大宗化学品细胞工厂的构建与应用

doi:10.12211/2096-8280.2020-049

摘要/Abstract

摘要：

随着合成生物学技术的发展，越来越多的大宗化学品可通过微生物细胞工厂发酵生产，为摆脱石油资源依赖、节能减排提供了可能。本文首先介绍了细胞工厂构建所需的关键技术，包括基因组编辑技术、多基因同时调控技术、蛋白骨架技术、基因动态调控技术、高通量筛选技术。随后结合丁二酸细胞工厂这一具体案例，从物质代谢、能量代谢及细胞生理代谢三方面阐述了如何解析微生物高效合成化学品的代谢调控机制，为高效细胞工厂创建奠定理论基础。并对近年来成功构建的大宗化学品细胞工厂作了举例介绍，包括L-丙氨酸、L-甲硫氨酸、丁二酸、D-乳酸、丙二酸、L-苹果酸、戊二酸、己二酸、1，3-丙二醇、1，4-丁二醇和异丁醇等。未来，进一步增加原料利用效率和拓宽产物范围是微生物细胞工厂的发展方向，但新酶设计与改造是限制代谢途径设计的主要瓶颈。相信随着研究的深入，微生物细胞工厂将在替代化学法生产大宗化学品方面有更广泛的应用。

关键词: 合成生物学, 大宗化学品, 细胞工厂, 代谢工程, 代谢调控, 基因组编辑

Abstract:

With the development of synthetic biology, more and more bulk chemicals can be produced through microbial cell factories, which can avoid the dependence on petroleum resources and decrease energy cost and pollution. In this review, the key technologies for construction of microbial cell factories were firstly introduced, including genome editing, simultaneous modulation of multiple genes, protein scaffold, gene dynamic modulation and high throughput screening technologies. How to characterize the metabolic regulation mechanisms for efficient production of chemicals was then introduced in three aspects: carbon metabolism, energy metabolism and physiological metabolism, and succinate cell factory was used as an example. Successful bulk chemical cell factories in recent years were then summarized, including L-alanine, L-methionine, succinate, D-lactate, malonate, L-malate, glutarate, adipate, 1,3-propanediol, 1,4-butanediol, isobutanol, etc. In the future, further increasing the efficiency of substrates utilization and broadening the range of products will be the directions of development of microbial cell factories, but the design and engineering of new enzymes are the key bottleneck limiting the design of metabolic pathways. It is believed that with the deepening of research, microbial cell factories will be more widely used in the production of bulk chemicals besides chemical methods.

Key words: synthetic biology, bulk chemicals, cell factories, metabolic engineering, metabolic regulation, genome editing

中图分类号:

于勇, 朱欣娜, 张学礼. 大宗化学品细胞工厂的构建与应用[J]. 合成生物学, 2020, 1(6): 674-684, doi: 10.12211/2096-8280.2020-049.

Yong YU, Xinna ZHU, Xueli ZHANG. Construction and application of microbial cell factories for production of bulk chemicals[J]. Synthetic Biology Journal, 2020, 1(6): 674-684, doi: 10.12211/2096-8280.2020-049.

图/表 5

图1

图2

图3

图4

图5

参考文献 55

1	于勇, 朱欣娜, 刘萍萍, 等. 微生物细胞工厂生产大宗化学品及其产业化进展[J]. 生物产业技术, 2019(1): 13-18.
	YU Y, ZHU X N, LIU P P, et al. Production of bulk chemicals through microbial cell factories and its Industrialization[J]. Biotechnology & Business, 2019(1):13-18.
2	MURPHY K C. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli[J]. Journal of Bacteriology, 1998, 180(8): 2063-2071.
3	ZHANG Y M, BUCHHOLZ F, MUYRERS J P P, et al. A new logic for DNA engineering using recombination in Escherichia coli[J]. Nature Genetics, 1998, 20(2): 123-128.
4	URNOV F D, REBAR E J, HOLMES M C, et al. Genome editing with engineered zinc finger nucleases[J]. Nature Reviews Genetics, 2010, 11(9): 636-646.
5	MILLER J C, TAN S, QIAO G, et al. A TALE nuclease architecture for efficient genome editing[J]. Nature Biotechnology, 2011, 29(2): 143-148.
6	何庆, 王茜, 朱清华, 等. 基因组工程在微生物细胞工厂构建中的应用进展 [J]. 生物产业技术, 2017(1): 31-36.
	HE Q, WANG Q, ZHU Q H, et al. Application progress of genome engineering for construction of microbial cell factories[J]. Biotechnology & Business, 2017(1): 31-36.
7	SHI S, LIANG Y, ZHANG M M, et al. A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2016, 33: 19-27.
8	HEO M J, JUNG H M, UM J, et al. Controlling citrate synthase expression by CRISPR/Cas9 genome editing for n-butanol production in Escherichia coli[J]. ACS Synthetic Biology, 2017, 6(2): 182-189.
9	BASSALO M C, GARST A D, HALWEG-EDWARDS A L, et al. Rapid and efficient one-step metabolic pathway integration in E. coli[J]. ACS Synthetic Biology, 2016, 5(7): 561-568.
10	WASELS F, JEAN-MARIE J, COLLAS F, et al. A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum[J]. Journal of Microbiol Methods, 2017, 140: 5-11.
11	CHO J S, CHOI K R, PRABOWO C P S, et al. CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum[J]. Metabolic Engineering, 2017, 42: 157-167.
12	ANZALONE A V, RANDOLPH P B, DAVIS J R, et al. Search-and-replace genome editing without double-strand breaks or donor DNA[J]. Nature, 2019, 576(7785): 149-157.
13	WANG H H, ISAACS F J, CARR P A, et al. Programming cells by multiplex genome engineering and accelerated evolution[J]. Nature, 2009, 460(7257): 894-898.
14	PFLEGER B F, PITERA D J, SMOLKE C D, et al. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes[J]. Nature Biotechnology, 2006, 24(8): 1027-1032.
15	WARNER J R, REEDER P J, KARIMPOUR-FARD A, et al. Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides[J]. Nature Biotechnology, 2010, 28(8): 856-862.
16	ISAACS F J, CARR P A, WANG H H, et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement[J]. Science, 2011, 333(6040): 348-353.
17	ZHU X N, ZHAO D D, QIU H N, et al. The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway[J]. Metabolic Engineering, 2017, 43: 37-45.
18	LEE M J, MANTELL J, HODGSON L, et al. Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm[J]. Nature Chemical Biology, 2018, 14(2): 142.
19	DUEBER J E, WU G C, MALMIRCHEGINI G R, et al. Synthetic protein scaffolds provide modular control over metabolic flux[J]. Nature Biotechnology, 2009, 27(8): 753.
20	KANG W, MA T, LIU M, et al. Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux[J]. Nature Communications, 2019, 10(1): 4248.
21	ZHANG F Z, CAROTHERS J M, KEASLING J D. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids[J]. Nature Biotechnology, 2012, 30(4): 354.
22	ZHANG X D. Optimal high-throughput screening: practical experimental design and data analysis for genome-scale RNAi research[M]. New York: Cambridge University Press, 2011.
23	SALIS H M, MIRSKY E A, VOIGT C A. Automated design of synthetic ribosome binding sites to control protein expression[J]. Nature Biotechnology, 2009, 27(10): 946-950.
24	KAN S B, LEWIS R D, CHEN K, et al. Directed evolution of cytochrome c for carbon-silicon bond formation: bringing silicon to life[J]. Science, 2016, 354(6315): 1048-1051.
25	SCHMIDL S R, EKNESS F, SOFJAN K, et al. Rewiring bacterial two-component systems by modular DNA-binding domain swapping[J]. Nature Chemical Biology, 2019, 15(7): 690-698.
26	SIKORA A E, TEHAN R, MCPHAIL K. Utilization of Vibrio cholerae as a model organism to screen natural product libraries for identification of new antibiotics[J]. Methods in Molecular Biology, 2018, 1839: 135-146.
27	LU J, TANG J L, LIU Y, et al. Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization[J]. Applied Microbiology and Biotechnology, 2012, 93(6): 2455-2462.
28	ZHANG X L, JANTAMA K, MOORE J C, et al. Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(48): 20180-20185.
29	CHEN J, ZHU X N, TAN Z G, et al. Activating C-4-dicarboxylate transporters DcuB and DcuC for improving succinate production[J]. Applied Microbiology and Biotechnology, 2014, 98(5): 2197-2205.
30	ZHU X N, TAN Z G, XU H T, et al. Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli[J]. Metabolic Engineering, 2014, 24: 87-96.
31	TAN Z G, CHEN J, ZHANG X L. Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production[J]. Biotechnology for Biofuels, 2016, 9: 13.
32	YU Y, ZHU X N, XU H T, et al. Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli[J]. Metabolic Engineering, 2019, 56: 181-189.
33	XIAO M Y, ZHU X N, FAN F Y, et al. Osmotolerance in Escherichia coli is improved by activation of copper efflux genes or supplementation with sulfur-containing amino acids[J]. Applied and Environmental Microbiology, 2017, 83(7): 11.
34	张学礼, 张冬竹. 一种高产L-丙氨酸的XZ-A26菌株及构建方法与应用: CN102329765A[P]. 2011-08-17.
	ZHANG X L, ZHANG D Z. High-yield L-alanine XZ-A26 and construction method and application thereof: CN102329765A[P]. 2011-08-17.
35	马桂燕. 2015年蛋氨酸市场回顾及2016年趋势展望[J]. 中国畜牧业, 2016(10): 50-51.
	MA G Y. Review of methionine market in 2015 and prospect of trend in 2016[J]. China Animal Industry, 2016(10): 50-51.
36	希杰L-蛋氨酸生物利用率高出40％[J]. 中国食品工业, 2015(2): 27.
	The bioavailability of CJ L-methionine was40% higher[J]. China Food, 2015(2): 27.
37	HUANG J F, SHEN Z Y, MAO Q L, et al. Systematic analysis of bottlenecks in a multibranched and multilevel regulated pathway: the molecular fundamentals of L-methionine biosynthesis in Escherichia coli[J]. ACS Synthetic Biology, 2018, 7(11): 2577-2589.
38	DIETRICH J A, FORTMAN J L, STEEN E J. Recombinant host cells for the production of malonate: US 10472653[P]. 2019-11-12.
39	KNUF C, NOOKAEW I, REMMERS I, et al. Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68[J]. Applied Microbiology and Biotechnology, 2014, 98(8): 3517-3527.
40	LI W N, MA L, SHEN X L, et al. Targeting metabolic driving and intermediate influx in lysine catabolism for high-level glutarate production[J]. Nature Communications, 2019, 10: 8.
41	ZHAO M, HUANG D X, ZHANG X J, et al. Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway[J]. Metabolic Engineering, 2018, 47: 254-262.
42	NAKAMURA C E, WHITED G M. Metabolic engineering for the microbial production of 1,3-propanediol[J]. Current Opinion in Biotechnology, 2003, 14(5): 454-459.
43	YIM H, HASELBECK R, NIU W, et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol[J]. Nature Chemical Biology, 2011, 7(7): 445-452.
44	DIEN S J VAN, BURGARD A P, HASELBECK R, et al. Microorganisms for the production of1,4-butanediol and related methods: US 10273508[P]. 2019-04-30.
45	ATSUMI S, HANAI T, LIAO J C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels[J]. Nature, 2008, 451(7174): 86-89.
46	SHELDON R A, PEREIRA P C. Biocatalysis engineering: the big picture[J]. Chemical Society Reviews, 2017, 46(10): 2678-2691.
47	曲戈, 朱彤, 蒋迎迎, 等. 蛋白质工程:从定向进化到计算设计[J]. 生物工程学报, 2019, 35(10): 1843-1856.
	QU G, ZHU T, JIANG Y Y, et al. Protein engineering: from directed evolution to computational design[J]. Chinese Journal of Biotechnology, 2019, 35(10): 1843-1856.
48	YU D, WANG J B, REETZ M T. Exploiting designed oxidase-peroxygenase mutual benefit system for asymmetric cascade reactions[J]. Journal of the American Chemical Society, 2019, 141(14): 5655-5658.
49	The runners-up[J]. Science, 2016, 354(6319): 1518-1523.
50	LEAVER-FAY A, TYKA M, LEWIS S M, et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules[J]. Methods in Enzymology, 2011, 487: 545-574.
51	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.
52	JIANG L, ALTHOFF E A, CLEMENTE F R, et al. De novo computational design of retro-aldol enzymes[J]. Science, 2008, 319(5868): 1387-1391.
53	SIEGEL J B, ZANGHELLINI A, LOVICK H M, et al. Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction[J]. Science, 2010, 329(5989): 309-313.
54	LU X Y, LIU Y W, YANG Y Q, et al. Constructing a synthetic pathway for acetylcoenzyme A from one-carbon through enzyme design[J]. Nature Communications, 2019, 10(1): 1-10.
55	LI R, WIJMA H J, SONG L, et al. Computational redesign of enzymes for regio- and enantioselective hydroamination[J]. Nature Chemical Biology, 2018, 14(7): 664-670.

[1]	卞佳豪, 杨广宇. 人工智能辅助的蛋白质工程[J]. 合成生物学, 2022, 3(3): 429-444.
[2]	冯晴晴, 张天鲛, 赵潇, 聂广军. 合成纳米生物学——合成生物学与纳米生物学的交叉前沿[J]. 合成生物学, 2022, 3(2): 260-278.
[3]	施茜, 吴园园, 杨洋. DNA纳米技术与合成生物学[J]. 合成生物学, 2022, 3(2): 302-319.
[4]	胥欣欣, 匡华. 基于合成受体的食品污染物生物检测进展[J]. 合成生物学, 2022, 3(2): 399-414.
[5]	武伟红, 李炜, 张先恩, 崔宗强. 合成生物学与荧光成像技术[J]. 合成生物学, 2022, 3(2): 369-384.
[6]	郑涵奇, 吴晴, 李洪军, 顾臻. 合成生物学与纳米生物学的交叉融合及其在生物医药领域的应用[J]. 合成生物学, 2022, 3(2): 279-301.
[7]	毕嘉成, 田志刚. 合成免疫学与未来NK细胞免疫治疗[J]. 合成生物学, 2022, 3(1): 22-34.
[8]	张亭, 冷梦甜, 金帆, 袁海. 合成生物研究重大科技基础设施概述[J]. 合成生物学, 2022, 3(1): 184-194.
[9]	郭姝媛, 吴良焕, 刘香健, 王博, 于涛. 微生物中一碳代谢网络构建的进展与挑战[J]. 合成生物学, 2022, 3(1): 116-137.
[10]	赵晓宇, 张浩, 李雪飞, 胡政. 进化视角下的定量生物学规律与人工生命合成[J]. 合成生物学, 2022, 3(1): 6-21.
[11]	褚亚东, 赵宗保. 小型集成化自动移液工作站系统及应用[J]. 合成生物学, 2022, 3(1): 195-208.
[12]	郭思敏, 叶斌, 徐飞. 美德伦理视角下的合成生物学技术伦理治理[J]. 合成生物学, 2022, 3(1): 224-237.
[13]	任师超, 孙秋艳, 冯旭东, 李春. 微生物细胞工厂合成五环三萜皂苷类化合物[J]. 合成生物学, 2022, 3(1): 168-183.
[14]	郭树奇, 焦子悦, 费强. 基于化学品生物合成的嗜甲烷菌人工细胞构建及应用进展[J]. 合成生物学, 2021, 2(6): 1017-1029.
[15]	陈久洲, 王钰, 蒲伟, 郑平, 孙际宾. 5-氨基乙酰丙酸生物合成技术的发展及展望[J]. 合成生物学, 2021, 2(6): 1000-1016.