Progress in metabolic engineering of microorganisms for the utilization of formate

doi:10.12211/2096-8280.2023-032

Abstract

Abstract:

One carbon resources are expected to become the next generation raw materials for the production of high value-added chemicals to achieve the recycling and utilization of carbon resources and promote the development of green industries. To achieve this aim, microbial utilization of formate produced from various one carbon resources, is one of the important strategies to build a green and sustainable platform for one-carbon biomanufacturing. However, there are many problems in the process of microbial utilization of formate, such as the low efficiency of formate utilization, the slow growth rate of formate-utilizing microorganisms, and the low yield of target metabolites and so on. To solve these problems, in this paper we systematically summarize and analyze the research progress in metabolic engineering of microorganisms for the utilization of formate from following three aspects: formate-utilizing microorganisms, metabolic pathways and metabolic engineering strategies. For formate-utilizing microorganisms, we summarize the metabolic characteristics and applicative advantages of natural formate-utilizing microorganisms, as well as the potential and advantages of model microorganisms for the application of metabolic engineering. For formate-utilizing metabolic pathways, we review the key steps, energy/reducing power consumption and characteristics of natural formate-utilizing pathways, reconstruction and optimization of formate-utilizing pathways and artificial formate-utilizing pathways, and then discuss the potential of metabolic engineering modification of these pathways. For formate-utilizing metabolic engineering strategies, we describe the useful strategies to improve the metabolic efficiency of formate assimilation pathways, such as optimizing the expression level of pathway genes, engineering key pathway enzymes, blocking the competitive pathways, reconstructing cofactor regeneration system, and modular pathway engineering, and then summarize the key approaches to improve the cell growth of formate-utilizing microorganisms, such as adaptive laboratory evolution, enhancing the cell growth of formatotrophs and improving the ability of microorganisms to synergistically utilize formate. Finally, we prospect the developmental direction of microbial utilization of formate from three aspects of formate-utilizing microorganisms, metabolic pathways and metabolic engineering strategies, which would lay the foundation for the development of formate bio-economy. {L-End}

Key words: formate, microorganisms, metabolic pathway, energy regeneration, metabolic engineering

摘要：

微生物利用甲酸生产高附加值产品，是实现碳资源回收利用与绿色产业发展的重要策略之一。然而，在微生物利用甲酸过程中存在甲酸利用效率偏低、细胞生长速率缓慢、目标代谢物产量不高等问题。为了解决上述问题，本文从甲酸利用的微生物、代谢路径与代谢工程策略三个方面，系统总结分析了代谢工程改造微生物利用甲酸的研究进展。在甲酸利用微生物方面，概述了天然甲酸利用微生物的代谢特点以及模式微生物的代谢工程改造潜能；在甲酸利用代谢路径方面，梳理了天然的甲酸利用路径、重构与优化的甲酸利用路径与人工的甲酸利用路径的关键步骤、能量/还原力消耗与特点；在甲酸利用代谢工程策略方面，阐述了提高甲酸同化效率与改善甲酸利用微生物细胞生长的关键方法。最后，从甲酸利用的微生物、代谢路径与代谢工程策略三个方面，展望了微生物利用甲酸的发展方向，为甲酸生物经济的发展奠定了基础。

关键词: 甲酸, 微生物, 代谢路径, 能量再生, 代谢工程

CLC Number:

Zhenzhen CHENG, Jian ZHANG, Cong GAO, Liming LIU, Xiulai CHEN. Progress in metabolic engineering of microorganisms for the utilization of formate[J]. Synthetic Biology Journal, 2023, 4(4): 756-778.

程真真, 张健, 高聪, 刘立明, 陈修来. 代谢工程改造微生物利用甲酸研究进展[J]. 合成生物学, 2023, 4(4): 756-778.

Figures/Tables 10

References 104

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微生物种类	需氧类型	模式菌株	天然甲酸代谢路径	应用优劣势	参考文献
产乙酸菌	专性厌氧	伍氏醋酸杆菌	还原性乙酰辅酶A途径	优势：能够天然利用甲酸和CO₂合成乙酸、乙醇等化合物劣势：在厌氧条件下生长，不利于工业应用；代谢研究尚不完善，代谢改造工具少	[20-22]
产甲烷菌	专性厌氧	氢营养型海沼甲烷球菌	还原性乙酰辅酶A途径	优势：能够天然利用甲酸和CO₂合成甲烷，在清洁能源产业方面具有应用潜能劣势：在厌氧条件下生长，不利于工业应用；代谢研究尚不完善，代谢改造工具少	[23-26]
硫酸盐还原菌	专型厌氧	普通脱硫弧菌	还原性乙酰辅酶A途径	优势：具有非常高的氢化酶活性，能够天然利用甲酸产氢，在清洁能源产业方面具有应用潜能劣势：厌氧条件下生长，不利于工业应用；代谢研究尚不完善，代谢改造工具少	[27-29]
甲基杆菌	好氧或兼性厌氧	扭脱甲基杆菌AM1	丝氨酸循环	优势：能够天然利用甲酸、CO₂与甲醇等一碳底物进行细胞生长；模式菌株的研究与改造相对充分，具有一定的改造潜能劣势：所具有的甲酸代谢路径的能量利用效率偏低，不利于工业应用	[30-32]
CBB循环依赖型微生物	好氧	钩虫贪铜菌H16	还原性磷酸戊糖循环	优势：具有甲酸脱氢酶，能够利用甲酸作为唯一碳源和能源；模式菌株的研究与改造相对充分，具有一定的应用潜能劣势：甲酸的氧化供能存在能量浪费，因此微生物的细胞生长速率与工业生产潜能均偏低	[33-36]

微生物种类	需氧类型	模式菌株	天然甲酸代谢路径	应用优劣势	参考文献
产乙酸菌	专性厌氧	伍氏醋酸杆菌	还原性乙酰辅酶A途径	优势：能够天然利用甲酸和CO₂合成乙酸、乙醇等化合物劣势：在厌氧条件下生长，不利于工业应用；代谢研究尚不完善，代谢改造工具少	[20-22]
产甲烷菌	专性厌氧	氢营养型海沼甲烷球菌	还原性乙酰辅酶A途径	优势：能够天然利用甲酸和CO₂合成甲烷，在清洁能源产业方面具有应用潜能劣势：在厌氧条件下生长，不利于工业应用；代谢研究尚不完善，代谢改造工具少	[23-26]
硫酸盐还原菌	专型厌氧	普通脱硫弧菌	还原性乙酰辅酶A途径	优势：具有非常高的氢化酶活性，能够天然利用甲酸产氢，在清洁能源产业方面具有应用潜能劣势：厌氧条件下生长，不利于工业应用；代谢研究尚不完善，代谢改造工具少	[27-29]
甲基杆菌	好氧或兼性厌氧	扭脱甲基杆菌AM1	丝氨酸循环	优势：能够天然利用甲酸、CO₂与甲醇等一碳底物进行细胞生长；模式菌株的研究与改造相对充分，具有一定的改造潜能劣势：所具有的甲酸代谢路径的能量利用效率偏低，不利于工业应用	[30-32]
CBB循环依赖型微生物	好氧	钩虫贪铜菌H16	还原性磷酸戊糖循环	优势：具有甲酸脱氢酶，能够利用甲酸作为唯一碳源和能源；模式菌株的研究与改造相对充分，具有一定的应用潜能劣势：甲酸的氧化供能存在能量浪费，因此微生物的细胞生长速率与工业生产潜能均偏低	[33-36]

微生物	非天然甲酸代谢路径	主要的应用优势	改造潜能	参考文献
大肠杆菌	重构的卡尔文循环；改良的丝氨酸循环；高丝氨酸循环； rTHF-rgcv途径及关键模块合成乙酰辅酶A途径等	生长周期短；遗传背景清晰；代谢工程改造工具种类丰富且高效	设计并构建更加高效的甲酸利用路径；通过实验室适应性进化与培养条件优化等策略提高菌株对甲酸的耐受性	[17-18,56-67]
酿酒酵母	rTHF-rgcv途径的关键模块	遗传背景清晰；分子遗传操作工具与技术成熟且高效；具有内源性甲酸脱氢酶，对甲酸的耐受性较高	通过代谢改造进一步提高rTHF-rgcv途径对甲酸的利用效率，开发以甲酸为唯一碳源和能源的工程菌株	[19]
毕赤酵母	以甲酸作为P _AOX1 的诱导物	能够利用甲醇作为唯一碳源和能源进行细胞生长；在生物医药产业和工业酶生产方面具有巨大潜力	通过代谢改造进一步提高菌株对甲酸的利用效率；基于菌株的代谢网络，设计并构建更加高效的甲酸利用路径	[68-69]
恶臭假单胞菌	rTHF-rgcv途径	能够编码多种天然甲酸脱氢酶；具有灵活的代谢机制，能够抵抗氧化应激和多种有毒化合物	开发更高效的分子遗传操作工具，进一步完善菌株的甲酸代谢网络	[70]

微生物	非天然甲酸代谢路径	主要的应用优势	改造潜能	参考文献
大肠杆菌	重构的卡尔文循环；改良的丝氨酸循环；高丝氨酸循环； rTHF-rgcv途径及关键模块合成乙酰辅酶A途径等	生长周期短；遗传背景清晰；代谢工程改造工具种类丰富且高效	设计并构建更加高效的甲酸利用路径；通过实验室适应性进化与培养条件优化等策略提高菌株对甲酸的耐受性	[17-18,56-67]
酿酒酵母	rTHF-rgcv途径的关键模块	遗传背景清晰；分子遗传操作工具与技术成熟且高效；具有内源性甲酸脱氢酶，对甲酸的耐受性较高	通过代谢改造进一步提高rTHF-rgcv途径对甲酸的利用效率，开发以甲酸为唯一碳源和能源的工程菌株	[19]
毕赤酵母	以甲酸作为P _AOX1 的诱导物	能够利用甲醇作为唯一碳源和能源进行细胞生长；在生物医药产业和工业酶生产方面具有巨大潜力	通过代谢改造进一步提高菌株对甲酸的利用效率；基于菌株的代谢网络，设计并构建更加高效的甲酸利用路径	[68-69]
恶臭假单胞菌	rTHF-rgcv途径	能够编码多种天然甲酸脱氢酶；具有灵活的代谢机制，能够抵抗氧化应激和多种有毒化合物	开发更高效的分子遗传操作工具，进一步完善菌株的甲酸代谢网络	[70]

菌株	路径酶来源	参考文献
Escherichia coli	Ftl、Fch、Mtd（源自Clostridium ljungdahalii） GCS、GlyA、Sda（内源酶）	[61]
Escherichia coli	Ftl、Fch、Mtd（源自Methylobacterium extorquens AM1） GCS、GlyA（内源酶）	[62]
Escherichia coli	Ftl、Fch、Mtd（源自Methylobacterium extorquens CM4） GCS、GlyA、Sda（内源酶） Fdh（源自Candida boidinii）	[63]
Escherichia coli	Ftl（源自Clostridium kluyveri） FolD（Fch/ Mtd）、GCS、GlyA（内源酶）	[64]
Escherichia coli	Ftl、Fch、Mtd（源自Methylobacterium extorquens AM1） GCS、GlyA、Sda（内源酶） Fdh（源自Pseudomonas sp.）	[17]
Escherichia coli （Bang等^[63]研究的进一步拓展）	Ftl、Fch、Mtd（源自Methylobacterium extorquens CM4） GCS、GlyA、Sda（内源酶） Fdh（源自Candida boidinii、Arabidopsis thaliana）	[18]
Escherichia coli （Döring等^[64]研究的进一步拓展）	Ftl（源自Clostridium kluyveri） FolD（Fch/ Mtd）、GCS、GlyA（内源酶）	[65]
Escherichia coli （Kim等^[17]研究的进一步拓展）	Ftl、Fch、Mtd（源自Methylobacterium extorquens AM1） GCS、GlyA、Sda（内源酶） Fdh（源自Pseudomonas sp. ）	[66]
Saccharomyces cerevisiae	MIS1（Ftl/ Fch/ Mtd）、GCS（内源酶） Fdh（内源酶）	[19]
Cupriavidus necator	Ftl、Fch、Mtd（源自Methylobacterium extorquens AM1） GCS、GlyA、Sda（内源酶） Fdh（内源酶）	[16]
Clostridium pasteurianum	GCS（源自Gottschalkia acidurici） Ftl、Fch、Mtd、GlyA、Sda（内源酶）	[89]
Pseudomonas putida	Ftl、Fch、Mtd（源自Methylobacterium extorquens AM1） GCS、GlyA、Sda（内源酶） Fdh（内源酶）	[70]