-α-D-glucosylglycerol, αGG) (图 4R) 是由一个甘油分子和一个葡萄糖分子通过糖苷键连接而成的一种糖苷类化合物, 是化妆品中的重要保湿成分、细胞保护渗透剂^[58]及蛋白质稳定剂^[59]。蔗糖磷酸化酶是一种转糖苷酶, 能催化蔗糖上的葡萄糖基转移到受体上, 但现有的蔗糖磷酸化酶具有热稳定性低的问题。为提高其热稳定性, Yang等^[60]采用半理性设计的方式构建筛选突变体。在50 ℃下测定残余酶活性获得耐热性和活性高的突变体V23L和S424R, 此外还构建了V23L/S424R组合型突变体, 其半衰期是野生型的2倍。在优化蔗糖磷酸化酶催化蔗糖和甘油产生甘油葡萄糖苷的反应条件后, 突变体V23L/S424R对蔗糖的转化率提高至76.3%, 实现了αGG的高水平合成。

硫酸软骨素A (chondroitin sulfate A, CSA) (图 4S) 是由软骨素通过硫转移酶介导的催化作用合成的, 被广泛用于抑制炎症^[61]、调节免疫^[62]和促进大脑发育^[63], 市场需求量很大。然而, 目前的合成方法受限于昂贵的硫酸基供体PAPS和低效的硫转移酶11 (CHST11)。Liu等^[64]利用基于机制的蛋白质工程技术, 提高了CHST11的热稳定性和催化效率, 使其T_m值和半衰期分别提高了6.9 ℃和3.5 h, 比活性提高了2.1倍, 并通过表面展示技术实现了CHST11的外膜表达, 构建了转化率为89.5%的CSA催化体系。这种催化工艺为CSA的工业化生产提供了一种很有前途的方法。辛伐他汀(图 4T) 是一种重要的降胆固醇药物, 一般通过化学或生物方法由中间体monacolin J转化而来, 而中间体monacolin J又由洛伐他汀水解酶PcEST水解洛伐他汀(图 4U) 而得到, Liang等^[65]通过结构分析对其进行蛋白质工程改造, 得到了一个热稳定更好的PcEST突变体D106A, 表明了通过蛋白质工程改造的洛伐他汀水解酶PcEST在辛伐他汀工业化生产方面具有较大的潜力。

利用蛋白质工程提高酶的热稳定性不仅意味着酶能在更高温度下保持活性进行天然产物生物转化, 较高温度还可以使底物分子具有更高的动力学能量和更频繁的碰撞机会, 而且酶的热稳定性提高后可以在比较宽泛的温度下进行反应, 从而适用不同的工艺要求, 有利于减少药用天然产物生物合成的生产成本。

2.1.7 缓解酶的底物或产物抑制

酶的底物或产物浓度抑制是酶重要的调节机制之一, 许多代谢步骤都受高浓度的底物或产物抑制^[66], 这也是通过微生物工厂生产药用天然产物的主要障碍之一。目前已通过蛋白质工程减少或消除了一些酶的底物及产物抑制作用, 并已成功地用于许多药用天然产物的生物合成。

Sephanopoulo课题组^[67]发现, 利用解脂耶氏酵母(Yarrowia lipolytica) 生物合成β-胡萝卜素(图 4V) 时, 底物番茄红素对番茄红素环化酶的抑制限制了β-胡萝卜素的产量。为了克服这一瓶颈, 他们通过结构导向的蛋白质工程得到了一个突变体Y27R, 其不再受底物抑制且保持了酶活性, 并结合代谢通量的调节, 使β-胡萝卜素的产量比初始菌株提高了1 441倍。

β-丙氨酸是生物体生长所必需的一种非蛋白氨基酸, 一般通过L-天冬氨酸-α-脱羧酶(L-aspartate-α-decarboxylase, ADC) 催化L-天冬氨酸脱羧生成β-丙氨酸。然而, 来源于谷氨酸棒杆菌的ADC在底物L-天冬氨酸浓度较高时, 全细胞生物催化系统会发生底物抑制, 这限制了β-丙氨酸的高产。Wang等^[68]采用epPCR方法建立突变文库, 从2 000个突变酶库中获得两个高活性突变体菌株panD-56 (ADCR12V)和panD-134 (ADCQ17A)。检测结果显示, 当L-天冬氨酸浓度达到100 g·L^-1时, 菌株panD-56的产率比野生型高出125%, 表明底物抑制获得极大缓解, 该突变体具有很强的工业生物催化生产β-丙氨酸应用潜力。

3-脱氧-阿拉伯庚酮糖-7-磷酸(3-deoxy-D-arabinoheptulosonate-7-phosphate, DAHP) 合酶是莽草酸途径的第一个关键酶, 主要受到下游产物酪氨酸和苯丙氨酸的反馈抑制, 从而限制了莽草酸途径的碳通量。酿酒酵母中存在两个DAHP合酶: Aro4和Aro3, 分别受到酪氨酸和苯丙氨酸的反馈抑制。Luo团队^[69]通过蛋白质工程得到了Aro3的D154N突变体, 其能够有效缓解苯丙氨酸的反馈抑制, 过表达D154N突变体可明显提高酵母中莽草酸途径的碳通量。这一解除反馈抑制突变策略推广至其他非模式酵母的DAHP合酶时也能有效缓解其反馈抑制。最后作者将D154N突变体应用于酪醇和红景天苷(图 4W) 的微生物生产, 酪醇和红景天苷产量分别提高了29.1%和43.6%, 尤其是红景天苷的产量达到2.4 g·L^-1, 是摇瓶发酵中报道的最高水平, 充分展示了这种反馈解锁策略在工业生产中的潜力。

2.1.8 提高酶的可溶性表达水平

优化细胞中的蛋白质折叠从而提高酶的可溶性表达, 有助于目标化合物的生物合成。改善蛋白质在体内折叠的最简单和最通用的方法是优化细胞生长和蛋白表达条件, 包括生长温度、诱导时间、启动子强度、诱导剂浓度和融合标签使用^[70], 但这些方法往往不足以将蛋白质的溶解度提高到所需的程度, 这是因为酶整体性质仍然受到天然序列的限制。因此, 通过蛋白质工程来改善酶的可溶性表达是一种更具有针对性的方法^[71]。

LovD是一种在洛伐他汀生物合成途径中发现的酰基转移酶, 在大肠杆菌中异源表达时, 一半以上的蛋白质以包涵体形式存在, 这阻碍了重组大肠杆菌细胞的生物催化活性。Xie等^[72]在LovD中发现了两个表面暴露的Cys40和Cys60残基, 它们形成的二硫键不是该酶活性所必需的, 将这两个位点进行突变, 显著提高了蛋白质的溶解度和全细胞生物催化活性, 其中C40A和C60N突变分别使全细胞催化活性增加27%和26%。

菊糖酶是一种果糖呋喃基水解酶, 可以将菊糖水解成果糖、葡萄糖和菊糖寡糖。Yadid课题组^[73]利用定向进化提高菊糖酶的表达量和活性, 得到的突变体酶的可溶性表达量比野生型酶提高了5倍, 为菊糖寡糖的工业化生产奠定了基础。分子伴侣可以改善蛋白质的折叠, 因此特异性地优化分子伴侣的表达也可以促进目标蛋白的可溶性表达。Wang等^[74]通过DNA改组技术得到了分子伴侣GroEL/S的突变体, 这些突变体增加了折叠腔的极性, 可以促进重组蛋白酶折叠并可溶性表达。

通过蛋白质工程改善酶性能的实例见表 1^{[34-40, 43-45, 48, 50, 52, 54, 56, 57, 60, 64, 65, 67-69, 72-74]}。

2.2 改善酶的作用环境

除了改善酶的特性, 蛋白质工程还可以为生物合成代谢提供更好的反应环境, 促进生物合成途径进行。蛋白质支架工程是一种利用结构信息(如蛋白质序列和接头结构域) 在空间上组织参与顺序反应的酶的方法。人参皂苷的生物合成途径中, 角鲨烯环氧化酶和达玛烯二醇-Ⅱ合成酶是决定达玛烷型人参皂苷前体达玛烯二醇-Ⅱ产率的两个关键酶。Ren课题组^[75]通过基于蛋白-蛋白相互作用的自组装, 开发了一种在毕赤酵母中促进达玛烯二醇-Ⅱ生物合成的策略。通过该方法成功实现了两种酶在细胞中的共定位, 使达玛烯二醇-Ⅱ的产率提高了2.1倍。

除了使催化连续反应的酶在空间上彼此靠近, 构建底物通道、制造多酶复合物也是提高酶在体内催化效率的有效途径。Wang课题组^[76]通过自组装在体内构建了多酶复合物反应器, 这种多酶复合物系统可以增加底物的局部浓度, 减少中间体的传递时间, 更好地增加代谢通量, 从而提高了黄芩素(图 4X) 和黄芩苷(图 4Y) 在大肠杆菌中的产量。Wang等^[77]发现蛋白质CipA和CipB在发光杆菌(Photorhabdus luminescens) 细胞质中会形成蛋白结晶包涵体, 其可以作为支架材料有效地将外源蛋白整合到包涵体中。为了进一步探索其在体内的应用, 研究人员利用CipA聚合了紫色杆菌素(图 4Z) 生物合成途径中的多种酶形成多酶复合物。结果显示, 与分散在溶液中的酶相比, 形成的多酶复合物催化产生的紫色杆菌素产量显著提高, 且副产物较少。这些结果表明了通过蛋白质工程连接多种酶在提高细胞工厂生产天然产物产量方面的优势和潜力。

2.3 促进合成生物学工具的开发

蛋白质工程在某种意义上还促进了合成生物学工具的发展, 这些工具又进一步增强了天然产物的生物合成^[78]。在合成生物学领域中, 各种各样的蛋白质工具扮演着重要的角色。通过蛋白质工程, 可以对蛋白质的结构和序列进行精准调控和改造, 进而创造出具备全新功能和特性的蛋白质工具, 为合成生物学工具的开发与创新提供了强有力的支撑。

蛋白质工程可以借助基于调控的生物传感器实现代谢途径的动态控制。变构转录因子(allosteric transcription factors, ATF) 是细菌转录调节网络中的重要组成部分, 其通过结合特定的小分子改变构象, 使其能够结合特定的DNA序列或从DNA上解离。RolR是一种变构转录因子, 它参与间苯二酚的代谢, 适合作为生物传感器开发。Kwan团队^[79]利用定向进化和高通量筛选的方法, 成功地将RolR进化为能够对多种不同芳香化合物产生特异响应的生物传感器突变体。这些工程化的生物传感器突变体对多种与代谢工程路径相关的中间产物或最终产物具有响应性, 例如邻苯二酚、左旋多巴、咖啡酸和甲基邻苯二酚等。此研究通过设计针对特定分子的ATF, 实现对多种分子的诱导和抑制响应, 扩展了代谢工程领域中动态控制生物传感器工具箱的应用, 为天然产物合成生物学带来了新的可能性。

生物传感器的动态控制也可以很好地应用在有毒化合物的生物合成过程中。为了平衡细胞生长和产物合成, Liang等^[80]通过定向进化开发出一种新的控制香兰素生物合成途径的调控蛋白, 该蛋白对产物香兰素和底物阿魏酸均有不同的响应能力, 反馈激活与级联动态控制相结合, 保证了菌株的充分生长和香兰素的产量, 显示了蛋白质工程在天然产物合成生物学中广泛应用的可能性。

众所周知, CRISPR-Cas9系统是强大的基因编辑工具, 来自化脓性链球菌(Streptococcus pyogenes) 的Cas9蛋白SpyCas9在gRNA的指导下识别结合目标DNA序列, 切割DNA产生双链断裂。SpyCas9蛋白在错配识别方面存在局限性, 但提高其特异性会影响蛋白活性。Rabinowitz等^[81]利用计算蛋白质工程平台, 模拟预测了SpyCas9的九个候选高保真突变体, 其中N692V实现了高活性和低脱靶的平衡, 且其几乎不会导致非特异性DNA损伤, 这一Cas9突变体在精确编辑基因方面具有巨大的应用潜力。

这些通过蛋白质工程改造得到的工具可以使合成生物学中的代谢工程、酶工程等更加简便和高效, 特别是ATF已被用作各种高通量生物传感器平台的基础, 以提高微生物菌株的优化效率。

3 结论与展望

收起

综上所述, 蛋白质工程技术在药用天然产物合成生物学中的应用具有广阔的发展前景。长期以来, 得到足够多具有良好生物活性的天然产物是药学界不断追求的目标。通过对蛋白质的结构和功能进行改造, 可以创造出具有理想特性的新型蛋白质, 从而实现药用天然产物的高效合成。这项技术不仅可以应用于药用天然产物合成生物学, 解决药用天然产物资源稀缺和含量低等问题, 还为新药研发提供了新的思路和工具。

然而, 蛋白质工程技术在药用天然产物合成生物学中的应用还面临一些亟待解决的挑战和问题。例如, 由于目前只有少数蛋白质的晶体结构被解析、缺乏详尽的结构认知, 对于蛋白质的理性设计存在一定的困难, 实际操作中多数酶在突变后的表现不尽人意。尽管人工智能技术的发展如AlphaFold2的问世为高精度建模提供了支撑, 为蛋白质工程在天然产物合成生物学中的应用提供了一定的指导, 但这些方法也具有局限性, 特别是面对较大的蛋白和结构复杂的化合物时, 序列-结构-功能关系往往难以准确预测。如何更好地预测和设计蛋白质的结构和功能、如何提高其对复杂天然产物的合成效率和选择性, 仍然是需要长期探索的问题。

此外, 尽管现代分子生物学方法和人工智能辅助计算工具使得获取大规模的突变文库相对容易, 但由于缺乏特异性高通量筛选方法, 快速简便地鉴定突变文库仍然是一个主要的难题。因此, 需要加快研发更多应用自动化设备和液相处理系统的高通量筛选方法, 以实现筛选过程的快速和高效。在未来的研究中需要不断探索和创新, 加强学科交叉合作, 抓住人工智能的发展机遇, 共同致力于解决这些问题, 以推动蛋白质工程技术在药用天然产物合成生物学中的应用进程。

根据Grand View Research, Inc.的一份报告^[82], 预计到2030年, 蛋白质工程市场规模将达到76.2亿美元。随着蛋白质工程技术的不断发展, 可以预见其在药用天然产物合成生物学领域乃至整个医药领域将出现更多的应用。首先, 进一步提高蛋白质工程技术的精准度和效率, 将为合成生物学的研究和实践提供更可靠和高效的工具。其次, 结合人工智能等新兴技术, 可以更好地设计和优化蛋白质的结构和功能, 从而进一步提升药用天然产物的合成效率, 并为新药研发带来重大突破和创新。

作者贡献: 孙晓燕负责调研文献、整理资料、执笔、作图; 杨金玲和朱平负责指导和修改; 陈晶晶、陈天娇和巩婷负责修改和审校。

利益冲突: 所有作者均不存在利益冲突。

基金

收起

“十四五”国家重点研发计划(2022YFF1100300)
国家自然科学基金资助项目(81673341)
北京市自然科学基金面上资助项目(7212158)

参考文献

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2024年第59卷第6期

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doi: 10.16438/j.0513-4870.2024-0105

接收时间：2024-02-01
首发时间：2025-11-26
出版时间：2024-06-12

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收稿日期：2024-02-01
修回日期：2024-03-11

基金

“十四五”国家重点研发计划(2022YFF1100300)

国家自然科学基金资助项目(81673341)

北京市自然科学基金面上资助项目(7212158)

作者信息

通讯作者:

^*杨金玲, Tel: 86-10-63165199, E-mail: yangjl@imm.ac.cn;

朱平, Tel: 86-10-63165199, E-mail: zhuping@imm.ac.cn

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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

Application	Chassis	Enzyme	Substrate	Product	Mutation	Result	Ref.
Improve the catalytic activity of enzymes	E. coli	PdxA, PdxJ	4-Phosphohydroxy-L-threonine, 3-phosphohydroxy-1-aminoacetone	Pyridoxine	PdxA2 (H136N), PdxJ1 (E104T/I218L/G194C)	Using two mutants combined with metabolic engineering to construct an engineering strain, the production of pyridoxine was increased to 1.4 g·L^-1	[34]
	S. cerevisiae	CnVS	Farnesyl pyrophosphate	Valencene	M560L	The production of (+)‐nootkatone in the strain constructed by utilizing mutants and combining with metabolic engineering increased to 804.96 mg·L^-1	[35]
	E. coli	SmpepD	β-Ala, L-His	L-Carnosine	T168S/G148D	A strain was constructed using this mutant and transporter engineering, resulting in an increase in the production yield of L-carnosine to 133.2 mmol·L^-1	[36]
	S. cerevisiae	ERG7	2, 3-Oxidosqualene	Lanosterol	F699T, I705K	Replacing wild-type ERG7 in yeast with mutants with reduced activity reduces lanosterol production and indirectly increases triterpenoid production	[37]
Changing the regioselectivity of enzymes towards substrates	E. coli	P450BM3	m-Alkylphenols	m-Alkylbenzene-1, 4-diols	R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V, R47L/Y51F/F87V/L181Q/L188P/I401P, R47I/F87V/L188P	Coexpression of mutants and cofactors in E. coli and biocatalysis for efficient synthesis of m-alkylbenzene-1, 4-diol	[38]
	—	AmGT8	Cycloastragenol, UDP-Glu	Cycloastragenol-3/6-O-glycoside, cycloastragenol-3-(2′-O-glycoside)-O-glycoside	A394F, T131V, P192E	By combining different mutants and utilizing different glycosyl donors, it synthesized the main saponin component, astragaloside Ⅲ and Ⅳ	[39]
	—	AmAT7-3	Astragaloside Ⅳ, UDP-Xyl	Isoastragaloside Ⅱ, cyclocephaloside Ⅱ, C3′, C4′-O acetylated astragaloside Ⅳ	A310G, A310W	Specific C3′-O and C4′-O acetylation of astragaloside Ⅳ was successfully achieved	[40]
Changing the substrate range of enzymes	E. coli	HpaBC	Tyrosol	Hydroxytyrosol	—	Mutant HpaBC can simultaneously catalyze tyrosol/tyramine hydroxylase, significantly improving the efficiency of hydroxytyramine biosynthesis	[43]
	E. coli	DEBS	(2S, 3R)-3-Hydroxy-2-methylpentanoic acid-SNAC thioester, methylmalonyl-CoA	(4S, 5R)-3-Oxo-2, 4-dimethyl-5-hydroxy-heptanoic acid-D-lactone	—	A novel polyketide compound was successfully synthesized	[44]
	E. coli	MpOMT	(2S)-Naringenin, (2S)-eriodictyol	(2S)-Eriodictyol, (2S)-hesperetin	S142V	The mutant S142V can mainly catalyzes (2S) - estrictyl, and its yield increased to 27.5 mg·L^-1 in E. coli	[45]
Changing the product spectrum of enzymes	—	β-1, 3-Xylanase	β-1, 3-Xylan	Xylose, β-1, 3-xylobiose, β-1, 3-xylotriose	—	Unnatral β-1, 3-xylanase AncXyl10 hydrolyzes xylose and only produces β-1, 3-xylobiose and β-1, 3-xylotriose without producing xylose	[48]
	E. coli	AlbC	aa-tRNAs	Diketopiperazine	F186L	F186L mutant can catalyze the synthesis of new diketopiperazine molecules that cannot be produced by wild enzymes	[50]
	—	OAC TKS	Malonyl, pentyltetra-β-ketide-CoA	Pentyltetra-β-ketide-CoA, olivetolic acid	OAC (F24I) TKS (L190G)	OAC and TKS mutants can catalyze the formation of cannabinoid cores with linear fatty acyl segments of up to 11 Cs	[52]
Improving the adaptability of enzymes to pH	C. glutamicum	GAD	L-Glutamate	GABA	E89Q and C-terminal deletion	Changed the pH preference of glutamic acid decarboxylase, resulting in a GABA production of 38 g·L^-1 in C. glutamicum	[54]
	—	PhDac	GlcNAc	GlcN	G74D/H152E/W232A/Q29E/K106E/N176ER221E/L271E	Mutant M20 maintains high catalytic efficiency under low pH conditions	[56]
	—	BsGDH	L-Glutamate	α-Ketoglutaric acid	N16D, K218D	Lowering the optimal catalytic pH range of BsGDH to its stable pH range	[57]
Improving the adaptability of enzymes to temperature	—	LmSP	Sucrose, glycerol	αGG	V23L/S424R	The half-life of the mutant at 50 ℃ is twice that of the wild-type, and the sucrose conversion rate is 76.3%	[60]
	E. coli	CHST11	Chondroitin, PAPS	CSA	E114D/A159N/P170E/I67D/L134V/I206T/K218R/S286A/S297D/Y298S/Y304S/A305G/T316A	The T_m and half-life of the mutant were increased by 6.9 ℃ and 3.5 h, respectively, and a whole cell CSA catalytic system with a conversion rate of 89.5% was established	[64]
	—	PcEST	Lovastatin	Monacolin J	D106A	Mutant D106A has better solubility and thermal stability	[65]
Relieve inhibition of substrate or product concentration	Y. lipolytica	CarRP	Geranylgeranyl pyrophosphate, lycopene	Phytoene, β-carotene	Y27R	The mutant Y27R is no longer inhibited by substrates and maintains enzyme activity	[67]
	E. coli	ADC	L-Aspartic acid	β-Alanine	R12V	When the concentration of L-aspartic acid reached 100 g·L^-1, the mutant yield remained at 0.45 g·g^-1, and substrate inhibition was greatly alleviated	[68]
	S. cerevisiae	Aro3	Erythrose 4-phosphate, phosphoenol pyruvate	DAHP	D154N	The D154N mutant can effectively alleviate feedback inhibition of phenylalanine, and the production of salidroside reaches 2.4 g·L^-1	[69]
Improve the soluble expression of enzymes	E. coli	LovD	Monacolin J	Simvastatin	C40A, C60N	Mutants C40A and C60N significantly increased protein solubility, resulting in a 27% and 26% increase in catalytic activity	[72]
	—	reTP	Inulin	Fructose, glucose, inulooligosaccharides	Y128H/A316T/E344K/T504M	The expression level of soluble enzymes in the mutant is five times higher than that of wild enzymes	[73]
	—	GroEL/S	—	—	GroES (Y71H, V26A/Y71R, V26A/Y71H/V83M), GroEL (A145V/D490G, A163V/D490G, D188G/M488V)	Mutants of GroEL/S can better promote the folding and soluble expression of recombinant proteins	[74]

Application

Chassis

Enzyme

Substrate

Product

Mutation

Result

Ref.

Improve the catalytic activity of enzymes

E. coli

PdxA, PdxJ

4-Phosphohydroxy-L-threonine, 3-phosphohydroxy-1-aminoacetone

Pyridoxine

PdxA2 (H136N), PdxJ1 (E104T/I218L/G194C)

Using two mutants combined with metabolic engineering to construct an engineering strain, the production of pyridoxine was increased to 1.4 g·L^-1

[34]

S. cerevisiae

CnVS

Farnesyl pyrophosphate

Valencene

M560L

The production of (+)‐nootkatone in the strain constructed by utilizing mutants and combining with metabolic engineering increased to 804.96 mg·L^-1

[35]

E. coli

SmpepD

β-Ala, L-His

L-Carnosine

T168S/G148D

A strain was constructed using this mutant and transporter engineering, resulting in an increase in the production yield of L-carnosine to 133.2 mmol·L^-1

[36]

S. cerevisiae

ERG7

2, 3-Oxidosqualene

Lanosterol

F699T, I705K

Replacing wild-type ERG7 in yeast with mutants with reduced activity reduces lanosterol production and indirectly increases triterpenoid production

[37]

Changing the regioselectivity of enzymes towards substrates

E. coli

P450BM3

m-Alkylphenols

m-Alkylbenzene-1, 4-diols

R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V, R47L/Y51F/F87V/L181Q/L188P/I401P, R47I/F87V/L188P

Coexpression of mutants and cofactors in E. coli and biocatalysis for efficient synthesis of m-alkylbenzene-1, 4-diol

[38]

—

AmGT8

Cycloastragenol, UDP-Glu

Cycloastragenol-3/6-O-glycoside, cycloastragenol-3-(2′-O-glycoside)-O-glycoside

A394F, T131V, P192E

By combining different mutants and utilizing different glycosyl donors, it synthesized the main saponin component, astragaloside Ⅲ and Ⅳ

[39]

—

AmAT7-3

Astragaloside Ⅳ, UDP-Xyl

Isoastragaloside Ⅱ, cyclocephaloside Ⅱ, C3′, C4′-O acetylated astragaloside Ⅳ

A310G, A310W

Specific C3′-O and C4′-O acetylation of astragaloside Ⅳ was successfully achieved

[40]

Changing the substrate range of enzymes

E. coli

HpaBC

Tyrosol

Hydroxytyrosol

—

Mutant HpaBC can simultaneously catalyze tyrosol/tyramine hydroxylase, significantly improving the efficiency of hydroxytyramine biosynthesis

[43]

E. coli

DEBS

(2S, 3R)-3-Hydroxy-2-methylpentanoic acid-SNAC thioester, methylmalonyl-CoA

(4S, 5R)-3-Oxo-2, 4-dimethyl-5-hydroxy-heptanoic acid-D-lactone

—

A novel polyketide compound was successfully synthesized

[44]

E. coli

MpOMT

(2S)-Naringenin, (2S)-eriodictyol

(2S)-Eriodictyol, (2S)-hesperetin

S142V

The mutant S142V can mainly catalyzes (2S) - estrictyl, and its yield increased to 27.5 mg·L^-1 in E. coli

[45]

Changing the product spectrum of enzymes

—

β-1, 3-Xylanase

β-1, 3-Xylan

Xylose, β-1, 3-xylobiose, β-1, 3-xylotriose

—

Unnatral β-1, 3-xylanase AncXyl10 hydrolyzes xylose and only produces β-1, 3-xylobiose and β-1, 3-xylotriose without producing xylose

[48]

E. coli

AlbC

aa-tRNAs

Diketopiperazine

F186L

F186L mutant can catalyze the synthesis of new diketopiperazine molecules that cannot be produced by wild enzymes

[50]

—

OAC
TKS

Malonyl, pentyltetra-β-ketide-CoA

Pentyltetra-β-ketide-CoA, olivetolic acid

OAC (F24I)
TKS (L190G)

OAC and TKS mutants can catalyze the formation of cannabinoid cores with linear fatty acyl segments of up to 11 Cs

[52]

Improving the adaptability of enzymes to pH

C. glutamicum

GAD

L-Glutamate

GABA

E89Q and C-terminal deletion

Changed the pH preference of glutamic acid decarboxylase, resulting in a GABA production of 38 g·L^-1 in C. glutamicum

[54]

—

PhDac

GlcNAc

GlcN

G74D/H152E/W232A/Q29E/K106E/N176ER221E/L271E

Mutant M20 maintains high catalytic efficiency under low pH conditions

[56]

—

BsGDH

L-Glutamate

α-Ketoglutaric acid

N16D, K218D

Lowering the optimal catalytic pH range of BsGDH to its stable pH range

[57]

Improving the adaptability of enzymes to temperature

—

LmSP

Sucrose, glycerol

αGG

V23L/S424R

The half-life of the mutant at 50 ℃ is twice that of the wild-type, and the sucrose conversion rate is 76.3%

[60]

E. coli

CHST11

Chondroitin, PAPS

CSA

E114D/A159N/P170E/I67D/L134V/I206T/K218R/S286A/S297D/Y298S/Y304S/A305G/T316A

The T_m and half-life of the mutant were increased by 6.9 ℃ and 3.5 h, respectively, and a whole cell CSA catalytic system with a conversion rate of 89.5% was established

[64]

—

PcEST

Lovastatin

Monacolin J

D106A

Mutant D106A has better solubility and thermal stability

[65]

Relieve inhibition of substrate or product concentration

Y. lipolytica

CarRP

Geranylgeranyl pyrophosphate, lycopene

Phytoene, β-carotene

Y27R

The mutant Y27R is no longer inhibited by substrates and maintains enzyme activity

[67]

E. coli

ADC

L-Aspartic acid

β-Alanine

R12V

When the concentration of L-aspartic acid reached 100 g·L^-1, the mutant yield remained at 0.45 g·g^-1, and substrate inhibition was greatly alleviated

[68]

S. cerevisiae

Aro3

Erythrose 4-phosphate, phosphoenol pyruvate

DAHP

D154N

The D154N mutant can effectively alleviate feedback inhibition of phenylalanine, and the production of salidroside reaches 2.4 g·L^-1

[69]

Improve the soluble expression of enzymes

E. coli

LovD

Monacolin J

Simvastatin

C40A, C60N

Mutants C40A and C60N significantly increased protein solubility, resulting in a 27% and 26% increase in catalytic activity

[72]

—

reTP

Inulin

Fructose, glucose, inulooligosaccharides

Y128H/A316T/E344K/T504M

The expression level of soluble enzymes in the mutant is five times higher than that of wild enzymes

[73]

—

GroEL/S

—

GroES (Y71H, V26A/Y71R, V26A/Y71H/V83M), GroEL (A145V/D490G, A163V/D490G, D188G/M488V)

Mutants of GroEL/S can better promote the folding and soluble expression of recombinant proteins

[74]