代谢工程改造大肠杆菌生产琥珀酸

doi:10.16085/j.issn.1000-6613.2021-0531

摘要/Abstract

摘要：

琥珀酸（succinic acid）是一种四碳二羧酸，在食品、医药、塑料和化工行业具有广泛的应用。目前，微生物法生产琥珀酸存在得率低、生产强度低、副产物积累等问题。为此，本研究通过复合诱变（ARTP和⁶⁰Co-γ射线）筛选到一株耐高渗突变株FMME-N-2，其琥珀酸得率为0.70g/g葡萄糖，同时积累18.8g/L乳酸、7.6g/L甲酸和17.3g/L乙酸。为了提高琥珀酸得率，通过敲除乳酸脱氢酶基因（ldhA）、丙酮酸-甲酸裂解酶-甲酸转运蛋白基因（pflB-focA）、磷酸转乙酰基基因（pta）、丙酸激酶基因（tdcD）和a-酮丁酸甲酸酯裂解酶基因（tdcE），阻断冗余代谢支路减少副产物积累，获得工程菌株FMME-N-13，琥珀酸得率增加到0.92g/g葡萄糖，同时副产物大大降低，积累0.6g/L乳酸、3.6g/L甲酸和12.3g/L乙酸。同时，通过调控RBS强度组合优化来自产琥珀酸放线杆菌的磷酸烯醇式丙酮酸羧激酶基因（AsPCK）和来自博伊丁假丝酵母的甲酸脱氢酶基因（CbFDH）的表达水平，调控胞内ATP和NADH的浓度，最优工程菌FMME-N-26（FMME-N-13-L-AsPCK-L-CbFDH）的琥珀酸得率增加至1.04g/g葡萄糖，仅积累5.5g/L乙酸；最终，对厌氧阶段葡萄糖浓度进行优化，当葡萄糖浓度控制在0~5g/L时，菌株FMME-N-26的琥珀酸浓度增加到111.9g/L，得率为1.11g/g葡萄糖（理论产率的99%），生产强度为1.76g/L/h，为琥珀酸的工业化生产奠定了良好的基础。

关键词: 琥珀酸, 大肠杆菌, 复合诱变, 副产物, 辅因子循环, 发酵优化

Abstract:

Succinic acid is a four-carbon dicarboxylic acid, which is widely used in food, medicine, plastics and chemical industries. Microbial production of succinic acid has problems such as low yield, low productivity and by-products accumulation. In this study, through compound mutagenesis (ARTP and ⁶⁰Co-γ), a high osmotic pressure-tolerant mutant strain FMME-N-2 was screened, with a succinic acid yield of 0.70g/g_glucose and an accumulation of 18.8g/L lactic acid, 7.6g/L formic acid and 17.3g/L acetic acid. To decrease the accumulation of by-products and further increase succinic acid yields, the strain FMME-N-13 with a yield of 0.92g/g_glucose was constructed by deleting the genesof ldhA, pflB-focA, pta, tdcD, and tdcE, accumulating 0.6g/L lactic acid, 3.6g/L formic acid and 12.3g/L acetic acid. At the same time, the control of RBS intensity combined with optimizing the level of AsPCK (Actinobacillus succinogenes phosphoenolpyruvate carboxykinase) and CbFDH (Candida boidinii formate dehydrogenase) was used to regulate the concentration of intracellular ATP and NADH, and the succinic acid yield of engineering strain FMME-N-26 (FMME-N-13-L-AsPCK-L-CbFDH) increased to 1.04g/g glucose only with an accumulation of 5.5g/L acetic acid. Finally, the glucose concentration of the anaerobic stage was optimized. When the glucose concentration was controlled at 0—5g/L, succinic acid titer of the strain FMME-N-26 was increased to 111.9g/L, with a yield of 1.11g/g glucose (99% of the theoretical yield) and a productivity of 1.76g/L/h, showing great potential for industrial production.

Key words: succinic acid, E. coli, combined mutagenesis, by-products, cofactors recycle, fermentation optimization

中图分类号:

Q815

唐文秀, 王学明, 郭亮, 季立豪, 高聪, 陈修来, 刘立明. 代谢工程改造大肠杆菌生产琥珀酸[J]. 化工进展, 2022, 41(2): 938-950.

TANG Wenxiu, WANG Xueming, GUO Liang, JI Lihao, GAO Cong, CHEN Xiulai, LIU Liming. Metabolic engineering of Escherichia coli to produce succinic acid[J]. Chemical Industry and Engineering Progress, 2022, 41(2): 938-950.

图/表 10

参考文献 31

1	DAI Z X, GUO F, ZHANG S J, et al. Bio-based succinic acid: an overview of strain development, substrate utilization, and downstream purification[J]. Biofuels, Bioproducts and Biorefining, 2020, 14(5): 965-985.
2	BOZELL J J, PETERSEN G R. Technology development for the production of biobased products from biorefinery carbohydrates—The US Department of Energy’s “Top 10” revisited[J]. Green Chemistry, 2010, 12(4): 539.
3	MIKA L T, CSÉFALVAY E, NÉMETH Á. Catalytic conversion of carbohydrates to initial platform chemicals: chemistry and sustainability[J]. Chemical Reviews, 2018, 118(2): 505-613.
4	GUARNIERI M T, CHOU Y C, SALVACHÚA D, et al. Metabolic engineering of actinobacillus succinogenes provides insights into succinic acid biosynthesis[J]. Applied and Environmental Microbiology, 2017, 83(17): 14-25..
5	AHN J H, SEO H, PARK W, et al. Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase[J]. Nature Communications, 2020, 11: 1970.
6	RAAB A M, GEBHARDT G, BOLOTINA N, et al. Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid[J]. Metabolic Engineering, 2010, 12(6): 518-525.
7	ZHU L W, TANG Y J. Current advances of succinate biosynthesis in metabolically engineered Escherichia coli[J]. Biotechnology Advances, 2017, 35(8): 1040-1048.
8	SÁNCHEZ A M, BENNETT G N, SAN K Y. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity[J]. Metabolic Engineering, 2005, 7(3): 229-239.
9	LIN H, BENNETT G N, SAN K Y. Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate[J]. Biotechnology and Bioengineering, 2005, 89(2): 148-156.
10	JANTAMA K, ZHANG X L, MOORE J C, et al. Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C[J]. Biotechnology and Bioengineering, 2008, 101(5): 881-893.
11	TAN Z G, ZHU X N, CHEN J, et al. Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improvement of succinate production[J]. Applied and Environmental Microbiology, 2013, 79(16): 4838-4844.
12	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, 2009, 106(48): 20180-20185.
13	VEMURI G N, EITEMAN M A, ALTMAN E. Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions[J]. Journal of Industrial Microbiology & Biotechnology, 2002, 28(6): 325-332.
14	WEI L N, ZHU L W, TANG Y J. Succinate production positively correlates with the affinity of the global transcription factor Cra for its effector FBP in Escherichia coli[J]. Biotechnology for Biofuels, 2016, 9: 264.
15	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(1): 262-275.
16	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.
17	JANTAMA K, HAUPT M J, SVORONOS S A, et al. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate[J]. Biotechnology and Bioengineering, 2008, 99(5): 1140-1153.
18	梁丽亚, 马江锋, 刘嵘明, 等. 过量表达苹果酸脱氢酶对大肠杆菌NZH111产丁二酸的影响[J]. 生物工程学报, 2011, 27(7): 1005-1012.
	LIANG Liya, MA Jiangfeng, LIU Rongming, et al. Effect of overexpression of malate dehydrogenase on succinic acid production in Escherichia coli NZN111[J]. Chinese Journal of Biotechnology, 2011, 27(7): 1005-1012.
19	SEONG W, HAN G H, LIM H S, et al. Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption[J]. Metabolic Engineering, 2020, 62: 249-259.
20	FERONE M, RAGANATI F, OLIVIERI G, et al. Bioreactors for succinic acid production processes[J]. Critical Reviews in Biotechnology, 2019, 39(4): 571-586.
21	HU G P, ZHOU J, CHEN X L, et al. Engineering synergetic CO₂-fixing pathways for malate production[J]. Metabolic Engineering, 2018, 47: 496-504.
22	ZHANG C, SONG W, LIU J, et al. Production of enantiopure (R)-or (S)-2-hydroxy-4-(methylthio) butanoic acid by multi-enzyme cascades[J]. Bioresources and Bioprocessing, 2019, 6(1): 12-20.
23	ANDERSSON C, HELMERIUS J, HODGE D, et al. Inhibition of succinic acid production in metabolically engineered Escherichia coli by neutralizing agent, organic acids, and osmolarity[J]. Biotechnology Progress, 2009, 25(1): 116-123.
24	LIU L M, XU Q L, LI Y, et al. Enhancement of pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata[J]. Biotechnology and Bioengineering, 2007, 97(4): 825-832.
25	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): e03050.
26	XIAO M Y, ZHU X N, XU H T, et al. A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli[J]. BMC Biotechnology, 2017, 17(1): 1-11.
27	ZHANG C, GOU D, MEI J, et al. Isolation of high osmotic-tolerant mutants of Escherichia coli for succinic acid production by metabolic evolution[J]. Chinese Journal of Biotechnology, 2012, 28(11): 1337-1345.
28	XU H T, ZHOU Z H, WANG C, et al. Enhanced succinic acid production in Corynebacterium glutamicum with increasing the available NADH supply and glucose consumption rate by decreasing H⁺-ATPase activity[J]. Biotechnology Letters, 2016, 38(7): 1181-1186.
29	LEE J W, YI J, KIM T Y, et al. Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens[J]. Metabolic Engineering, 2016, 38: 409-417.
30	CHEN X Z, ZHOU L, TIAN K M, et al. Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production[J]. Biotechnology Advances, 2013, 31(8): 1200-1223.
31	LI J J, LI Y K, CUI Z Y, et al. Enhancement of succinate yield by manipulating NADH/NAD⁺ ratio and ATP generation[J]. Applied Microbiology and Biotechnology, 2017, 101(8): 3153-3161.

菌株/质粒	相关特性	来源
质粒
pKD4	oriR6Kγ，Kan，rgnB（Ter）	本研究室
pKD46	pSC101ts ori，bla，PBAD，gam，bet，exo	本研究室
pCP20	pSC101ts ori，Cm，Flp	本研究室
pETM6R1	ColE1 ori，Amp，P_Tac	本研究构建
pETM6R1-AsPCK	ColE1 ori，Amp，P_Tac，AsPCK	本研究构建
pETM6R1-CbFDH	ColE1 ori，Amp，P_Tac，CbFDH	本研究构建
pETM6R1-AsPCK- CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，	本研究构建
pETM6R1-RBS10（H）-AsPCK-RBS09（M）-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS10，RBS09	本研究构建
pETM6R1-RBS10（H）-AsPCK-RBS03（L）-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS10，RBS03	本研究构建
pETM6R1-H-AsPCK-H-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS10，RBS10	本研究构建
pETM6R1-M-AsPCK-M-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS09，RBS09	本研究构建
pETM6R1-M-AsPCK-L-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS09，RBS03	本研究构建
pETM6R1-M-AsPCK-H-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS09，RBS10	本研究构建
pETM6R1-L-AsPCK-M-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS03，RBS09	本研究构建
pETM6R1-L-AsPCK-L-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS03，RBS03	本研究构建
pETM6R1-L-AsPCK-H-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS03，RBS10	本研究构建
菌株
E. coli JM109	General cloning host	Addgene
E. coli FMME-N	Wild type screened from rumen of camel（preservation number，CCTCC M2018568）	本研究室
FMME-N-1	After ARTP mutagenesis breeding from FMME-N	本研究室筛选
FMME-N-2	After⁶⁰Co-γ irradiation mutagenesis breeding from FMME-N-1	本研究室筛选
FMME-N-3	FMME-N-2 ΔpflB-focA	本研究构建
FMME-N-4	FMME-N-2 ΔldhA	本研究构建
FMME-N-5	FMME-N-2 Δpta	本研究构建
FMME-N-6	FMME-N-2 ΔackA	本研究构建
FMME-N-7	FMME-N-2 ΔpflB-focAΔldhA	本研究构建
FMME-N-8	FMME-N-2 ΔpflB-focAΔpta	本研究构建
FMME-N-9	FMME-N-2 ΔpflB-focAΔackA	本研究构建
FMME-N-10	FMME-N-2 ΔpflB-focAΔldhAΔpta	本研究构建
FMME-N-11	FMME-N-2 ΔpflB-focAΔldhAΔackA	本研究构建
FMME-N-12	FMME-N-2 ΔpflB-focAΔldhAΔptaΔackA	本研究构建
FMME-N-13	FMME-N-2 ΔpflB-focAΔldhAΔptaΔtdcDΔtdcE	本研究构建
FMME-N-14	FMME-N-13 pETM6R1-AsPCK	本研究构建
FMME-N-15	FMME-N-13 pETM6R1-CbFDH	本研究构建
FMME-N-16	FMME-N-13 pETM6R1-AsPCK-CbFDH	本研究构建
FMME-N-17	FMME-N-13 pETM6R1-H-AsPCK-M-CbFDH	本研究构建
FMME-N-18	FMME-N-13 pETM6R1-H-AsPCK-L-CbFDH	本研究构建
FMME-N-19	FMME-N-13 pETM6R1-H-AsPCK-H-CbFDH	本研究构建
FMME-N-20	FMME-N-13 pETM6R1-M-AsPCK-M-CbFDH	本研究构建
FMME-N-21	FMME-N-13 pETM6R1-M-AsPCK-L-CbFDH	本研究构建
FMME-N-22	FMME-N-13 pETM6R1-M-AsPCK-H-CbFDH	本研究构建
FMME-N-23	FMME-N-13 pETM6R1-L-AsPCK-M-CbFDH	本研究构建
FMME-N-24	FMME-N-13 pETM6R1-L-AsPCK-L-CbFDH	本研究构建
FMME-N-25	FMME-N-13 pETM6R1-L-AsPCK-H-CbFDH	本研究构建
FMME-N-26	FMME-N-13-L-AsPCK-L-CbFDH	本研究构建

菌株/质粒	相关特性	来源
质粒
pKD4	oriR6Kγ，Kan，rgnB（Ter）	本研究室
pKD46	pSC101ts ori，bla，PBAD，gam，bet，exo	本研究室
pCP20	pSC101ts ori，Cm，Flp	本研究室
pETM6R1	ColE1 ori，Amp，P_Tac	本研究构建
pETM6R1-AsPCK	ColE1 ori，Amp，P_Tac，AsPCK	本研究构建
pETM6R1-CbFDH	ColE1 ori，Amp，P_Tac，CbFDH	本研究构建
pETM6R1-AsPCK- CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，	本研究构建
pETM6R1-RBS10（H）-AsPCK-RBS09（M）-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS10，RBS09	本研究构建
pETM6R1-RBS10（H）-AsPCK-RBS03（L）-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS10，RBS03	本研究构建
pETM6R1-H-AsPCK-H-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS10，RBS10	本研究构建
pETM6R1-M-AsPCK-M-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS09，RBS09	本研究构建
pETM6R1-M-AsPCK-L-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS09，RBS03	本研究构建
pETM6R1-M-AsPCK-H-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS09，RBS10	本研究构建
pETM6R1-L-AsPCK-M-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS03，RBS09	本研究构建
pETM6R1-L-AsPCK-L-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS03，RBS03	本研究构建
pETM6R1-L-AsPCK-H-CbFDH	ColE1 ori，Amp，P_Tac，AsPCK，CbFDH，RBS03，RBS10	本研究构建
菌株
E. coli JM109	General cloning host	Addgene
E. coli FMME-N	Wild type screened from rumen of camel（preservation number，CCTCC M2018568）	本研究室
FMME-N-1	After ARTP mutagenesis breeding from FMME-N	本研究室筛选
FMME-N-2	After⁶⁰Co-γ irradiation mutagenesis breeding from FMME-N-1	本研究室筛选
FMME-N-3	FMME-N-2 ΔpflB-focA	本研究构建
FMME-N-4	FMME-N-2 ΔldhA	本研究构建
FMME-N-5	FMME-N-2 Δpta	本研究构建
FMME-N-6	FMME-N-2 ΔackA	本研究构建
FMME-N-7	FMME-N-2 ΔpflB-focAΔldhA	本研究构建
FMME-N-8	FMME-N-2 ΔpflB-focAΔpta	本研究构建
FMME-N-9	FMME-N-2 ΔpflB-focAΔackA	本研究构建
FMME-N-10	FMME-N-2 ΔpflB-focAΔldhAΔpta	本研究构建
FMME-N-11	FMME-N-2 ΔpflB-focAΔldhAΔackA	本研究构建
FMME-N-12	FMME-N-2 ΔpflB-focAΔldhAΔptaΔackA	本研究构建
FMME-N-13	FMME-N-2 ΔpflB-focAΔldhAΔptaΔtdcDΔtdcE	本研究构建
FMME-N-14	FMME-N-13 pETM6R1-AsPCK	本研究构建
FMME-N-15	FMME-N-13 pETM6R1-CbFDH	本研究构建
FMME-N-16	FMME-N-13 pETM6R1-AsPCK-CbFDH	本研究构建
FMME-N-17	FMME-N-13 pETM6R1-H-AsPCK-M-CbFDH	本研究构建
FMME-N-18	FMME-N-13 pETM6R1-H-AsPCK-L-CbFDH	本研究构建
FMME-N-19	FMME-N-13 pETM6R1-H-AsPCK-H-CbFDH	本研究构建
FMME-N-20	FMME-N-13 pETM6R1-M-AsPCK-M-CbFDH	本研究构建
FMME-N-21	FMME-N-13 pETM6R1-M-AsPCK-L-CbFDH	本研究构建
FMME-N-22	FMME-N-13 pETM6R1-M-AsPCK-H-CbFDH	本研究构建
FMME-N-23	FMME-N-13 pETM6R1-L-AsPCK-M-CbFDH	本研究构建
FMME-N-24	FMME-N-13 pETM6R1-L-AsPCK-L-CbFDH	本研究构建
FMME-N-25	FMME-N-13 pETM6R1-L-AsPCK-H-CbFDH	本研究构建
FMME-N-26	FMME-N-13-L-AsPCK-L-CbFDH	本研究构建

培养基	成分
LB培养基	蛋白胨10g?L^-1，酵母粉5g?L^-1，氯化钠10g?L^-1
固体筛选培养基	葡萄糖16g?L^-1，酵母粉5g?L^-1，蛋白胨5g?L^-1，NaCl 1.5g?L^-1，KH₂PO₄ 1.16g?L^-1，K₂HPO₄ 0.5g?L^-1，MgSO₄·7H₂O 0.3g?L^-1，NaHCO₃ 0.4g?L^-1，溴甲酚紫0.05g?L^-1，琼脂粉20g?L^-1，添加相应浓度的NaCl
发酵培养基	葡萄糖20g?L^-1，玉米浆5g?L^-1，（NH₄）₂SO₄ 3.3g?L^-1，KH₂PO₄ 0.6g?L^-1，K₂HPO₄ 1.4g?L^-1，NaCl 1g?L^-1，MgSO₄·7H₂O 0.4g?L^-1
抗生素	氨苄青霉素（Amp），硫酸卡那霉素（Kan），氯霉素（Chl）
诱导剂	IPTG，阿拉伯糖

培养基	成分
LB培养基	蛋白胨10g?L^-1，酵母粉5g?L^-1，氯化钠10g?L^-1
固体筛选培养基	葡萄糖16g?L^-1，酵母粉5g?L^-1，蛋白胨5g?L^-1，NaCl 1.5g?L^-1，KH₂PO₄ 1.16g?L^-1，K₂HPO₄ 0.5g?L^-1，MgSO₄·7H₂O 0.3g?L^-1，NaHCO₃ 0.4g?L^-1，溴甲酚紫0.05g?L^-1，琼脂粉20g?L^-1，添加相应浓度的NaCl
发酵培养基	葡萄糖20g?L^-1，玉米浆5g?L^-1，（NH₄）₂SO₄ 3.3g?L^-1，KH₂PO₄ 0.6g?L^-1，K₂HPO₄ 1.4g?L^-1，NaCl 1g?L^-1，MgSO₄·7H₂O 0.4g?L^-1
抗生素	氨苄青霉素（Amp），硫酸卡那霉素（Kan），氯霉素（Chl）
诱导剂	IPTG，阿拉伯糖

参数	处理条件
载气	He
输出功率	100W
辐照距离	2mm
气体流量	10 SLM
辐照时间	0s、15s、30s、45s、46s、75s、90s