环脂肽生物合成的研究进展

doi:10.12211/2096-8280.2021-008

摘要/Abstract

摘要：

环脂肽化合物是一类结构新颖的环状肽类，其两亲性的物化特性决定了其独特的生物活性，可作为抗生素、生物表面活性剂等。在生物防治、药物开发、环境修复和疾病治疗等方面广泛应用，具有迫切的市场需求和广阔的发展前景。环脂肽类天然产物主要由非核糖体肽合成途径合成，由于环脂肽合成复杂的代谢网络和前体需求、专一且严格的合成途径、多种同系物的共存，制约着环脂肽合成的微生物开发和产品价值提升。本文主要介绍了来源于细菌界的环脂肽类物质的结构特性，非核糖体肽合成途径及非核糖体肽合成酶（non-ribosomal peptide synthetase，NRPS）的结构域特点，天然产物底盘菌株开发现状，通过基因工程、代谢工程方法进行同系物调控和生物合成策略，混菌对脂肽生物合成的影响，以及合成生物学在脂肽合成中的应用。随着合成生物技术的迅速发展和运用，环脂肽类天然产物的微生物合成也有望实现“质”和“量”的提升，以及促进新型脂肽的开发。

关键词: 环脂肽, 非核糖体肽合成途径, NRPS, 模块结构域, 同系物调控, 合成生物学

Abstract:

As antibiotics and bio-surfactants,cyclic lipopeptides have unique molecular structure and biological activity and are widely applied in the fields of biological control, drug development, environmental remediation and disease treatment. It has vigorous market demand and promising future. Cyclic lipopeptides are a class of antibiotics synthesized from non-ribosomal peptide pathways by microorganisms. However, the complex metabolic network and precursor requirements, specific and strict synthetic pathway, and the coexistence of multiple homologues are restricting our capability of developing the lipopeptide syntheses potential of microorganism and promoting the product value of lipopeptide of bacteria. In this paper, we summarize the types of chassis cells for producing cyclic lipopeptides. We also introduce the structural characteristics of cyclic lipopeptides based on bacterial origin, synthesis pathway of non-ribosomal peptide, and structural domain characteristics of non-ribosomal peptide synthetase. In addition, the strategies for homologues regulation and biosynthetic yield improvement through genetic and metabolic engineering methods were reviewed, as well as the development status of natural product chassis strains. The synthesis of lipopeptide products can be effectively improved by optimizing the precursor metabolism, enhancing the expression of lipopeptide synthesis gene cluster, blocking the competitive pathway of lipopeptide synthesis, and the modification of various regulatory factors. The non-ribosomal peptide synthetase structural domain can be modified to obtain higher value lipopeptide and new lipopeptide drugs. We also review the effects of mixed-culture on lipopeptide syntheses, the development status of chassis strains for producing natural product, and the application of synthetic biology for improving lipopeptide biosynthesis. With the rapid development and application of synthetic biotechnology, the quality and quantity of natural lipopeptide from microorganisms will be improved rapidly. It also boosts the development of novel cyclic lipopeptides. And a better understanding of the synthesis, modification and mechanism of action of antimicrobial peptides will restart its commercial development.

Key words: cyclic lipopeptide, non-ribosomal peptide synthesis pathway, NRPS, module domain, homologous regulation, synthetic biology

中图分类号:

Q815

侯正杰, 孙慧中, 白松, 陈新月, 曹春阳, 程景胜. 环脂肽生物合成的研究进展[J]. 合成生物学, 2021, 2(4): 577-597.

Zhengjie HOU, Huizhong SUN, Song BAI, Xinyue CHEN, Chunyang CAO, Jingsheng CHENG. Research progress of cyclic lipopeptide biosynthesis[J]. Synthetic Biology Journal, 2021, 2(4): 577-597.

图/表 11

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脂肽	方法	结果	参考文献
actinomycin	N-甲基缬氨酸活化结构域替换	导致催化非甲基化和N-甲基化酰基二肽的合成	[52]
bacillibactin	DhbE-A结构域突变、文库筛选	3-羟基苯甲酸和2-氨基苯甲酸酰基化的bacillibactin	[53]
bacitracin	Cy结构域插入	产生新的杂环二肽	[54]
CDA	CdaPS3-A结构域点突变	产生含Gln、mGln的CDA变体	[55]
clorobiocin	调控相关酶表达	表达卤化酶HrmQ导致形成两个有5-氯吡咯的clorobiocin衍生物	[56]
daptomycin	DptBC引入新的模块	21种改造菌产生超过60种新型物质	[57]
enterobactin	EntF-AThr结构域替换、定向进化	产生中间体，得到两种不同的NRPS变体，酶活性和产物产量提高了10倍	[58]
fusaricidins	FusA-A3结构域点突变	增加了fusaricidin类似物（LI-F07）的量	[59]
gramicidin	GrsA-Aphe结构域替换	将编码不同特异性的亚结构域移植到GrsA-Aphe结构域中，所有嵌合体表达	[60]
luminmide	A结构域点突变	luminmide B在几种突变体中变得显著丰富（超过90%）	[61]
polymyxin	PmxA-ALeu与PmxA-Athr替换	通过替换polymyxin A的A结构域合成polymyxin B、D、P	[62]
pyoverdine	PvdD-C/A结构域替换	产生变短或者被修饰的多肽	[63]
pyoverdine	PvdD-T结构域替换	产生了两个重组pyoverdine	[64]
surfactin	SrfAC-Aleu结构域替换	得到surfactin结构中亮氨酸改变的5种surfactin类似物	[65]
surfactin	SrfA模块的替换	创建一个完全活跃的杂交酶，以高产率形成一个新的肽	[66]
surfactin	SrfAA-AGlu点突变与模块的替换	产生未知的七肽Asn5表面活性素	[67]
surfactin	SrfAA-Aleu模块删除	产生缺少一个亮氨基酸的表面活性素变体	[68]
tyrocidine	TE结构域替换	来源不同的TE结构域都可以水解二肽	[69]
tyrocidine	模块融合	合成不同三肽	[70]

脂肽	方法	结果	参考文献
actinomycin	N-甲基缬氨酸活化结构域替换	导致催化非甲基化和N-甲基化酰基二肽的合成	[52]
bacillibactin	DhbE-A结构域突变、文库筛选	3-羟基苯甲酸和2-氨基苯甲酸酰基化的bacillibactin	[53]
bacitracin	Cy结构域插入	产生新的杂环二肽	[54]
CDA	CdaPS3-A结构域点突变	产生含Gln、mGln的CDA变体	[55]
clorobiocin	调控相关酶表达	表达卤化酶HrmQ导致形成两个有5-氯吡咯的clorobiocin衍生物	[56]
daptomycin	DptBC引入新的模块	21种改造菌产生超过60种新型物质	[57]
enterobactin	EntF-AThr结构域替换、定向进化	产生中间体，得到两种不同的NRPS变体，酶活性和产物产量提高了10倍	[58]
fusaricidins	FusA-A3结构域点突变	增加了fusaricidin类似物（LI-F07）的量	[59]
gramicidin	GrsA-Aphe结构域替换	将编码不同特异性的亚结构域移植到GrsA-Aphe结构域中，所有嵌合体表达	[60]
luminmide	A结构域点突变	luminmide B在几种突变体中变得显著丰富（超过90%）	[61]
polymyxin	PmxA-ALeu与PmxA-Athr替换	通过替换polymyxin A的A结构域合成polymyxin B、D、P	[62]
pyoverdine	PvdD-C/A结构域替换	产生变短或者被修饰的多肽	[63]
pyoverdine	PvdD-T结构域替换	产生了两个重组pyoverdine	[64]
surfactin	SrfAC-Aleu结构域替换	得到surfactin结构中亮氨酸改变的5种surfactin类似物	[65]
surfactin	SrfA模块的替换	创建一个完全活跃的杂交酶，以高产率形成一个新的肽	[66]
surfactin	SrfAA-AGlu点突变与模块的替换	产生未知的七肽Asn5表面活性素	[67]
surfactin	SrfAA-Aleu模块删除	产生缺少一个亮氨基酸的表面活性素变体	[68]
tyrocidine	TE结构域替换	来源不同的TE结构域都可以水解二肽	[69]
tyrocidine	模块融合	合成不同三肽	[70]

外源添加	目标脂肽	影响	参考文献
肉豆蔻酸、十五烷酸、棕榈酸等烷酸	iturin、fengycin、 surfactin	脂肽合成基因的转录水平上调，碳链长度为奇数的烷酸对脂肽产生靶基因上调的影响大于碳链长度为偶数的烷酸	[97]
棕榈酸	iturin A	通过引入棕榈酸酯的烷基来促进iturin A的形成	[105]
碳源、氮源和氨基酸	C₁₄ iturin W、 C₁₅ iturin W	除淀粉外，碳源能显著提高C₁₄ iturin W的产量，尤其是山梨醇，但不同碳源使C₁₅ iturin W产量受到抑制；氮源除了硫酸铵外，对C₁₄ iturin W的产量都有促进，而C₁₅ iturin W的产量除胰蛋白胨外都略有抑制；不同氨基酸对C₁₄ iturin W和C₁₅ iturin W的影响很大且不同	[106]
酪氨酸、谷氨酰胺、天冬酰胺和脯氨酸、丝氨酸	iturin A	复合氨基酸的产量最高，天冬酰胺和脯氨酸的加入略微提高iturin A产量，丝氨酸可显著促进合成	[99]
异亮氨酸、缬氨酸	bacillomycin F	促进枯草芽孢杆菌产生bacillomycin F	[107]
L-亮氨酸、D-亮氨酸	surfactin	L-Leu使surfactin产量显著增加，添加D-Leu后surfactin产量急剧下降	[98]
碳源、金属离子	surfactin	不同碳源的添加（木糖除外）对表面活性素同系物的相对产量无显著影响；不同碳源的添加对不同脂肪酸链长的分子基团的比例有影响；金属离子的加入可能选择性地影响了脂肽第5位AME5残基的氨基酸序列	[101]
亚铁离子	iturin A	通过添加亚铁离子提高了解淀粉芽孢杆菌产iturin A的能力	[103]
锰和铁盐	surfactin	提高了surfactin的产量	[104]
氧化锌纳米颗粒	surfactin、iturin	增加脂肽surfactin和iturin的合成，增强菌株的抗真菌活性	[108]

外源添加	目标脂肽	影响	参考文献
肉豆蔻酸、十五烷酸、棕榈酸等烷酸	iturin、fengycin、 surfactin	脂肽合成基因的转录水平上调，碳链长度为奇数的烷酸对脂肽产生靶基因上调的影响大于碳链长度为偶数的烷酸	[97]
棕榈酸	iturin A	通过引入棕榈酸酯的烷基来促进iturin A的形成	[105]
碳源、氮源和氨基酸	C₁₄ iturin W、 C₁₅ iturin W	除淀粉外，碳源能显著提高C₁₄ iturin W的产量，尤其是山梨醇，但不同碳源使C₁₅ iturin W产量受到抑制；氮源除了硫酸铵外，对C₁₄ iturin W的产量都有促进，而C₁₅ iturin W的产量除胰蛋白胨外都略有抑制；不同氨基酸对C₁₄ iturin W和C₁₅ iturin W的影响很大且不同	[106]
酪氨酸、谷氨酰胺、天冬酰胺和脯氨酸、丝氨酸	iturin A	复合氨基酸的产量最高，天冬酰胺和脯氨酸的加入略微提高iturin A产量，丝氨酸可显著促进合成	[99]
异亮氨酸、缬氨酸	bacillomycin F	促进枯草芽孢杆菌产生bacillomycin F	[107]
L-亮氨酸、D-亮氨酸	surfactin	L-Leu使surfactin产量显著增加，添加D-Leu后surfactin产量急剧下降	[98]
碳源、金属离子	surfactin	不同碳源的添加（木糖除外）对表面活性素同系物的相对产量无显著影响；不同碳源的添加对不同脂肪酸链长的分子基团的比例有影响；金属离子的加入可能选择性地影响了脂肽第5位AME5残基的氨基酸序列	[101]
亚铁离子	iturin A	通过添加亚铁离子提高了解淀粉芽孢杆菌产iturin A的能力	[103]
锰和铁盐	surfactin	提高了surfactin的产量	[104]
氧化锌纳米颗粒	surfactin、iturin	增加脂肽surfactin和iturin的合成，增强菌株的抗真菌活性	[108]

多黏菌素	AA-3	AA-6	AA-7	脂肪酰基尾巴
polymyxins A
P-A1	D-Dab	D-Leu	L-Thr	6-MOA
P-A2	D-Dab	D-Leu	L-Thr	6-MHA
polymyxin B
P-B1	L-Dab	D-Phe	L-Leu	6-MOA
P-B2	L-Dab	D-Phe	L-Leu	6-MHA
P-B3	L-Dab	D-Phe	L-Leu	OA
P-B4	L-Dab	D-Phe	L-Leu	HA
P-B5	L-Dab	D-Phe	L-Leu	NA
P-B6	L-Dab	D-Phe	L-Leu	3-OH-6-MOA
Ile-P-B1	L-Dab	D-Phe	L-Ile	6-MOA
polymyxin C
P-C	L-Dab	D-Phe	L-Thr	6-MOA
polymyxin D
P-D1	D-Ser	D-Leu	L-Thr	6-MOA
P-D2	D-Ser	D-Leu	L-Thr	6-MHA
polymyxin E
P-E1	L-Dab	D-Leu	D-Leu	6-MOA
P-E2	L-Dab	D-Leu	D-Leu	6-MHA
P-E3	L-Dab	D-Leu	D-Leu	OA
P-E4	L-Dab	D-Leu	D-Leu	HA
P-E7	L-Dab	D-Leu	D-Leu	7-MOA
Ile-P-E1	L-Dab	D-Leu	L-Ile	6-MOA
Ile-P-E2	L-Dab	D-Leu	L-Ile	6-MHA
Ile-P-E8	L-Dab	D-Leu	L-Ile	7-MNA
Nval-E1	L-Dab	D-Leu	L-Nval	6-MOA
Val-E1	L-Dab	D-Leu	L-Val	6-MOA
Val-E2	L-Dab	D-Leu	L-Val	6-MHA
polymyxin M
P-M	L-Dab	D-Leu	L-Thr	6-MOA
polymyxin P
P-P1	D-Dab	D-Phe	L-Thr	6-MOA
P-P2	D-Dab	D-Phe	L-Thr	6-MHA