Enhanced l-ornithine production from glucose and sucrose via manipulation of the fructose metabolic pathway in Corynebacterium glutamicum

Abstract

l-Ornithine, an important non-essential amino acid, has considerable medicinal value in the treatment of complex liver diseases. Microbial fermentation strategies using robust engineered strains have remarkable potential for producing l-ornithine. We showed that glucose and sucrose co-utilization accumulate more l-ornithine in Corynebacterium glutamicum than glucose alone. Further manipulating the expression of intracellular fructose-1-phosphate kinase through the deletion of pfkB1resulted in the engineered strain C. glutamicum SO30 that produced 47.6 g/L of l-ornithine, which represents a 32.8% increase than the original strain C. glutamicum SO26 using glucose as substrate (35.88 g/L). Moreover, fed-batch cultivation of C. glutamicum SO30 in 5-L fermenters produced 78.0 g/L of l-ornithine, which was a 78.9% increase in yield compared with that produced by C. glutamicum SO26. These results showed that manipulating the fructose metabolic pathway increases l-ornithine accumulation and provides a reference for developing C. glutamicum to produce valuable metabolites.

Keywords

Green biomanufacturing / Corynebacterium glutamicum / l-Ornithine')">l-Ornithine / Sucrose

References

[1] Becker J, Wittmann C. Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products. Angew Chem Int Ed Engl, 2015, 54: 3328-3350.

[2] Blombach B, Seibold GM. Carbohydrate metabolism in Corynebacterium glutamicum and applications for the metabolic engineering of l-lysine production strains. Appl Microbiol Biotechnol, 2010, 86: 1313-1322.

[3] Dong J, Kan B, Liu H, Zhan M, Wang S, Xu G, Han R, Ni Y. CRISPR-Cpf1-assisted engineering of Corynebacterium glutamicum SNK118 for enhanced l-ornithine production by NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and NADH-dependent glutamate dehydrogenase. Appl Biochem Biotechnol, 2020, 191: 955-967.

[4] Georgi T, Rittmann D, Wendisch VF. Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. Metab Eng, 2005, 7: 291-301.

[5] Hasegawa S, Tanaka Y, Suda M, Jojima T, Inui M. Enhanced glucose consumption and organic acid production by engineered Corynebacterium glutamicum based on analysis of a pfkB1 deletion mutant. Appl Environ Microbiol, 2017, 83: e02638-e12616.

[6] Jiang LY, Zhang YY, Li Z, Liu JZ. Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability. J Ind Microbiol Biotechnol, 2013, 40: 1143-1151.

[7] Jiang Y, Huang MZ, Chen XL, Zhang B. Proteome analysis guided genetic engineering of Corynebacterium glutamicum S9114 for tween 40-triggered improvement in L-ornithine production. Microb Cell Fact, 2020, 19: 2.

[8] Jover-Cobos M, Noiret L, Lee K, Sharma V, Habtesion A, Romero-Gomez M, Davies N, Jalan R. Ornithine phenylacetate targets alterations in the expression and activity of glutamine synthase and glutaminase to reduce ammonia levels in bile duct ligated rats. J Hepatol, 2014, 60: 545-553.

[9] Lee YJ, Cho JY. Genetic manipulation of a primary metabolic pathway for L-ornithine production in Escherichia coli. Biotechnol Lett, 2006, 28: 1849-1856.

[10] Martins GB, Giacomelli G, Goldbeck O, Seibold GM, Bramkamp M. Substrate-dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases. Mol Microbiol, 2019, 111: 1335-1354.

[11] Mitsuhashi S. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides. Curr Opin Biotechnol, 2014, 26: 38-44.

[12] Park SH, Kim HU, Kim TY, Park JS, Kim S-S, Lee SY. Metabolic engineering of Corynebacterium glutamicum for l-arginine production. Nat Commun, 2014, 5: 1-9.

[13] Pérez-García F, Max Risse J, Friehs K, Wendisch VF. Fermentative production of l-pipecolic acid from glucose and alternative carbon sources. Biotechnol J, 2017, 12: 1600646.

[14] Qin J, Zhou YJ, Krivoruchko A, Huang M, Liu L, Khoomrung S, Siewers V, Jiang B, Nielsen J. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of l-ornithine. Nat Commun, 2015, 6: 8224.

[15] Ramp P, Lehnert A, Matamouros S, Wirtz A, Baumgart M, Bott M. Metabolic engineering of Corynebacterium glutamicum for production of scyllo-inositol, a drug candidate against Alzheimer's disease. Metab Eng, 2021, 67: 173-185.

[16] Rosen H. A modified ninhydrin colorimetric analysis for amino acids. Arch Biochem Biophys, 1957, 67: 10-15.

[17] Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene, 1994, 145: 69-73.

[18] Sgobba E, Stumpf AK, Vortmann M, Jagmann N, Krehenbrink M, Dirks-Hofmeister ME, Moerschbacher B, Philipp B, Wendisch VF. Synthetic Escherichia coli-Corynebacterium glutamicum consortia for l-lysine production from starch and sucrose. Bioresour Technol, 2018, 260: 302-310.

[19] Wang Z, Chan SHJ, Sudarsan S, Blank LM, Jensen PR, Solem C. Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum. Metab Eng, 2016, 38: 344-357.

[20] Wu XY, Guo XY, Zhang B, Jiang Y, Ye BC. Recent advances of l-ornithine biosynthesis in metabolically engineered Corynebacterium glutamicum. Front Bioeng Biotechnol, 2020, 7: 440.

[21] Xu JZ, Ruan HZ, Yu HB, Liu LM, Zhang W. Metabolic engineering of carbohydrate metabolism systems in Corynebacterium glutamicum for improving the efficiency of l-lysine production from mixed sugar. Microb Cell Fact, 2020, 19: 39.

[22] Zhang B, Zhou N, Liu YM, Liu C, Lou CB, Jiang CY, Liu SJ. Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum. Microb Cell Fact, 2015, 14: 71.

[23] Zhang YY, Bu YF, Liu JZ. Production of L-ornithine from sucrose and molasses by recombinant Corynebacterium glutamicum. Folia Microbiol (praha), 2015, 60: 393-398.

[24] Zhang B, Yu M, Zhou Y, Li Y, Ye BC. Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production. Microb Cell Fact, 2017, 16: 158.

[25] Zhang B, Ren LQ, Yu M, Zhou Y, Ye BC. Enhanced l-ornithine production by systematic manipulation of l-ornithine metabolism in engineered Corynebacterium glutamicum S9114. Bioresourc Technol, 2018, 250: 60-68.

[26] Zhang B, Yu M, Wei WP, Ye BC. Optimization of l-ornithine production in recombinant Corynebacterium glutamicum S9114 by cg3035 overexpression and manipulating the central metabolic pathway. Microb Cell Fact, 2018, 17: 91.

[27] Zhang J, Wang P, Wang X, Feng J, Sandhu HS, Wang Y. Enhancement of sucrose metabolism in Clostridium saccharoperbutylacetonicum N1–4 through metabolic engineering for improved acetone-butanol-ethanol (ABE) fermentation. Bioresour Technol, 2018, 270: 430-438.

[28] Zhang X, Xu G, Shi J, Koffas MAG, Xu Z. Microbial production of l-serine from renewable feedstocks. Trends Biotechnol, 2018, 36: 700-712.

[29] Zhang X, Zhang X, Xu G, Zhang X, Shi J, Xu Z. Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum. Appl Microbiol Biotechnol, 2018, 102: 5939-5951.

[30] Zhang B, Gao G, Chu XH, Ye BC. Metabolic engineering of Corynebacterium glutamicum S9114 to enhance the production of l-ornithine driven by glucose and xylose. Bioresour Technol, 2019, 284: 204-213.

[31] Zhang B, Jiang Y, Li Z, Wang F, Wu XY. Recent progress on chemical production from non-food renewable feedstocks using Corynebacterium glutamicum. Front Bioeng Biotechnol, 2020, 8.

[32] Zhang X, Gao Y, Chen Z, Xu G, Zhang X, Li H, Shi J, Koffas MAG, Xu Z. High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum. Microb Cell Fact, 2020, 19: 115.

[33] Zhao C, Zhang Y, Li Y. Metabolic engineering for the production of butanol, a potential advanced biofuel, from renewable resources. Biochem Soc Trans, 2020, 48: 2283-2293.

Funding

Natural Science Foundation of Jiangxi Province(20202BAB213023)

Major Discipline Academic and Technical Leaders Training Program of Jiangxi Province(20212BCJ23012)

National Natural Science Foundation of China(32000057)