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:2021-04-30 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 ZHANG^1,², Yaomeng YUAN^3,⁴, Chong ZHANG^3,⁴, Song YANG^1,^2,⁵, Xinhui XING^3,⁴

^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:2021-04-30 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.

参考文献 68

1	CLOMBURG J M, CRUMBLEY A M, GONZALEZ R. Industrial biomanufacturing: the future of chemical production[J]. Science, 2017, 355(6320): 38.
2	ZHU T, ZHAO T, BANKEFA O E, et al. Engineering unnatural methylotrophic cell factories for methanol-based biomanufacturing: challenges and opportunities[J]. Biotechnology Advances, 2020, 39: 107467.
3	MARTENS J A, BOGAERTS A, DE KIMPE N, et al. The chemical route to a carbon dioxide neutral world[J]. Chemsuschem, 2017, 10(6): 1039-1055.
4	OLAH G A. Beyond oil and gas: the methanol economy[J]. Angewandte Chemie International Edition, 2005, 44(18): 2636-2639.
5	ZHANG M, YUAN X J, ZHANG C, et al. Bioconversion of methanol into value-added chemicals in native and synthetic methylotrophs[J]. Current Issues Molecular Biology, 2019, 33: 225-236.
6	SCHRADER J, SCHILLING M, HOLTMANN D, et al. Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria[J]. Trends in Biotechnology, 2009, 27(2):107-115.
7	ZHANG W M, SONG M, YANG Q, et al. Current advance in bioconversion of methanol to chemicals[J]. Biotechnology for Biofuels, 2018, 11: 260.
8	CHISTOSERDOVA L, KALYUZHNAYA M G. Current trends in methylotrophy[J]. Trends in Microbiology, 2018, 26(8): 703-714.
9	CUI J, GOOD N M, HU B, et al. Metabolomics revealed an association of metabolite changes and defective growth in Methylobacterium extorquens AM1 overexpressing ecm during growth on methanol[J]. PLoS One, 2016, 11(4): e0154043.
10	ANTHONY C, GHOSH M. The structure and function of the PQQ-containing quinoprotein dehydrogenases[J]. Progress in Biophysics and Molecular Biology, 1998, 69(1): 1-21.
11	KROG A, HEGGESET T M, MÜLLER J E, et al. Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD⁺ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties[J]. PLoS One, 2013, 8(3): e59188.
12	GUNKEL K, DIJK R VAN, VEENHUIS M, et al. Routing of Hansenula polymorpha alcohol oxidase: an alternative peroxisomal protein-sorting machinery[J]. Molecular Biology of the Cell, 2004, 15(3): 1347-1355.
13	ZHU W L, CUI J Y, CUI L Y, et al. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway[J]. Applied Microbiology and Biotechnology, 2016, 100(5): 2171-2182.
14	LIANG W F, CUI L Y, CUI J Y, et al. Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply[J]. Metabolic Engineering, 2017, 39: 159-168.
15	YANG Y M, CHEN W J, YANG J, et al. Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route[J]. Microbial Cell Factories, 2017, 16(1): 179.
16	SONNTAG F, KRONER C, LUBUTA P, et al. Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol[J]. Metabolic Engineering, 2015, 32: 82-94.
17	LENNART S V B, REMUS-EMSERMANN M, WEISHAUPT R, et al. A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in Methylobacterium extorquens AM1 and other alphaproteobacteria[J]. ACS Synthetic Biology, 2015, 4(4): 430.
18	CARRILLO M, WAGNER M, PETIT F, et al. Design and control of extrachromosomal elements in Methylorubrum extorquens AM1[J]. ACS Synthetic Biology, 2019, 8(11): 2451-2456.
19	CHISTOSERDOVA L. Modularity of methylotrophy, revisited[J]. Environmental Microbiology, 2011, 13(10): 2603-2622.
20	MÜLLER J E, MEYER F, LITSANOV B, et al. Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress[J]. Molecular Microbiology, 2015, 98(6): 1089-1100.
21	WHITAKER W B, SANDOVAL N R, BENNETT R K, et al. Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization[J]. Current Opinion in Biotechnology, 2015, 33: 165-175.
22	WHITAKER W B, JONES J A, BENNETT R K, et al. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli[J]. Metabolic Engineering, 2017, 39: 49-59.
23	WOOLSTON B M, KING J R, REITER M, et al. Improving formaldehyde consumption drives methanol assimilation in engineered E. coli[J]. Nature Communications, 2018, 9(1): 2387.
24	CHEN F Y H, JUNG H W, TSUEI C Y, et al. Converting Escherichia coli to a synthetic methylotroph growing solely on methanol[J]. Cell, 2020, 182(4): 933-946. e14.
25	WITTHOFF S, SCHMITZ K, NIEDENFÜHR S, et al. Metabolic engineering of Corynebacterium glutamicum for methanol metabolism[J]. Applied and Environmental Microbiology, 2015, 81(6): 2215-2225.
26	MÜLLER J E N, MEYER F, LITSANOV B, et al. Engineering Escherichia coli for methanol conversion[J]. Metabolic Engineering, 2015, 28: 190-201.
27	WU T Y, CHEN C T, LIU J T J, et al. Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1[J]. Applied Microbiology and Biotechnology, 2016, 100(11): 4969-4983.
28	ROTH T B, WOOLSTON B M, STEPHANOPOULOS G, et al. Phage-assisted evolution of Bacillus methanolicus methanol dehydrogenase 2[J]. ACS Synthetic Biology, 2019, 8(4): 796-806.
29	SIEGEL J B, SMITH A L, POUST S, et al. Computational protein design enables a novel one-carbon assimilation pathway[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(12): 3704-3709.
30	LU X, LIU Y, YANG Y, et al. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design[J]. Nature Communications, 2019, 10(1): 1378.
31	PRICE J V, CHEN L, WHITAKER W B, et al. Scaffoldless engineered enzyme assembly for enhanced methanol utilization[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(45): 12691-12696.
32	FAN L, WANG Y, TUYISHIME P, et al. Engineering artificial fusion proteins for enhanced methanol bioconversion[J]. 2018, 19(23): Chembiochem, 2465-2471.
33	OSMAN D, PIERGENTILI C, CHEN J, et al. The effectors and sensory sites of formaldehyde-responsive regulator FrmR and metal-sensing variant[J]. The J ournal of Biological Chemistry, 2016, 291(37): 19502-19516.
34	DENBY K J, IWIG J, BISSON C, et al. The mechanism of a formaldehyde-sensing transcriptional regulator[J]. Scientific Reports, 2016, 6: 38879.
35	ROHLHILL J, SANDOVAL N R, PAPOUTSAKIS E T. Sort-seq approach to engineering a formaldehyde-inducible promoter for dynamically regulated Escherichia coli growth on methanol[J]. ACS Synthetic Biology, 2017, 6(8): 1584-1595.
36	BENNETT R K, GONZALEZ J E, WHITAKER W B, et al. Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph[J]. Metabolic Engineering, 2018, 45: 75-85.
37	BRAUTASET T, JAKOBSEN M Ø M, FLICKINGER M C, et al. Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus[J]. Journal of Bacteriology, 2004, 186(5): 1229-1238.
38	MÜLLER J E, HEGGESET T M, WENDISCH V F, et al. Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol[J]. Applied Microbiology and Biotechnology, 2015, 99(2): 535-551.
39	ROHLHILL J, GERALD HAR J R, ANTONIEWICZ M R, et al. Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation[J]. Metabolic Engineering, 2020, 57: 247-255.
40	KELLER P, NOOR E, MEYER F, et al. Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle[J]. Nature Communications, 2020, 11(1): 5403.
41	MEYER F, KELLER P, HARTL J, et al. Methanol-essential growth of Escherichia coli[J]. Nature Communications, 2018, 9(1): 1508.
42	BENNETT R K, DILLON M, GERALD HAR J R, et al. Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production[J]. Metabolic Engineering, 2020, 60: 45-55.
43	CHEN C T, CHEN F Y, BOGORAD I W, et al. Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production[J]. Metabolic Engineering, 2018, 49: 257-266.
44	TUYISHIME P, WANG Y, FAN L, et al. Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[J]. Metabolic Engineering, 2018, 49: 220-231.
45	GONZALEZ J E, BENNETT R K, PAPOUTSAKIS E T, et al. Methanol assimilation in Escherichia coli is improved by co-utilization of threonine and deletion of leucine-responsive regulatory protein[J]. Metabolic Engineering, 2018, 45: 67-74.
46	LEßMEIER L, PFEIFENSCHNEIDER J, CARNICER M, et al. Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[J]. Applied Microbiology and Biotechnology, 2015, 99(23): 10163-10176.
47	WANG X, WANG X L, LU X L, et al. Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli[J]. Biotechnology for Biofuels, 2019, 12(1): 17.
48	WANG C, REN J, ZHOU L, et al. An aldolase-catalyzed new metabolic pathway for the assimilation of formaldehyde and methanol to synthesize 2-keto-4-hydroxybutyrate and 1, 3-propanediol in Escherichia coli[J]. ACS Synthetic Biology, 2019, 8(11): 2483-2493.
49	YU H, LIAO J C. A modified serine cycle in Escherichia coli coverts methanol and CO₂ to two-carbon compounds[J]. Nature Communications, 2018, 9(1): 3992.
50	HE H, HOPER R, DODENHOFT M, et al. An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli[J]. Metabolic Engineering, 2020, 60: 1-13.
51	DAI Z, GU H, ZHANG S, et al. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae[J]. Bioresource Technology, 2017, 245(pt b): 1407-1412.
52	BOGORAD I W, CHEN C T, THEISEN M K, et al. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(45): 15928-15933.
53	CHOU A, CLOMBURG J M, QIAN S, et al. 2-Hydroxyacyl-CoA lyase catalyzes acyloin condensation for one-carbon bioconversion[J]. Nature Chemical Biology, 2019, 15(9): 900-906.
54	YANG X, YUAN Q, LUO H, et al. Systematic design and in vitro validation of novel one-carbon assimilation pathways[J]. Metabolic Engineering, 2019, 56: 142-153.
55	ZHANG J, JENSEN M K, KEASLING J D. Development of biosensors and their application in metabolic engineering[J]. Current Opinion in Chemical Biology, 2015, 28: 1-8.
56	ROGERS J K, TAYLOR N D, CHURCH G M. Biosensor-based engineering of biosynthetic pathways[J]. Current Opinion in Biotechnology, 2016, 42: 84-91.
57	CHEN N H, DJOKO K Y, VEYRIER F J, et al. Formaldehyde stress responses in bacterial pathogens[J]. Frontiers in Microbiology, 2016, 7: 257.
58	CUI L Y, WANG S S, GUAN C G, et al. Breeding of methanol-tolerant Methylobacterium extorquens AM1 by atmospheric and room temperature plasma mutagenesis combined with adaptive laboratory evolution[J]. Biotechnology Journal, 2018, 13(6): e1700679.
59	HU B, YANG Y M, BECK D A, et al. Comprehensive molecular characterization of Methylobacterium extorquens AM1 adapted for 1-butanol tolerance[J]. Biotechnology for Biofuels, 2016, 9: 84.
60	HU B, LIDSTROM M E. Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production[J]. Biotechnology for Biofuels, 2014, 7(1): 1-10.
61	WANG T M, GUAN C G, GUO J H, et al. Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance[J]. Nature Communications, 2018, 9(1): 2475.
62	MO X H, ZHANG H, WANG T M, et al. Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis[J]. Applied Microbiology and Biotechnology, 2020, 104(10): 4515-4532.
63	GLEIZER S, BEN-NISSAN R, BAR-ON Y M, et al. Conversion of Escherichia coli to generate all biomass carbon from CO₂[J]. Cell, 2019, 179(6): 1255-1263.
64	FABARIUS J T, WEGAT V, ROTH A, SIEBER V. Synthetic methylotrophy in yeasts: towards a circular bioeconomy[J]. Trends in Biotechnology, 2020, 39(4):348-358.
65	DONG C, FONTANA J, PATEL A, et al. Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria[J]. Nature Communications, 2018, 9(1): 2489.
66	GARST A D, BASSALO M C, PINES G, et al. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering[J]. Nature Biotechnology, 2017, 35(1): 48-55.
67	WONG B G, MANCUSO C P, KIRIAKOV S, et al. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER[J]. Nature Biotechnology, 2018, 36(7): 614-623.
68	JIAN X J, GUO X J, WANG J, et al. Microbial microdroplet culture system (MMC): An integrated platform for automated, high-throughput microbial cultivation and adaptive evolution[J]. Biotechnology and Bioengineering, 2020, 117(6): 1724-1737.

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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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