Metabolic engineering of Escherichia coli for chondroitin production

doi:10.11949/0438-1157.20221582

Abstract

Abstract:

The current research strategies for biosynthesizing chondroitin mainly focus on the construction of synthetic pathways, lacking the fine regulation of the supply of precursor substances, thus limiting the synthesis efficiency of chondroitin. To solve the above problems, in this study, the biosynthetic pathway for chondroitin synthesis was reconstructed and optimized in Escherichia coli by different metabolic engineering strategies, obtaining a microbial cell factory for chondroitin synthesis by fermentation. A complete synthesis pathway for chondroitin was constructed in E. coli BL21 STAR (DE3) by expressing UDP-glucose dehydrogenase (KfoF), UDP-glucosamine isomerase (KfoA) and chondroitin polymerase (KfoC). The supply of chondroitin precursor UDP-GalNAc was improved by optimizing the gene expression levels of aminotransferase (GlmS) and phosphoglucosamine mutase (GlmM). The supply efficiency of chondroitin synthesis precursor UDP-GlcA was improved by optimizing the gene expression level of KfoF. Finally, with the optimized strain E. coli GZ17, chondroitin production was increased to 2.95 g/L in a 5 L fermenter. The above research strategy laid the foundation for the construction and application of chondroitin sulfate strains, and also provided a reference for metabolic engineering to produce other glycosaminoglycans.

Key words: chondroitin, Escherichia coli, metabolic engineering, pathway optimization

摘要：

目前生物法合成软骨素的研究策略主要侧重于构建合成路径，缺乏对前体物质供应的精细化调控，从而限制了软骨素的合成效率。为了解决上述问题，本研究借助代谢工程策略，在大肠杆菌(Escherichia coli)中重构与优化了软骨素合成路径，获得了合成软骨素的微生物细胞工厂，实现了发酵法生产软骨素。通过在E. coli BL21 STAR (DE3)中表达UDP-葡萄糖脱氢酶(KfoF)、UDP-氨基葡萄糖异构酶(KfoA)和软骨素聚合酶(KfoC)，构建了完整的软骨素合成路径。通过氨基转移酶(GlmS)和磷酸葡萄糖胺变位酶(GlmM)的基因表达水平优化，提高了软骨素合成前体UDP-GalNAc的供给量；优化KfoF的基因表达水平，改善了软骨素合成前体UDP-GlcA的供给效率。在5 L发酵罐上，最优工程菌株E. coli GZ17的软骨素产量达到了2.95 g/L。上述研究策略为硫酸软骨素菌株的构建与应用奠定了基础，也为代谢工程改造生产其他糖胺聚糖提供了借鉴。

关键词: 软骨素, 大肠杆菌, 代谢工程, 路径优化

CLC Number:

Q 815

Chunlei ZHAO, Liang GUO, Cong GAO, Wei SONG, Jing WU, Jia LIU, Liming LIU, Xiulai CHEN. Metabolic engineering of Escherichia coli for chondroitin production[J]. CIESC Journal, 2023, 74(5): 2111-2122.

赵春雷, 郭亮, 高聪, 宋伟, 吴静, 刘佳, 刘立明, 陈修来. 代谢工程改造大肠杆菌生产软骨素[J]. 化工学报, 2023, 74(5): 2111-2122.

Figures/Tables 10

References 32

1	Laezza A, Iadonisi A, de Castro C, et al. Chemical fucosylation of a polysaccharide: a semisynthetic access to fucosylated chondroitin sulfate[J]. Biomacromolecules, 2015, 16(7): 2237-2245.
2	Zhang X, Liu H Y, Yao W, et al. Semisynthesis of chondroitin sulfate oligosaccharides based on the enzymatic degradation of chondroitin[J]. The Journal of Organic Chemistry, 2019, 84(11): 7418-7425.
3	Legendre F, Bauge C, Roche R, et al. Chondroitin sulfate modulation of matrix and inflammatory gene expression in IL-1β-stimulated chondrocytes—study in hypoxic alginate bead cultures[J]. Osteoarthritis and Cartilage, 2008, 16(1): 105-114.
4	Maruyama T, Toida T, Imanari T, et al. Conformational changes and anticoagulant activity of chondroitin sulfate following its O-sulfonation[J]. Carbohydrate Research, 1998, 306(1/2): 35-43.
5	Martins J R M, Gadelha M E C, Fonseca S M, et al. Patients with head and neck tumors excrete a chondroitin sulfate with a low degree of sulfation: a new tool for diagnosis and follow-up of cancer therapy[J]. Otolaryngology-Head and Neck Surgery, 2000, 122(1): 115-118.
6	Volpi N. Chondroitin sulfate safety and quality[J]. Molecules, 2019, 24(8): 1447.
7	Couto M R, Rodrigues J L, Rodrigues L R. Heterologous production of chondroitin[J]. Biotechnology Reports, 2022, 33: e00710.
8	Shi Y G, Meng Y C, Li J R, et al. Chondroitin sulfate: extraction, purification, microbial and chemical synthesis[J]. Journal of Chemical Technology & Biotechnology, 2014, 89(10): 1445-1465.
9	Abdallah M M, Fernandez N, Matias A A, et al. Hyaluronic acid and chondroitin sulfate from marine and terrestrial sources: extraction and purification methods[J]. Carbohydrate Polymers, 2020, 243: 116441.
10	Xu S Q, Qiu M L, Zhang Q, et al. Chain structure and immunomodulatory activity of a fructosylated chondroitin from an engineered Escherichia coli K4[J]. International Journal of Biological Macromolecules, 2019, 133: 702-711.
11	Badri A, Williams A, Awofiranye A, et al. Complete biosynthesis of a sulfated chondroitin in Escherichia coli [J]. Nature Communications, 2021, 12: 1389.
12	Kobayashi S, Morii H, Itoh R, et al. Enzymatic polymerization to artificial hyaluronan: a novel method to synthesize a glycosaminoglycan using a transition state analogue monomer[J]. Journal of the American Chemical Society, 2001, 123(47): 11825-11826.
13	Li J E, Sparkenbaugh E M, Su G W, et al. Enzymatic synthesis of chondroitin sulfate E to attenuate bacteria lipopolysaccharide-induced organ damage[J]. ACS Central Science, 2020, 6(7): 1199-1207.
14	Li J E, Su G W, Liu J. Enzymatic synthesis of homogeneous chondroitin sulfate oligosaccharides[J]. Angewandte Chemie, 2017, 56(39): 11784-11787.
15	Sugiura N, Shimokata S, Minamisawa T, et al. Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants[J]. Glycoconjugate Journal, 2008, 25(6): 521-530.
16	Gottschalk J, Zaun H, Eisele A, et al. Key factors for A one-pot enzyme cascade synthesis of high molecular weight hyaluronic acid[J]. International Journal of Molecular Sciences, 2019, 20(22): 5664.
17	Zhang Q, Yao R, Chen X L, et al. Enhancing fructosylated chondroitin production in Escherichia coli K4 by balancing the UDP-precursors[J]. Metabolic Engineering, 2018, 47: 314-322.
18	Bedini E, De Castro C, De Rosa M, et al. A microbiological-chemical strategy to produce chondroitin sulfate A, C[J]. Angewandte Chemie, 2011, 50(27): 6160-6163.
19	Cimini D, Restaino O F, Catapano A, et al. Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications[J]. Applied Microbiology and Biotechnology, 2010, 85(6): 1779-1787.
20	Restaino O F, Cimini D, De Rosa M, et al. High-performance CE of Escherichia coli K4 cell surface polysaccharides[J]. Electrophoresis, 2009, 30(22): 3877-3883.
21	He W Q, Fu L, Li G Y, et al. Production of chondroitin in metabolically engineered E. coli [J]. Metabolic Engineering, 2015, 27: 92-100.
22	Cheng F Y, Luozhong S J, Yu H M, et al. Biosynthesis of chondroitin in engineered Corynebacterium glutamicum [J]. Journal of Microbiology and Biotechnology, 2019, 29(3): 392-400.
23	Jin P, Zhang L P, Yuan P H, et al. Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis [J]. Carbohydrate Polymers, 2016, 140: 424-432.
24	Zhou Z X, Li Q, Huang H, et al. A microbial-enzymatic strategy for producing chondroitin sulfate glycosaminoglycans[J]. Biotechnology and Bioengineering, 2018, 115(6): 1561-1570.
25	Karim A S, Curran K A, Alper H S. Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications[J]. FEMS Yeast Research, 2013, 13(1): 107-116.
26	Kang C W, Lim H G, Yang J, et al. Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number[J]. Metabolic Engineering, 2018, 48: 121-128.
27	Zhao M, Huang D, Zhang X, et al. Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway[J]. Metabolic Engineering, 2018, 47: 254-262.
28	Zhang C, Liu L, Teng L, et al. Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor[J]. Metabolic Engineering, 2012, 14(5): 521-527.
29	Jin X R, Zhang W J, Wang Y, et al. Biosynthesis of non-animal chondroitin sulfate from methanol using genetically engineered Pichia pastoris [J]. Green Chemistry, 2021, 23(12): 4365-4374.
30	Cimini D, Iacono I D, Carlino E, et al. Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest[J]. AMB Express, 2017, 7(1): 61.
31	Rouches M V, Xu Y S, Cortes L B G, et al. A plasmid system with tunable copy number[J]. Nature Communications, 2022, 13: 3908.
32	Lemuth K, Steuer K, Albermann C. Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin[J]. Microbial Cell Factories, 2011, 10: 29.

质粒	描述	来源
pEM	T5 promoter, Amp^R, f1 ori	实验室储存
pCDR	Trc promoter, Str^R, CloDF13 ori	实验室储存
pEM-kfoA	pEM连接基因kfoA	本研究构建
pCDR-kfoF	pCDR连接基因kfoF	本研究构建
pEM-RBS_L	pEM使用低表达强度RBS	本研究构建
pEM-RBS_M	pEM使用中表达强度RBS	本研究构建
pEM-RBS_H	pEM使用高表达强度RBS	本研究构建
pCDR-kfoF-kfoC-kfoA	pCDR连接基因kfoA、kfoC、kfoF	本研究构建
pEM-RBS_L-glmS	pEM连接低表达强度的glmS基因	本研究构建
pEM-RBS_M-glmS	pEM连接中表达强度的glmS基因	本研究构建
pEM-RBS_H-glmS	pEM连接高表达强度的glmS基因	本研究构建
pEM-RBS_L-glmM	pEM连接低表达强度的glmM基因	本研究构建
pEM-RBS_M-glmM	pEM连接中表达强度的glmM基因	本研究构建
pEM-RBS_H-glmM	pEM连接高表达强度的glmM基因	本研究构建
pEM-RBS_L-kfoF	pEM连接低表达强度的kfoF基因	本研究构建
pEM-RBS_M-kfoF	pEM连接中表达强度的kfoF基因	本研究构建
pEM-RBS_H-kfoF	pEM连接高表达强度的kfoF基因	本研究构建

质粒	描述	来源
pEM	T5 promoter, Amp^R, f1 ori	实验室储存
pCDR	Trc promoter, Str^R, CloDF13 ori	实验室储存
pEM-kfoA	pEM连接基因kfoA	本研究构建
pCDR-kfoF	pCDR连接基因kfoF	本研究构建
pEM-RBS_L	pEM使用低表达强度RBS	本研究构建
pEM-RBS_M	pEM使用中表达强度RBS	本研究构建
pEM-RBS_H	pEM使用高表达强度RBS	本研究构建
pCDR-kfoF-kfoC-kfoA	pCDR连接基因kfoA、kfoC、kfoF	本研究构建
pEM-RBS_L-glmS	pEM连接低表达强度的glmS基因	本研究构建
pEM-RBS_M-glmS	pEM连接中表达强度的glmS基因	本研究构建
pEM-RBS_H-glmS	pEM连接高表达强度的glmS基因	本研究构建
pEM-RBS_L-glmM	pEM连接低表达强度的glmM基因	本研究构建
pEM-RBS_M-glmM	pEM连接中表达强度的glmM基因	本研究构建
pEM-RBS_H-glmM	pEM连接高表达强度的glmM基因	本研究构建
pEM-RBS_L-kfoF	pEM连接低表达强度的kfoF基因	本研究构建
pEM-RBS_M-kfoF	pEM连接中表达强度的kfoF基因	本研究构建
pEM-RBS_H-kfoF	pEM连接高表达强度的kfoF基因	本研究构建

菌株	描述	来源
E. coli JM109	Wild-type E. coli strain	实验室储存
E. coli BL21 STAR (DE3)(简称STAR)	Wild-type E. coli strain	Addgene公司购买
E. coli GZ00	STAR带有重组质粒pCDR-kfoF-kfoC-kfoA	本研究构建
E. coli GZ01	STAR基因组整合kfoF-kfoC-kfoA	本研究构建
E. coli GZ02	GZ00带有重组质粒pEM-RBSL-glmS	本研究构建
E. coli GZ03	GZ00带有重组质粒pEM-RBSM-glmS	本研究构建
E. coli GZ04	GZ00带有重组质粒pEM-RBSH-glmS	本研究构建
E. coli GZ05	GZ01增加一个拷贝glmS	本研究构建
E. coli GZ06	GZ01增加两个拷贝glmS	本研究构建
E. coli GZ07	GZ01增加三个拷贝glmS	本研究构建
E. coli GZ08	GZ00带有重组质粒pEM-RBSL-glmM	本研究构建
E. coli GZ09	GZ00带有重组质粒pEM-RBSM-glmM	本研究构建
E. coli GZ10	GZ00带有重组质粒pEM-RBSH-glmM	本研究构建
E. coli GZ11	GZ06增加一个拷贝glmM	本研究构建
E. coli GZ12	GZ06增加两个拷贝glmM	本研究构建
E. coli GZ13	GZ06增加三个拷贝glmM	本研究构建
E. coli GZ14	GZ00带有重组质粒pEM-RBSL-kfoF	本研究构建
E. coli GZ15	GZ00带有重组质粒pEM-RBSM-kfoF	本研究构建
E. coli GZ16	GZ00带有重组质粒pEM-RBSH-kfoF	本研究构建
E. coli GZ17	GZ12增加一个拷贝kfoF	本研究构建
E. coli GZ18	GZ12增加两个拷贝kfoF	本研究构建

菌株	描述	来源
E. coli JM109	Wild-type E. coli strain	实验室储存
E. coli BL21 STAR (DE3)(简称STAR)	Wild-type E. coli strain	Addgene公司购买
E. coli GZ00	STAR带有重组质粒pCDR-kfoF-kfoC-kfoA	本研究构建
E. coli GZ01	STAR基因组整合kfoF-kfoC-kfoA	本研究构建
E. coli GZ02	GZ00带有重组质粒pEM-RBSL-glmS	本研究构建
E. coli GZ03	GZ00带有重组质粒pEM-RBSM-glmS	本研究构建
E. coli GZ04	GZ00带有重组质粒pEM-RBSH-glmS	本研究构建
E. coli GZ05	GZ01增加一个拷贝glmS	本研究构建
E. coli GZ06	GZ01增加两个拷贝glmS	本研究构建
E. coli GZ07	GZ01增加三个拷贝glmS	本研究构建
E. coli GZ08	GZ00带有重组质粒pEM-RBSL-glmM	本研究构建
E. coli GZ09	GZ00带有重组质粒pEM-RBSM-glmM	本研究构建
E. coli GZ10	GZ00带有重组质粒pEM-RBSH-glmM	本研究构建
E. coli GZ11	GZ06增加一个拷贝glmM	本研究构建
E. coli GZ12	GZ06增加两个拷贝glmM	本研究构建
E. coli GZ13	GZ06增加三个拷贝glmM	本研究构建
E. coli GZ14	GZ00带有重组质粒pEM-RBSL-kfoF	本研究构建
E. coli GZ15	GZ00带有重组质粒pEM-RBSM-kfoF	本研究构建
E. coli GZ16	GZ00带有重组质粒pEM-RBSH-kfoF	本研究构建
E. coli GZ17	GZ12增加一个拷贝kfoF	本研究构建
E. coli GZ18	GZ12增加两个拷贝kfoF	本研究构建

菌株	OD₆₀₀	CH/ (mg/L)	CH对细胞得率/ (mg/g)	CH对甘油得率/ (mg/g)
GZ01	11.0	7.1	1.6	0.5
GZ05	11.2	29.9	6.5	2.0
GZ06	11.8	46.2	9.5	3.1
GZ07	11.2	45.1	9.8	3.0
GZ11	12.2	54.4	10.8	3.6
GZ12	12.8	80.1	15.2	5.3
GZ13	12.2	79.9	15.9	5.3
GZ17	12.1	108.8	21.8	7.3
GZ18	11.6	70.7	14.8	4.7