Modifying Corynebacterium glutamicum by metabolic engineering for efficient synthesis of l-lysine

Abstract

This study described that a low-producing mutagenic strain was transformed to a l-lysine high-producing recombinant strain by optimizing the l-lysine metabolic pathway of Corynebacterium glutamicum. The nucleotide sequence results revealed that the lysC of mutant strain CgK37 mutated at 279th codon. Based on this site, site-directed saturation mutation was performed to screen for the mutant with better effect in relieving aspartate kinase feedback inhibition. Then, the supply of oxaloacetate and nicotinamide adenine dinucleotide phosphate was increased via knockout and overexpression of related genes. In order to solve the problem of low utilization efficiency of culture medium, fructokinase gene gmuE was heterologous expressed in CgK37, which could directly use intracellular fructose to improve the growth rate. In addition, the synthesis of partial by-products was weakened at the gene transcription level to avoid carbon excessively flowing into the branch metabolism. Finally, a large-scale fermentation experiment was conducted in 5 L jar fermenter. The l-lysine yield of CgK37-11 was 196.58 ± 1.68 g/L, which was 83.24% original higher than CgK37, and the productivity reached 2.46 g/L/h.

References

[1] Cheng J, et al.. Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives. J Ind Microbiol Biotechnol, 2018, 45(8): 719-734

[2] Felix F, et al.. l-lysine production improvement: a review of the state of the art and patent landscape focusing on strain development and fermentation technologies. Crit Rev Biotechnol, 2019, 39(8): 1031-1055

[3] Sagong H, Hong H, Kim K. Structural basis for redox sensitivity in Corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis. FEBS J, 2017, 284: 230-230

[4] Vetrova OY, et al.. Sorption-catalytic determination of rutin, lysine, and collagen in pharmaceuticals and cosmetics. Pharm Chem J, 2015, 48(11): 755-760

[5] Liu J, et al.. Industrial production of l-lysine in Corynebacterium glutamicum: progress and prospects. Microbiol Res, 2022, 262 127101

[6] Fei X, et al.. Breeding of high l-isoleucine producing strains. China Brewing, 2014, 33(11): 98-100

[7] Matsui D, Oikawa T. Detection of d-ornithine extracellularly produced by Corynebacterium glutamicum ATCC 13032::argF. Biosci Biotechnol Biochem, 2010, 74(12): 2507-2510

[8] Wang X. Metabolic engineering in Corynebacterium glutamicum to increase l-valine production. J Food Sci Biotechnol, 2012, 31(3): 225-231

[9] Liang L, Yanlln QIU, Jintao XU. Breeding of l-histidine producing bacterium by composite mutation with UV and NAN0_2. J Mirobiol, 2008, 28(2): 27-29

[10] Rastegari H, et al.. Improvement in the production of l-Lysine by overexpression of Aspartokinase (ASK) in C. glutamicum ATCC 21799. Trop J Pharmaceut Res, 2013, 12(1): 51-56

[11] Yoshida A, et al.. Mechanism of concerted Inhibition of alpha(2)beta(2)-type hetero-oligomeric aspartate kinase from Corynebacterium glutamicum. J Biol Chem, 2010, 285(35): 27477-27486

[12] Binder S, et al.. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Genome Biol, 2012, 13(5): R40

[13] Dong X, et al.. Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of l-isoleucine biosynthesis. J Ind Microbiol Biotechnol, 2016, 43(6): 873-885

[14] Han C, et al.. The mutant T379L of novel aspartokinase from Corynebacterium pekinense: a combined experimental and molecular dynamics simulation study. Process Biochem, 2019, 83: 77-85

[15] Hoffmann SL, et al.. Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering. Metab Eng, 2021, 67: 293-307

[16] Koffas MAG, et al.. Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum. Appl Environ Microbiol, 2002, 68(11): 5422-5428

[17] Ohnishi J, et al.. A novel methodology employing Corynebacterium glutamicum genome information to generate a new l-lysine-producing mutant. Appl Microbiol Biotechnol, 2002, 58(2): 217-223

[18] Becker J, et al.. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol, 2005, 71(12): 8587-8596

[19] Kabus A, et al.. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation. Appl Microbiol Biotechnol, 2007, 75(1): 47-53

[20] Liu J, et al.. L-lysine production by systems metabolic engineering of an NADPH auto-regulated Corynebacterium glutamicum. Bioresour Technol, 2023, 387: 129701

[21] Zhan M, et al.. Metabolic engineering of Corynebacterium glutamicum for improved l-arginine synthesis by enhancing NADPH supply. J Ind Microbiol Biotechnol, 2019, 46(1): 45-54

[22] Qinglan LI, Chun CUI, Wei W. Research progress on the main active components and physiological functions of sugarcane molasses and beet molasses. Food Mach., 2014, 37(4): 207-211

[23] Ke X, Ping X. Betaine and beet molasses enhance l-lactic acid production by Bacillus coagulans. PLoS ONE, 2014, 9(6): e100731-e100731

[24] Sarka E, et al.. Molasses as a by-product of sugar crystallization and a perspective raw material. Proc Eng, 2012, 42: 1219-1228

[25] Moon M-W, et al.. The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation. J Mol Microbiol Biotechnol, 2007, 12(1–2): 43-50

[26] Parche S, et al.. Corynebacterium glutamicum : a dissection of the PTS. J Mol Microbiol Biotechnol, 2001, 3(3): 423-428

[27] Wittmann C, Kiefer P, Zelder O. Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source. Appl Environ Microbiol, 2004, 70(12): 7277-7287

[28] Moon M-W, et al.. Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicumATCC 13032. FEMS Microbiol Lett, 2005, 244(2): 259-266

[29] Xu G, et al. Production of l-serine and its derivative l-cysteine from renewable feedstocks using Corynebacterium glutamicum: advances and perspectives. Crit Rev Biotechnol. 2023.

[30] Xu JZ, et al.. Rational modification of tricarboxylic acid cycle for improving l-lysine production in Corynebacterium glutamicum. Microb Cell Fact, 2018, 17(1): 105

[31] Korös A, Varga Z, Molnár-Perl I. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A, 2008, 1203(2): 146-152

[32] MartinezBarajas E, et al.. Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill) fructokinase expressed in Escherichia coli. Protein Expression Purificat, 1997, 11(1): 41-46

[33] Chen Z, et al.. Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production. Appl Environ Microbiol, 2011, 77(13): 4352-4360

[34] Ball J, et al.. Steric hindrance controls pyridine nucleotide specificity of a flavin-dependent NADH:quinone oxidoreductase. Protein Sci, 2019, 28(1): 167-175

[35] Wu K, et al.. Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element. Catal Sci Technol, 2020, 10(6): 1650-1660

[36] Becker J, et al.. From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for l-lysine production. Metab Eng, 2011, 13(2): 159-168

[37] Peters-Wendisch PG, et al.. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol, 2001, 3(2): 295-300

[38] Ikeda M, et al.. A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient l-lysine production. J Ind Microbiol Biotechnol, 2006, 33(7): 610-615

[39] Petersen S, et al.. In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum *. J Biol Chem, 2000, 275(46): 35932-35941

[40] Becker J, et al.. Metabolic flux engineering of l-lysine production in Corynebacterium glutamicum—over expression and modification of G6P dehydrogenase. J Biotechnol, 2007, 132(2): 99-109

[41] 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(4): 291-301

[42] El Asri O, Farag MA. The potential of molasses from different dietary sources in industrial applications: a source of functional compounds and health attributes, a comprehensive review. Food Biosci, 2023, 56 103263

[43] Palmonari A, et al.. Short communication: characterization of molasses chemical composition. J Dairy Sci, 2020, 103(7): 6244-6249

[44] Xu J, et al.. Improvement of cell growth and l-lysine production by genetically modified Corynebacterium glutamicum during growth on molasses. J Ind Microbiol Biotechnol, 2013, 40(12): 1423-1432

[45] Xu JZ, et al.. 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(1): 39

[46] Zhang X, et al.. Enhancement of fructose utilization from sucrose in the cell for improved l-serine production in engineered Corynebacterium glutamicum. Biochem Eng J, 2017, 118: 113-122

[47] Li H, et al.. Metabolic engineering of Escherichia coli W3110 for l-homoserine production. Process Biochem, 2016, 51(12): 1973-1983

[48] Liu P, et al.. O-Succinyl-l-homoserine overproduction with enhancement of the precursor succinyl-CoA supply by engineered Escherichia coli. J Biotechnol, 2021, 325: 164-172

[49] Man Z, et al. Systems pathway engineering of Corynebacterium crenatum for improved l-arginine production. Sci Rep. 2016; 6.

[50] Chen Y-J, et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods. 2013; 10(7): 659.

[51] Jin-Yu W, et al.. Enhancing beta-alanine production from glucose in genetically modified Corynebacterium glutamicum by metabolic pathway engineering. Appl Microbiol Biotechnol, 2021, 105(24): 9153-9166

[52] Eggeling L, Bott M. A giant market and a powerful metabolism: l-lysine provided by Corynebacterium glutamicum. Appl Microbiol Biotechnol, 2015, 99(8): 3387-3394

[53] Hirao T, et al.. l-Lysine production in continuous culture of an l-lysine hyperproducing mutant of Corynebacterium glutamicum. Appl Microbiol Biotechnol, 1989, 32(3): 269-273

[54] Hadj Sassi A, et al.. Effect of medium composition on l-Lysine production by a variant strain of Corynebacterium clutamicum ATCC 21513. Biotech Lett, 1990, 12(4): 295-298

[55] Kiss RD, Stephanopoulos G. Metabolic-activity control of the l-lysine fermentation by restrained growth fed-batch strategies. Biotechnol Prog, 1991, 7(6): 501-509

[56] Oldiges M, Eikmanns BJ, Blombach B. Application of metabolic engineering for the biotechnological production of l-valine. Appl Microbiol Biotechnol, 2014, 98(13): 5859-5870

[57] Park JH, Lee SY. Metabolic pathways and fermentative production of l-aspartate family amino acids. Biotechnol J, 2010, 5(6): 560-577

[58] Chen Z, et al.. Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum. Appl Environ Microbiol, 2014, 80(4): 1388-1393

[59] Jiang L-Y, et al.. Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability. J Ind Microbiol Biotechnol, 2013, 40(10): 1143-1151

[60] Takeno S, et al.. l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene. Metab Eng, 2016, 37: 1-10

Funding

Important Amino Acid Industrial Strain System Transformation and Industrial Demonstration(2021YFC2100900)

RIGHTS & PERMISSIONS

Jiangnan University