Production of N-acetylneuraminic acid in Escherichia coli using different carbon sources

  • YI Jinhang ,
  • ZONG Yuan ,
  • SUN Chao ,
  • ZHANG Xiaoyu ,
  • GENG Zihao ,
  • SUN Wenchao ,
  • ZHANG Chunyue ,
  • LIU Zhengkai ,
  • XIONG Wenwen ,
  • TANG Yulin ,
  • MA Qian
Expand
  • (College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)

Received date: 2024-01-23

  Revised date: 2024-02-28

  Online published: 2025-01-23

Abstract

N-acetylneuramic acid, the most common type of sialic acids, generally locates at the terminal of glycoprotein and glycolipid on the surface of cell membrane, where it performs a variety of biological functions.Because of its unique physiological and biochemical properties, it is more and more widely used in pharmaceutical, cosmetic and food industries, and its demand will continue to increase.The heterologous biosynthesis of N-acetylneuraminic acid in Escherichia coli needs to compete precursors with the process of central carbon metabolism.Therefore, the efficiency of carbon source utilization and the distribution of carbon metabolic flux in cell growth and N-acetylneuraminic acid synthesis directly affect the synthesis efficiency of N-acetylneuraminic acid.In this study, the effects of glucose, glycerol and their mixture on the synthesis of N-acetylneuraminic acid were compared from the point of view of carbon source utilization regulation.When using single glucose carbon source, the knockout of pfkA in strain NEU5AC-2 weakened the intensity of central carbon metabolism, reduced the accumulation of by-product acetic acid, and coordinated the supply of precursors GlcNAc and PEP.NEU5AC-2 produced 23.8 g/L N-acetylneuraminic acid after 38 h of fed-batch fermentation in a 5-L bioreactor, which obtained higher level of fermentation production and strong industrial application potential.This study shed light on similar studies using metabolites in EMP as precursors for the biosynthesis of heterologous chemicals.

Cite this article

YI Jinhang , ZONG Yuan , SUN Chao , ZHANG Xiaoyu , GENG Zihao , SUN Wenchao , ZHANG Chunyue , LIU Zhengkai , XIONG Wenwen , TANG Yulin , MA Qian . Production of N-acetylneuraminic acid in Escherichia coli using different carbon sources[J]. Food and Fermentation Industries, 2025 , 51(1) : 18 -25 . DOI: 10.13995/j.cnki.11-1802/ts.038677

References

[1] HU S Y, CHEN J, YANG Z Y, et al.Coupled bioconversion for preparation of N-acetyl-D-neuraminic acid using immobilized N-acetyl-D-glucosamine-2-epimerase and N-acetyl-D-neuraminic acid lyase[J].Applied Microbiology and Biotechnology, 2010, 85(5):1383-1391.
[2] KRAGL U, GYGAX D, GHISALBA O, et al.Enzymatic two-step synthesis of N-acetyl-neuraminic acid in the enzyme membrane reactor[J].Angewandte Chemie International Edition in English, 1991, 30(7):827-828.
[3] ZHANG X L, LIU Y F, LIU L, et al.Microbial production of sialic acid and sialylated human milk oligosaccharides:Advances and perspectives[J].Biotechnology Advances, 2019, 37(5):787-800.
[4] LEE Y C, CHIEN H C R, HSU W H.Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp.CH1 N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase[J].Journal of Biotechnology, 2007, 129(3):453-460.
[5] MARU I, OHNISHI J, OHTA Y, et al.Why is sialic acid attracting interest now? Complete enzymatic synthesis of sialic acid with N-acylglucosamine 2-epimerase[J].Journal of Bioscience and Bioengineering, 2002, 93(3):258-265.
[6] ZHANG L, WEI T T, LI Y, et al.Functional metabolomics characterizes a key role for N-acetylneuraminic acid in coronary artery diseases[J].Circulation, 2018, 137(13):1374-1390.
[7] BONDIOLI L, RUOZI B, BELLETTI D, et al.Sialic acid as a potential approach for the protection and targeting of nanocarriers[J].Expert Opinion on Drug Delivery, 2011, 8(7):921-937.
[8] GAO X Y, ZHANG F F, WU M, et al.Production of N-acetyl-D-neuraminic acid by whole cells expressing Bacteroides thetaiotaomicron N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid aldolase[J].Journal of Agricultural and Food Chemistry, 2019, 67(22):6285-6291.
[9] WEINER M, ALBERMANN C, GOTTLIEB K, et al.Fed-batch production of L-phenylalanine from glycerol and ammonia with recombinant Escherichia coli[J].Biochemical Engineering Journal, 2014, 83:62-69.
[10] MURARKA A, DHARMADI Y, YAZDANI S S, et al.Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals[J].Applied and Environmental Microbiology, 2008, 74(4):1124-1135.
[11] LI Y F, LIN Z Q, HUANG C, et al.Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing[J].Metabolic Engineering, 2015, 31:13-21.
[12] 侯正杰. 大肠杆菌N-乙酰神经氨酸合成途径的构建与优化[D].天津:天津科技大学, 2020.
HOU Z J, Construction and optimization of N-acetylneuraminic acid pathway in Escherichia coli[D].Tianjin:Tianjin University of Science and Technology, 2020.
[13] 张颖. 代谢工程改造大肠杆菌高效生产N-乙酰神经氨酸[D].天津:天津科技大学, 2022.
ZHANG Y.Metabolic engineering transformation of Escherichia coli for efficient production of N-acetylneuraminic acid[D].Tianjin:Tianjin University of Science & Technology, 2022.
[14] CHENG K K, LEE B S, MASUDA T, et al.Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol[J].Nature Communications, 2014, 5:3233.
[15] GOTTLIEB K, ALBERMANN C, SPRENGER G A.Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains:The role of extra copies of glpK, glpX, and tktA genes[J].Microbial Cell Factories, 2014, 13(1):96.
[16] PETTIGREW D W, MA D P, CONRAD C A, et al.Escherichia coli glycerol kinase.Cloning and sequencing of the glpK gene and the primary structure of the enzyme[J].Journal of Biological Chemistry, 1988, 263(1):135-139.
[17] LI Y J, YAN F Q, WU H Y, et al.Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli[J].Journal of Industrial Microbiology & Biotechnology, 2019, 46(1):81-90.
[18] MA Q, SUN Q W, TAN M, et al.Highly efficient production of N-acetyl-glucosamine in Escherichia coli by appropriate catabolic division of labor in the utilization of mixed glycerol/glucose carbon sources[J].Journal of Agricultural and Food Chemistry, 2021, 69(21):5966-5975.
[19] MEISWINKEL T M, RITTMANN D, LINDNER S N, et al.Crude glycerol-based production of amino acids and putrescine by Corynebacterium glutamicum[J].Bioresource Technology, 2013, 145:254-258.
[20] BLANKSCHIEN M D, CLOMBURG J M, GONZALEZ R.Metabolic engineering of Escherichia coli for the production of succinate from glycerol[J].Metabolic Engineering, 2010, 12(5):409-419.
[21] ZHANG X M, ZHANG D, ZHU J F, et al.High-yield production of L-serine from glycerol by engineered Escherichia coli[J].Journal of Industrial Microbiology & Biotechnology, 2019, 46(2):221-230.
[22] UTRILLA J, LICONA-CASSANI C, MARCELLIN E, et al.Engineering and adaptive evolution of Escherichia coli for D-lactate fermentation reveals GatC as a xylose transporter[J].Metabolic Engineering, 2012, 14(5):469-476.
[23] WANG Z K, GAO C J, WANG Q, et al.Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering[J].Biochemical Engineering Journal, 2012, 67:126-131.
[24] WU M K, GUAN Z, WANG Y J, et al.Efficient succinic acid production by engineered Escherichia coli using ammonia as neutralizer[J].Journal of Chemical Technology & Biotechnology, 2016, 91(9):2412-2418.
Outlines

/