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.

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.

图/表 5

图1 微生物高效合成化学品的代谢调控机制

Fig. 1 Mechanisms of metabolic regulation for efficient production of chemicals

图2 丁二酸合成的4个限速步骤

Fig. 2 Four rate-limiting steps for synthesis of succinate

图3 调控葡萄糖能量代谢模式解决丁二酸合成中的还原力问题

Fig. 3 Regulation of glucose energy metabolism to solve reducing equivalent problem for synthesis of succinate

图4 调控甘油能量代谢模式解决丁二酸合成中的能量问题

Fig. 4 Regulation of glycerol energy metabolism to solve energy problem for synthesis of succinate

图5 大肠杆菌细胞耐渗透胁迫的新机制(a)在正常渗透压条件下（5%葡萄糖），铜离子大多以低毒的二价离子形式存在，Cus系统不激活；（b)在高渗透压条件下（12%葡萄糖），周质空间中的一价铜离子含量升高，激活了copA、cusS、cusR和cusCFAB表达量提高，转运一价铜离子的能力增强；（c）在高渗透压条件下（12%葡萄糖），突变后的cusS及cusCFBA表达量提高，转运一价铜离子的能力增强;(d)甲硫氨酸、半胱氨酸等渗透保护物种可螯合一价铜离子

Fig. 5 New osmotolerance mechanism of Escherichia coli(a) Under normal conditions (5% glucose), copper exists mostly in the form of less-toxic Cu(Ⅱ), and the CopA and Cus system was not induced; (b) Under high osmotic pressure (12% glucose), increasing free Cu(Ⅰ) in the periplasmic space activates the expression of copA, cusS, cusR and cusCFAB to expel toxic Cu(Ⅰ); (c) Under high osmotic pressure (12% glucose), expression levels of cusS and cusCFAB are increased to expel more Cu(Ⅰ) from the periplasmic space; (d) Osmotic protective substances such as methionine and cysteine can chelate valence copper ions.

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]	孙梦楚, 陆亮宇, 申晓林, 孙新晓, 王佳, 袁其朋. 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建[J]. 合成生物学, 2023, 4(5): 947-965.
[2]	刁志钿, 王喜先, 孙晴, 徐健, 马波. 单细胞拉曼光谱测试分选装备研制及应用进展[J]. 合成生物学, 2023, 4(5): 1020-1035.
[3]	卢挥, 张芳丽, 黄磊. 合成生物学自动化装置iBioFoundry的构建与应用[J]. 合成生物学, 2023, 4(5): 877-891.
[4]	白仲虎, 任和, 聂简琪, 孙杨. 高通量平行发酵技术的发展与应用[J]. 合成生物学, 2023, 4(5): 904-915.
[5]	吴玉洁, 刘欣欣, 刘健慧, 杨开广, 随志刚, 张丽华, 张玉奎. 基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展[J]. 合成生物学, 2023, 4(5): 1000-1019.
[6]	胡哲辉, 徐娟, 卞光凯. 自动化高通量技术在天然产物生物合成中的应用[J]. 合成生物学, 2023, 4(5): 932-946.
[7]	刘欢, 崔球. 原位电离质谱技术在微生物菌株筛选中的应用进展[J]. 合成生物学, 2023, 4(5): 980-999.
[8]	陈永灿, 司同, 张建志. 自动化合成生物技术在DNA组装与微生物底盘操作中的应用[J]. 合成生物学, 2023, 4(5): 857-876.
[9]	王雁南, 孙宇辉. 碱基编辑技术及其在微生物合成生物学中的应用[J]. 合成生物学, 2023, 4(4): 720-737.
[10]	刘晚秋, 季向阳, 许慧玲, 卢屹聪, 李健. 限制性内切酶的无细胞快速制备研究[J]. 合成生物学, 2023, 4(4): 840-851.
[11]	孙美莉, 王凯峰, 陆然, 纪晓俊. 解脂耶氏酵母底盘细胞的工程改造及应用[J]. 合成生物学, 2023, 4(4): 779-807.
[12]	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.
[13]	孙智, 杨宁, 娄春波, 汤超, 杨晓静. 功能拓扑的理性设计及其在合成生物学中的应用[J]. 合成生物学, 2023, 4(3): 444-463.
[14]	赖奇龙, 姚帅, 查毓国, 白虹, 宁康. 微生物组生物合成基因簇发掘方法及应用前景[J]. 合成生物学, 2023, 4(3): 611-627.
[15]	孟巧珍, 郭菲. “可折叠性”在酶智能设计改造中的应用研究——以AlphaFold2为例[J]. 合成生物学, 2023, 4(3): 571-589.