合成甲基营养细胞工厂同化甲醇的研究进展及未来展望

doi:10.12211/2096-8280.2020-048

合成生物学 ›› 2021, Vol. 2 ›› Issue (2): 222-233.DOI: 10.12211/2096-8280.2020-048

合成甲基营养细胞工厂同化甲醇的研究进展及未来展望

张卉¹^,², 袁姚梦³^,⁴, 张翀³^,⁴, 杨松¹^,²^,⁵, 邢新会³^,⁴

^1.青岛农业大学生命科学学院，山东青岛　266109
^2.青岛农业大学，山东省应用真菌重点实验室，山东青岛　266109
^3.清华大学化学工程系生物化工研究所，工业生物催化教育部重点实验室，北京 100084
^4.清华大学合成与系统生物学研究中心，北京　100084
^5.青岛农业大学，青岛生物沼气环境微生物国际合作基地，山东青岛　266109

收稿日期:2020-06-15 修回日期:2021-01-11 出版日期:2021-04-29 发布日期:2021-04-30
通讯作者: 杨松,邢新会
作者简介:张卉（1996─），女，在读硕士。主要研究方向为微生物代谢工程。E-mail：zhanghui_96@126.com
杨松（1977─），男，博士，教授。主要研究方向为代谢工程和系统组学。E-mail：yangsong1209@163.com
邢新会（1963─），男，博士，教授。主要研究方向为生物化工，合成生物学，生物育种。E-mail：xhxing@tsinghua.edu.cn
基金资助:
国家重点研发计划(2018YFA0901500)

Research progresses and future prospects of synthetic methylotrophic cell factory for methanol assimilation

Hui ZHANG¹^,², Yaomeng YUAN³^,⁴, Chong ZHANG³^,⁴, Song YANG¹^,²^,⁵, Xinhui XING³^,⁴

^1.School of Life Sciences，Qingdao Agricultural University，Qingdao 266109，Shandong，China
^2.Shandong Province Key Laboratory of Applied Mycology，Qingdao Agricultural University，Qingdao 266109，Shandong，China
^3.Key Lab of Industrial Biocatalysis，Ministry of Education，Institute of Biochemical Engineering，Department of Chemical Engineering，Tsinghua University，Beijing 100084，China
^4.Center for Synthetic and Systems Biology，Tsinghua University，Beijing 100084，China
^5.Qingdao International Center on Microbes Utilizing Biogas，Qingdao Agricultural University，Qingdao 266109，Shandong，China

Received:2020-06-15 Revised:2021-01-11 Online:2021-04-29 Published:2021-04-30
Contact: Song YANG, Xinhui XING

摘要/Abstract

摘要：

甲醇具有来源广、易储存运输、原料价格竞争力强等优势，被视为极具潜力的生物制造非糖基碳源资源。常用的模式底盘微生物研究历史长、认知清楚、操作工具多，在工程化改造中具有显著优势。近年来，通过借鉴天然甲基营养型微生物的甲醇利用途径对模式底盘进行改造，获得具备高效利用甲醇能力的合成甲基营养细胞工厂的研究日益受到关注。本文系统综述合成甲基营养细胞工厂的甲醇氧化、同化基因及其调控元件，和以共用糖基碳源结合适应性进化策略为主的核酮糖单磷酸途径重构及甲醇同化途径设计构建的研究现状，进而结合合成甲基营养细胞工厂面临的挑战进行展望，提出基于全基因组靶向基因编辑技术结合实验室适应性进化策略构建更高效合成甲基营养细胞工厂的研究方向。

关键词: 甲醇, 合成甲基营养细胞工厂, 生物元件, 核酮糖单磷酸途径, 实验室适应性进化

Abstract:

Methanol is an important and attractive non-sugar carbon source for industry biotechnology due to its advantages of available source, easy storage, convenient transportation and competitive price. The widely-studied microorganisms such as Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae have a long research history, clear genetic background and a number of matured genetic tools, showing their potential in engineering cell factories for bioproduction. With the accumulated knowledge of native methylotrophs in recent years, these traditional industrial microorganisms have been engineered as methylotrophic cell factories (MeCFs) capable of using methanol as the major carbon and energy source. In this artical, we review the recent research progress including genes involved in the methanol oxidation, assimilation and regulatory elements of MeCFs. We also summarized the strategies based on the adaptive laboratory evolution which applies sugar as the co-substrate to construct the ribulose monophosphate (RuMP) cycle, as well as strategies for designing and tuning methanol assimilation pathway. In the synthetic MeCFs, the construction of the RuMP cycle requires the introduction of three heterologous enzymes, i.e., methanol dehydrogenase encoded by mdh, 3-hexulose 6-phosphate synthase encoded by hps and 6-phospho 3-hexuloisomerase encoded by phi. These enzymes can be further engineered to improve activities through protein engineering and directed evolution. Meanwhile, genes involved in methanol oxidation and assimilation pathways can be finely tuned to exhibit dynamic expression. To further improve methanol utilization of the synthetic MeCFs, the co-substrate for supporting growth has been developed to propel methanol assimilation, which rationally designs a methanol dependent growth model, and thus provides an ideal starting point for subsequent long-term adaptive laboratory evolution experiments. At the end, we discuss the challenges of engineering the synthetic MeCFs, and summarize the future prospects for improving the efficiency of methanol utilization through the combined strategies of genome-wide targeted gene editing and adaptive laboratory evolution.

Key words: methanol, synthetic methylotrophic cell factory, biological elements, ribulose monophosphate pathway, adaptive laboratory evolution

中图分类号:

Q815

张卉, 袁姚梦, 张翀, 杨松, 邢新会. 合成甲基营养细胞工厂同化甲醇的研究进展及未来展望[J]. 合成生物学, 2021, 2(2): 222-233.

Hui ZHANG, Yaomeng YUAN, Chong ZHANG, Song YANG, Xinhui XING. Research progresses and future prospects of synthetic methylotrophic cell factory for methanol assimilation[J]. Synthetic Biology Journal, 2021, 2(2): 222-233.

图/表 4

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碳源	宿主	Mdh、Hps和Phi来源	甲醇同化进展	文献
甲醇和酵母抽提物	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	39%三羧酸循环中间产物和53%糖酵解中间产物被¹³C甲醇标记，实现柚皮素合成	[22]
	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	增强核酮糖-5-磷酸再生和甲醇同化，增加3-磷酸甘油酸和磷酸烯醇式丙酮酸的¹³C甲醇标记量	[38]
	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	P_frm控制动态调控酶的活性，增加大肠杆菌在甲醇中的生长速率	[39]
甲醇和葡萄糖	谷氨酸棒状杆菌	甲醇芽孢杆菌，枯草芽孢杆菌，枯草芽孢杆菌	甲醇消耗速率为1.7 mmol/(L·h)	[25]
甲醇和葡萄糖	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	增加甲醇进入中间代谢物的碳通量	[38]
甲醇和葡萄糖酸盐	大肠杆菌	甲醇芽孢杆菌，甲基鞭毛杆菌，甲基鞭毛杆菌	通过实验室适应性进化，24%甲醇进入中心碳代谢，甲醇可作为主要生长碳源	[41]
甲醇和木糖	大肠杆菌	钩虫贪铜菌，甲醇芽孢杆菌，甲基鞭毛杆菌	通过实验室适应性进化，甲醇和木糖大约以1∶1的摩尔比共同消耗，进化菌株的生长速率为（0.17±0.006） h^－1，并利用甲醇生产乙醇和丁醇	[43]
	大肠杆菌	甲醇芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	进化的Mdh2突变体最大反应速度增加3.5倍，甲醇进入突变菌株中心代谢物的碳通量是未突变菌株的2倍	[28]
	谷氨酸棒状杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	高达63%的¹³C甲醇进入细胞内代谢物	[44]
甲醇和核糖	大肠杆菌	甲醇芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	40%的甲醇进入中心碳代谢途径	[26]
甲醇和核糖	谷氨酸棒状杆菌	甲醇芽孢杆菌，枯草芽孢杆菌，枯草芽孢杆菌	25%的¹³C标记出现在糖酵解和磷酸戊糖途径中间代谢物	[46]

碳源	宿主	Mdh、Hps和Phi来源	甲醇同化进展	文献
甲醇和酵母抽提物	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	39%三羧酸循环中间产物和53%糖酵解中间产物被¹³C甲醇标记，实现柚皮素合成	[22]
	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	增强核酮糖-5-磷酸再生和甲醇同化，增加3-磷酸甘油酸和磷酸烯醇式丙酮酸的¹³C甲醇标记量	[38]
	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	P_frm控制动态调控酶的活性，增加大肠杆菌在甲醇中的生长速率	[39]
甲醇和葡萄糖	谷氨酸棒状杆菌	甲醇芽孢杆菌，枯草芽孢杆菌，枯草芽孢杆菌	甲醇消耗速率为1.7 mmol/(L·h)	[25]
甲醇和葡萄糖	大肠杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	增加甲醇进入中间代谢物的碳通量	[38]
甲醇和葡萄糖酸盐	大肠杆菌	甲醇芽孢杆菌，甲基鞭毛杆菌，甲基鞭毛杆菌	通过实验室适应性进化，24%甲醇进入中心碳代谢，甲醇可作为主要生长碳源	[41]
甲醇和木糖	大肠杆菌	钩虫贪铜菌，甲醇芽孢杆菌，甲基鞭毛杆菌	通过实验室适应性进化，甲醇和木糖大约以1∶1的摩尔比共同消耗，进化菌株的生长速率为（0.17±0.006） h^－1，并利用甲醇生产乙醇和丁醇	[43]
	大肠杆菌	甲醇芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	进化的Mdh2突变体最大反应速度增加3.5倍，甲醇进入突变菌株中心代谢物的碳通量是未突变菌株的2倍	[28]
	谷氨酸棒状杆菌	嗜热脂肪芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	高达63%的¹³C甲醇进入细胞内代谢物	[44]
甲醇和核糖	大肠杆菌	甲醇芽孢杆菌，甲醇芽孢杆菌，甲醇芽孢杆菌	40%的甲醇进入中心碳代谢途径	[26]
甲醇和核糖	谷氨酸棒状杆菌	甲醇芽孢杆菌，枯草芽孢杆菌，枯草芽孢杆菌	25%的¹³C标记出现在糖酵解和磷酸戊糖途径中间代谢物	[46]

宿主	基因名称	基因功能	基因突变类型	依赖甲醇生长的预测功能	文献
大肠杆菌	frmA	谷胱甘肽依赖的甲醛脱氢酶	移码突变	促进甲醛氧化为CO₂，提供额外的NADH	[24]
	fdoG	甲酸脱氢酶	移码突变	促进甲酸氧化为CO₂，提供额外的NADH
	gltA	柠檬酸合成酶	转座子插入	降低三羧酸循环的碳通量，插入转座子元件
	ptsH	编码Hpr的蛋白酶	转座子插入	破坏磷酸糖转移酶系统，插入转座子元件
	pgi	磷酸葡萄糖异构酶	基因内缺失	提高磷酸葡萄糖异构酶酶活，增加细胞生长所需的NADPH
大肠杆菌	gntR	DNA结合转录抑制子	碱基置换	改变葡萄糖酸摄取量	[41]
	frmA	谷胱甘肽依赖的甲醛脱氢酶	移码突变	失活甲醛氧化途径，甲醛用于同化途径
	nadR	DNA结合转录抑制子/烟酰胺单核苷酸腺苷转移酶	碱基置换	改变NAD⁺/NADH比值
大肠杆菌	ptsI	组成磷酸转移酶系统的酶I	基因内缺失	降低葡萄糖消耗速率	[42]
	icd	异柠檬酸脱氢酶	基因内缺失	降低三羧酸循环的碳通量，以维持细胞氧化还原平衡	[42]
大肠杆菌	zwf	6-磷酸葡萄糖脱氢酶	碱基置换	调低Entner-Doudoroff途径通量	[43]
	pykF	丙酮酸激酶I	基因内插入	调低糖酵解途径通量
	cyaA	腺苷酸环化酶	基因内插入	降低三羧酸循环相关酶的转录水平，改变NAD⁺/NADH比值
	deoD	嘌呤核苷磷酸化酶	基因内插入	提供甲醛固定受体
	frmAB, yaiO	甲醛脱毒操纵子，外膜蛋白	基因间缺失	增加细胞内甲醛浓度
谷氨酸棒状杆菌	atlR	碳水化合物代谢的多功能调节子	碱基置换	提高醇脱氢酶和木糖激酶的催化活性以强化甲醇和木糖的利用效率	[44]
	metY	邻乙酰高丝氨酸巯基化酶	碱基置换	提高宿主对甲醇的耐受性
	mtrA	参与细胞形态、抗生素易感性、渗透保护在内的基因功能的多重调控子	碱基置换	通过调节类谷氧还蛋白和NAD⁺合成酶，参与维持细胞内氧化还原状态
	cgl2030, ctaE	预测的ATP激酶，细胞色素C氧化酶亚基蛋白酶	碱基置换	改变能量代谢

宿主	基因名称	基因功能	基因突变类型	依赖甲醇生长的预测功能	文献
大肠杆菌	frmA	谷胱甘肽依赖的甲醛脱氢酶	移码突变	促进甲醛氧化为CO₂，提供额外的NADH	[24]
	fdoG	甲酸脱氢酶	移码突变	促进甲酸氧化为CO₂，提供额外的NADH
	gltA	柠檬酸合成酶	转座子插入	降低三羧酸循环的碳通量，插入转座子元件
	ptsH	编码Hpr的蛋白酶	转座子插入	破坏磷酸糖转移酶系统，插入转座子元件
	pgi	磷酸葡萄糖异构酶	基因内缺失	提高磷酸葡萄糖异构酶酶活，增加细胞生长所需的NADPH
大肠杆菌	gntR	DNA结合转录抑制子	碱基置换	改变葡萄糖酸摄取量	[41]
	frmA	谷胱甘肽依赖的甲醛脱氢酶	移码突变	失活甲醛氧化途径，甲醛用于同化途径
	nadR	DNA结合转录抑制子/烟酰胺单核苷酸腺苷转移酶	碱基置换	改变NAD⁺/NADH比值
大肠杆菌	ptsI	组成磷酸转移酶系统的酶I	基因内缺失	降低葡萄糖消耗速率	[42]
	icd	异柠檬酸脱氢酶	基因内缺失	降低三羧酸循环的碳通量，以维持细胞氧化还原平衡	[42]
大肠杆菌	zwf	6-磷酸葡萄糖脱氢酶	碱基置换	调低Entner-Doudoroff途径通量	[43]
	pykF	丙酮酸激酶I	基因内插入	调低糖酵解途径通量
	cyaA	腺苷酸环化酶	基因内插入	降低三羧酸循环相关酶的转录水平，改变NAD⁺/NADH比值
	deoD	嘌呤核苷磷酸化酶	基因内插入	提供甲醛固定受体
	frmAB, yaiO	甲醛脱毒操纵子，外膜蛋白	基因间缺失	增加细胞内甲醛浓度
谷氨酸棒状杆菌	atlR	碳水化合物代谢的多功能调节子	碱基置换	提高醇脱氢酶和木糖激酶的催化活性以强化甲醇和木糖的利用效率	[44]
	metY	邻乙酰高丝氨酸巯基化酶	碱基置换	提高宿主对甲醇的耐受性
	mtrA	参与细胞形态、抗生素易感性、渗透保护在内的基因功能的多重调控子	碱基置换	通过调节类谷氧还蛋白和NAD⁺合成酶，参与维持细胞内氧化还原状态
	cgl2030, ctaE	预测的ATP激酶，细胞色素C氧化酶亚基蛋白酶	碱基置换	改变能量代谢

[1]	王俊婷, 郭潇佳, 李青, 万里, 赵宗保. 创制非天然辅酶偏好型甲醇脱氢酶[J]. 合成生物学, 2021, 2(4): 651-661.
[2]	王也, 王昊晨, 晏明皓, 胡冠华, 汪小我. 生物分子序列的人工智能设计[J]. 合成生物学, 2021, 2(1): 1-14.
[3]	高教琪, 周雍进. 甲醇生物转化的机遇与挑战[J]. 合成生物学, 2020, 1(2): 158-173.
[4]	张博, 马永硕, 尚轶, 黄三文. 植物合成生物学研究进展[J]. 合成生物学, 2020, 1(2): 121-140.

合成甲基营养细胞工厂同化甲醇的研究进展及未来展望

Research progresses and future prospects of synthetic methylotrophic cell factory for methanol assimilation

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