Enhancing nicotinamide mononucleotide production from glucose in Escherichia coli by genetic engineering

Abstract

Nicotinamide mononucleotide (NMN), as a healthcare product, plays positive effects on the human body. In this study, an engineered Escherichia coli (E. coli) strain was constructed to efficiently produce NMN from glucose and nicotinamide (NAM) through whole-cell biotransformation. First, a highly active nicotinamide phosphoribosyltransferase (NAMPT/NadV) from Vibrio phage KVP40 was screened, which showed the best productivity in catalyzing phosphoribosyl pyrophosphate (PRPP) and NAM to NMN in E. coli. Next, enzymatic activity of E. coli ribose-phosphate diphosphokinase (EcPRS) was improved and its expression level was optimized, which increased the supply of precursor PRPP. The results showed that mutant EcPRS^D115S had optimal activity and that the promoter and RBS combination (P_J23119 and RBS_III) optimized for EcPRS^D115S further accumulated NMN. With the systematic modification, such as the deletion of pfkA and pncC genes, and the expression of Cgzwf ^A243T and Cggnd ^S361F genes from Corynebacterium glutamicum, the biosynthesis of NMN was improved. Finally, NMN production was further increased after the introduction of NAM and NMN transporters. The strain ZY17 achieved NMN production of 1.61 ± 0.02 g/L in the shake flask level (OD600 = 10), and 13.3 ± 0.35 g/L in the 2-L bioreactor with a productivity of 1.11 g/(L.h) and an NAM conversion (α) of 85.3%. In conclusion, we propose a strategy that can effectively enhance the production of NMN in E. coli, which may bring new ideas for the biosynthesis of other nucleotide compounds.

Keywords

Nicotinamide mononucleotide / Biocatalysis / Escherichia coli / PRS / Metabolic engineering

References

1.	Yamamoto T, Byun J, Zhai P, Ikeda Y, Oka S, Sadoshima J. Nicotinamide mononucleotide, an intermediate of NAD+ synthesis, protects the heart from ischemia and reperfusion. PLoS ONE, 2014, 9(6): e98972, pmcid: 4048236
2.	Belenky P, Bogan K, Brenner C. NAD+ metabolism in health and disease. Trends Biochem Sci, 2007, 32(1): 12-19,
3.	Yoshino J, Mills K, Yoon M, Imai S. Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab, 2011, 14(4): 528-536, pmcid: 3204926
4.	Caton P, Kieswich J, Yaqoob M, Holness M, Sugden M. Nicotinamide mononucleotide protects against pro-inflammatory cytokine-mediated impairment of mouse islet function. Diabetologia, 2011, 54(12): 3083-3092,
5.	Ramanathan C, Lackie T, Williams D, Simone P, Zhang Y, Bloomer R. Oral administration of nicotinamide mononucleotide increases nicotinamide adenine dinucleotide level in an animal brain. Nutrients, 2022, pmcid: 8778478
6.	Maharjan A, Singhvi M, Kim BS. Biosynthesis of a therapeutically important nicotinamide mononucleotide through a phosphoribosyl pyrophosphate synthetase 1 and 2 engineered strain of Escherichia coli. ACS Synth Biol, 2021, 10(11): 3055-3065,
7.	Revollo JR, Grimm AA, Imai S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J Biol Chem, 2004, 279(49): 50754-50763,
8.	Houtkooper R, Canto C, Wanders R, Auwerx J. The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways. Endocr Rev, 2010, 31(2): 194-223,
9.	Black WB, Aspacio D, Bever D, King E, Zhang L, Li H. Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor. Microb Cell Fact, 2020, 19(1): 150, pmcid: 7384224
10.	Hove-Jensen B, Andersen KR, Kilstrup M, Martinussen J, Switzer RL, Willemoes M. Phosphoribosyl diphosphate (PRPP): biosynthesis, enzymology, utilization, and metabolic significance. Microbiol Mol Biol Rev, 2017,
11.	Kadziola A, Jepsen C, Johansson E, McGuire J, Larsen S, Hove-Jensen B. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschII. J Mol Biol, 2005, 354(4): 815-828,
12.	Hove-Jensen B, Harlow KW, King CJ, Switzer LR. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene. J Biol Chem, 1986,
13.	Becker M, Smith P, Taylor W, Mustafi R, Switzer R. The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity. J Clin Invest, 1995, 96(5): 2133-2141, pmcid: 185862
14.	Zakataeva NP, Romanenkov DV, Skripnikova VS, Vitushkina MV, Livshits VA, Kivero AD, Novikova AE. Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from Bacillus amyloliquefaciens: purification, characterization, and application to increase purine nucleoside production. Appl Microbiol Biotechnol, 2012, 93(5): 2023-2033,
15.	Marinescu GC, Popescu RG, Stoian G, Dinischiotu A. beta-nicotinamide mononucleotide (NMN) production in Escherichia coli. Sci Rep, 2018, 8(1): 12278, pmcid: 6095924
16.	Shoji S, Yamaji T, Makino H, IshII J, Kondo A. Metabolic design for selective production of nicotinamide mononucleotide from glucose and nicotinamide. Metab Eng, 2021, 65: 167-177,
17.	Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol, 2015, 81(7): 2506-2514, pmcid: 4357945
18.	Zhang RY, Qin Y, Lv XQ, Wang P, Xu TY, Zhang L, Miao CY. A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase. Anal Biochem, 2011, 412(1): 18-25,
19.	Dong W, Sun C, Zhu G, Hu S, Xiang L, Shao J. New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD+ salvage from nicotinamide. BMC Microbiol, 2014, 14: 29, pmcid: 3923242
20.	Huang Z, Li N, Yu S, Zhang W, Zhang T, Zhou J. Systematic engineering of Escherichia coli for efficient production of nicotinamide mononucleotide from nicotinamide. ACS Synth Biol, 2022, pmcid: 9680020
21.	Willemoes M, Hove-Jensen B, Larsen S. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase. J Biol Chem, 2000, 275(45): 35408-35412,
22.	Borujeni A, Channarasappa A, Salis H. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res, 2014, 42(4): 2646-2659,
23.	Markley AL, Begemann MB, Clarke RE, Gordon GC, Pfleger BF. Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. ACS Synth Biol, 2015, 4(5): 595-603,
24.	Gupta J, Srivastava S. The effect of promoter and RBS combination on the growth and glycogen productivity of sodium-dependent bicarbonate transporter (SbtA) overexpressing Synechococcus sp. PCC 7002 Cells. Front Microbiol, 2021, 12: 607411, pmcid: 8076525
25.	Nowroozi FF, Baidoo EE, Ermakov S, Redding-Johanson AM, Batth TS, Petzold CJ, Keasling JD. Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly. Appl Microbiol Biotechnol, 2014, 98(4): 1567-1581,
26.	Becker J, Klopprogge C, Herold A, Zelder O, Bolten CJ, Wittmann C. Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum——over expression and modification of G6P dehydrogenase. J Biotechnol, 2007, 132(2): 99-109,
27.	Ohnishi J, Katahira R, Mitsuhashi S, Kakita S, Ikeda M. A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum. FEMS Microbiol Lett, 2005, 242(2): 265-274,
28.	Sprenger GA. Genetics of pentose-phosphate pathway enzymes of Escherichia coli K-12. Arch Microbiol, 1995, 164(5): 324-330,
29.	Goormans AR, Snoeck N, Decadt H, Vermeulen K, Peters G, Coussement P, Van Herpe D, Beauprez JJ, De Maeseneire SL, Soetaert WK. Comprehensive study on Escherichia coli genomic expression: does position really matter. Metab Eng, 2020, 62: 10-19,
30.	Wang Z, Chen T, Ma X, Shen Z, Zhao X. Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum. Bioresour Technol, 2011, 102(4): 3934-3940,
31.	Gazzaniga F, Stebbins R, Chang S, McPeek M, Brenner C. Microbial NAD metabolism: lessons from comparative genomics. Microbiol Mol Biol Rev, 2009, 73(3): 529-541, pmcid: 2738131
32.	Black W, Zhang L, Mak W, Maxel S, Cui Y, King E, Fong B, Martinez A, Siegel JB, Li H. Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis. Nat Chem Biol, 2020, 16(1): 87-94,
33.	Liu Y, Yasawong M, Yu B. Metabolic engineering of Escherichia coli for biosynthesis of beta-nicotinamide mononucleotide from nicotinamide. Microb Biotechnol, 2021, 14(6): 2581-2591, pmcid: 8601175
34.	Lucks JB, Qi L, Mutalik VK, Wang D, Arkin AP. Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proc Natl Acad Sci U S A, 2011, 108(21): 8617-8622, pmcid: 3102349
35.	Xu D, Tan Y, Li Y, Wang X. Construction of a novel promoter-probe vector and its application for screening strong promoter for Brevibacterium flavum metabolic engineering. World J Microbiol Biotechnol, 2010, 27(4): 961-968,
36.	Wang B, Eckert C, Maness P, Yu J. A genetic toolbox for modulating the expression of heterologous genes in the cyanobacterium Synechocystis sp. PCC 6803. ACS Synth Biol, 2018, 7(1): 276-286,

Funding

the Green Bio-fabrication Program of China(No. 2021YFC2100900)