Recent advances in the construction strategy of methylotrophic Escherichia coli

doi:10.16085/j.issn.1000-6613.2020-1607

Abstract

Abstract:

The use of microbial cell factories to realize the transformation of raw materials and the synthesis of products is the core of green biological manufacturing. However, the current bio-manufacturing mainly uses sugar-rich grains as the raw material, leading to a controversy of "competing with the people for food". As a result, there is an urgent need to develop non-food raw materials. As a main product of coal chemical industry, methanol has the advantages of abundant sources, low price, and high degree of reduction, indicating that it has the potential to replace food raw materials. Natural methylotrophs have been used to produce single-cell protein and various amino acids from methanol. However, there are many disadvantages such as low theoretic yield and inefficient genetic tool. With the development of synthetic biology, the use of model organisms such as Escherichia coli as chassis cells to construct artificial methylotrophic bacteria and realize the production of methanol to various chemicals has become a research hotspot. In this review, we firstly summarized the construction strategies of a variety of methylotrophic Escherichia coli. Then, the key factors affecting the metabolism of natural pathways and important intermediate metabolites were clarified. Moreover, various methods for optimizing natural methanol metabolic pathways and constructing new artificial pathways were discussed. And we put forward a prospect for the optimization of methanol utilization by engineering strains.

Key words: methanol, Escherichia coli, synthetic biology, industrial biotechnology, metabolic pathway, methylotrophic

摘要：

利用微生物细胞工厂实现原料转化和产品合成是绿色生物制造的核心。然而，当前生物制造仍以富含糖类的谷物粮食为主要原料，存在“与民争粮”的争议，亟需开发非粮原料。甲醇作为煤化工产业中的重要产品，具有来源广、价格低、还原性强等优势，有替代粮食原料的潜力。天然甲基营养菌可利用甲醇生产单细胞蛋白和各种氨基酸，但存在理论收率低、遗传改造工具不足等缺点。随着合成生物学的发展，以大肠杆菌等模式生物作为底盘细胞构建人工甲基营养菌，实现甲醇到各种化学品的生产已成为研究热点。本文总结了多种甲基营养型大肠杆菌的构建策略，明确了影响天然路径代谢的关键因素与代谢过程中的重要中间产物，概括了各种天然路径在大肠杆菌中的优化策略与人工新路径的构建方法，并对工程菌株的优化提出了展望。

关键词: 甲醇, 大肠杆菌, 合成生物学, 工业生物技术, 代谢途径, 甲基营养型

CLC Number:

TAO Yuxuan, ZHANG Shangjie, JING Yiwen, XIN Fengxue, DONG Weiliang, ZHOU Jie, JIANG Yujia, ZHANG Wenming, JIANG Min. Recent advances in the construction strategy of methylotrophic Escherichia coli[J]. Chemical Industry and Engineering Progress, 2021, 40(7): 3932-3941.

陶雨萱, 张尚杰, 景艺文, 信丰学, 董维亮, 周杰, 蒋羽佳, 章文明, 姜岷. 甲基营养型大肠杆菌构建策略的研究进展[J]. 化工进展, 2021, 40(7): 3932-3941.

Figures/Tables 7

底物	宿主	改造方法	改造结果	文献
酵母提取物+甲醇	E. coli ΔfrmA	引入甲醇芽孢杆菌RuMP途径酶表达了密码子优化的甲醇芽孢杆菌HPS和PHI基因	甲醛消耗量从（0.058±0.013）mm增加到（0.074±0.001）mm	［13］
甲醇	E. coliΔfrmA	利用SH3-配体组装工程超分子酶复合物	甲醇转化为F6P的能力提高了50倍	［15］
甲醇+氨基酸	E. coliΔlrp	苏氨酸共利用敲除亮氨酸反馈调节蛋白（LRP）	利用酵母提取物和甲醇使生物量最终浓度增加33% Δlrp菌株的生物量在有甲醇的情况下比不加甲醇的高34%	［16］
甲醇+葡萄糖酸盐	MeSV1	敲除磷酸葡萄糖酸脱水酶（EDD）和5-磷酸核糖异构酶（RPIAB）	高达24%的甲醇进入中心代谢不加酵母粉培养的MeSV2.1 第九代最大生长量达到OD600=1.34±0.03	［17］
甲醇+葡萄糖	E. coliΔfrmA	表达甲醇芽孢杆菌的PPP途径敲除磷酸葡萄糖异构酶基因（PGI）和甲醛脱氢酶基因（FRMA）	甲基营养大肠杆菌ΔfrmAΔpgi的甲醇利用率比甲基营养大肠杆菌ΔfrmA提高了219%	［19］
甲醇+木糖	E. coli	碘乙酸酯抑制糖酵解通量过量表达景天庚酮糖二磷酸酶（GLPX）	Ru5P相应增加(4.0±1.2)倍，甲醛降幅为2.50%±0.7%	［20］
甲醇	E. coli	拷贝数变异（CNVs）适应性进化	构建菌株能够以甲醇为唯一碳源生长在8h的倍增时间内高效生长	［21］
甲醇+葡萄糖	E. coli	敲除葡萄糖6-磷酸异构酶（PGI）、磷酸葡萄糖酸脱水酶（EDD）和核糖5-磷酸异构酶（RPIAB）	进化菌株在含甲醇的葡萄糖微量培养基中的最大生长速率为0.15/h	［22］
甲醇+木糖	E. coli	将菌株对甲醇的利用与在五碳（C₅）糖上的生长相结合敲除用于戊糖利用的戊糖磷酸途径中的必要基因并表达来自RuMP途径的异源酶	菌株能够以(0.17±0.006)h^-1的速率利用甲醇，甲醇和木糖以大约1∶1的摩尔比共同化	［23］

E. coliΔserA ΔgcvP

改进丝氨酸循环

使用甲醛脱氢酶（FALDH）来简化甲醛氧化成甲酸盐的过程

最快的甲醇同化速率为0.7mm·h^-1（OD600），产生乙醇约6.7mmol·L^-1

［24］

甲酸+葡萄糖

E. coli strain DH5α

合成丝氨酸-苏氨酸循环

证明甲酸盐可以作为细胞碳一的唯一来源（倍增时间为2.3h）

底物	宿主	改造方法	改造结果	文献
甲酸盐	fdoG MG1655-derived E. coli strain	计算机设计甲醛酶（FLS）实现碳一到碳三化合物的合成	很高的化学推动力（>3kcal·mol^-1，1cal=4.1840J） FLS活性增加了100倍	［33］
甲醇+酵母膏	BW25113frmA（EC-ΔfrmA）	高效表达MDH和人工FLS 组装人工线性甲醇同化途径	甲醇消耗量从原始1g·L^-1增加到1.45g·L^-1	［29］
甲醛+甲醇+乙醇醛	体外到E. coli	合成乙酰辅酶A（SACA）途径	热力学有利（Δ_rG $G m ’$ 约为-96.7kJ·mol^-1）乙醇醛的产率随甲醛底物浓度的增加而增加（2g·L^-1时，转化率为80%）	［30］
甲醛	体外	体外构建醇醛同化（GAA）途径	碳转化率达到了88%	［31］
葡萄糖+甲醇	E. coli Suc360 E. coli Suc460 E. coli Suc560 E. coli Suc660	引入甲醇芽孢杆菌的甲醇脱氢酶和不同供体生物的单磷酸核酮糖途径	丁二酸产率从(0.91±0.08)g·g^-1提升至(0.98±0.11)g·g^-1	［34］
200mmol·L^-1甲醇	无细胞体系	甲醇缩合循环（MCC）	碳转化率为 80%（消耗33.5mm甲醇）（天然RuMP途径理论转化率为66%）	［35］
葡萄糖+甲酸盐	E. coli SBS550MGCms243	将念珠菌依赖NAD⁺的甲酸脱氢酶基因（FDH1）与乳酸乳球菌丙酮酸羧化酶（PycA）共表达	改造后平均葡萄糖消耗速率为2g·L^-1·h^-1，琥珀酸生产强度为2g·L^-1·h^-1，副产物甲酸盐浓度为0~3mmol·L^-1	［36］
甲醇+葡萄糖	E. coliΔfrmA	共表达ACT蛋白提高MDH的活性	MDH活性从22.1mU·mg^-1 提升到29.1mU·mg^-1	［37］

底物	宿主	改造方法	改造结果	文献
甲酸盐	fdoG MG1655-derived E. coli strain	计算机设计甲醛酶（FLS）实现碳一到碳三化合物的合成	很高的化学推动力（>3kcal·mol^-1，1cal=4.1840J） FLS活性增加了100倍	［33］
甲醇+酵母膏	BW25113frmA（EC-ΔfrmA）	高效表达MDH和人工FLS 组装人工线性甲醇同化途径	甲醇消耗量从原始1g·L^-1增加到1.45g·L^-1	［29］
甲醛+甲醇+乙醇醛	体外到E. coli	合成乙酰辅酶A（SACA）途径	热力学有利（Δ_rG $G m ’$ 约为-96.7kJ·mol^-1）乙醇醛的产率随甲醛底物浓度的增加而增加（2g·L^-1时，转化率为80%）	［30］
甲醛	体外	体外构建醇醛同化（GAA）途径	碳转化率达到了88%	［31］
葡萄糖+甲醇	E. coli Suc360 E. coli Suc460 E. coli Suc560 E. coli Suc660	引入甲醇芽孢杆菌的甲醇脱氢酶和不同供体生物的单磷酸核酮糖途径	丁二酸产率从(0.91±0.08)g·g^-1提升至(0.98±0.11)g·g^-1	［34］
200mmol·L^-1甲醇	无细胞体系	甲醇缩合循环（MCC）	碳转化率为 80%（消耗33.5mm甲醇）（天然RuMP途径理论转化率为66%）	［35］
葡萄糖+甲酸盐	E. coli SBS550MGCms243	将念珠菌依赖NAD⁺的甲酸脱氢酶基因（FDH1）与乳酸乳球菌丙酮酸羧化酶（PycA）共表达	改造后平均葡萄糖消耗速率为2g·L^-1·h^-1，琥珀酸生产强度为2g·L^-1·h^-1，副产物甲酸盐浓度为0~3mmol·L^-1	［36］
甲醇+葡萄糖	E. coliΔfrmA	共表达ACT蛋白提高MDH的活性	MDH活性从22.1mU·mg^-1 提升到29.1mU·mg^-1	［37］

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