Ferulic acid production by metabolically engineered Escherichia coli

Abstract

Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics. Production of FA by eco‐friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p-coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as caffeic acid O-methytransferase. Fermentation result revealed JM109(DE3) as a more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 or p15A), and the combination p15a-T5 works best. To further improve FA production, E. coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also overexpressed. Based on the flask fermentation data which show that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E. coli K-12 strains reported to the best of our knowledge.

Keywords

Ferulic acid / E. coli / Biosynthesis / NADPH / SAM

References

[1] Ajikumar PK, Xiao WH, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichiacoli. Science, 2010, 330(6000): 70-74.

[2] Berner M, Krug D, Bihlmaier C, Vente A, Muller R, Bechthold A. Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrixespanaensis. J Bacteriol, 2006, 188(7): 2666-2673.

[3] Braga A, Faria N. Bioprocess optimization for the production of aromatic compounds with metabolically engineered hosts: recent developments and future challenges. Front Bioeng Biotechnol, 2020, 8: 96.

[4] Cabulong RB, Valdehuesa KNG, Banares AB, Ramos KRM, Nisola GM, Lee WK, Chung WJ. Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase. J Ind Microbiol Biotechnol, 2019, 46(2): 159-169.

[5] Choi O, Wu C-Z, Kang SY, Ahn JS, Uhm T-B, Hong Y-S. Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in Escherichiacoli. J Ind Microbiol Biotechnol, 2011, 38(10): 1657-1665.

[6] Cui Y-Y, Ling C, Zhang Y-Y, Huang J, Liu J-Z. Production of shikimic acid from Escherichiacoli through chemically inducible chromosomal evolution and cofactor metabolic engineering. Microb Cell Fact, 2014, 13: 21-21.

[7] Fowler ZL, Koffas MA. Biosynthesis and biotechnological production of flavanones: current state and perspectives. Appl Microbiol Biotechnol, 2009, 83(5): 799-808.

[8] Goris T, Perez-Valero A, Martinez I, Yi D, Fernandez-Calleja L, San Leon D, Bornscheuer UT, Magadan-Corpas P, Lombo F, Nogales J. Repositioning microbial biotechnology against COVID-19: the case of microbial production of flavonoids. Microb Biotechnol, 2020

[9] Han G, Hu X, Wang X. Overexpression of methionine adenosyltransferase in Corynebacteriumglutamicum for production of S-adenosyl-L-methionine. Biotechnol Appl Biochem, 2016, 63(5): 679-689.

[10] Harris PJ, Trethewey JAK. The distribution of ester-linked ferulic acid in the cell walls of angiosperms. Phytochem Rev, 2009, 9(1): 19-33.

[11] Haslinger K, Prather KLJ. Heterologous caffeic acid biosynthesis in Escherichiacoli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450. Microb Cell Fact, 2020, 19(1): 26.

[12] Heo KT, Kang SY, Hong YS. Denovo biosynthesis of pterostilbene in an Escherichiacoli strain using a new resveratrol O-methyltransferase from Arabidopsis. Microb Cell Fact, 2017, 16(1): 30.

[13] Huang Q, Lin Y, Yan Y. Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichiacoli strain. Biotechnol Bioeng, 2013, 110(12): 3188-3196.

[14] Huang D, Yang K, Liu J, Xu Y, Wang Y, Wang R, Liu B, Feng L. Metabolic engineering of Escherichiacoli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement. Metab Eng, 2017, 41: 23-38.

[15] Jendresen CB, Stahlhut SG, Li M, Gaspar P, Siedler S, Forster J, Maury J, Borodina I, Nielsen AT. Highly active and specific tyrosine ammonia-lyases from diverse origins enable enhanced production of aromatic compounds in bacteria and Saccharomycescerevisiae. Appl Environ Microbiol, 2015, 81(13): 4458-4476.

[16] Jones KL, Kim SW, Keasling JD. Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. Metab Eng, 2000, 2(4): 328-338.

[17] Kabus A, Georgi T, Wendisch VF, Bott M. Expression of the Escherichiacoli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacteriumglutamicum improves L-lysine formation. Appl Microbiol Biotechnol, 2007, 75(1): 47-53.

[18] Kang S-Y, Choi O, Lee JK, Hwang BY, Uhm T-B, Hong Y-S. Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichiacoli strain. Microb Cell Fact, 2012, 11(1): 153.

[19] Kunjapur AM, Hyun JC, Prather KL. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E.colidenovo vanillin biosynthesis pathway. Microb Cell Fact, 2016, 15: 61.

[20] Lerner CG, Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichiacoli with blue/white insert screening capability. Nucleic Acids Res, 1990, 18(15): 4631.

[21] Liang Y, Xi X, Liu Q, Huang P, Li J, Lin Q. Research progress on the physiological activity and application of ferulic acid and its derivatives. J Food Sci Biotechnol, 2018, 37(05): 449-454.

[22] Lin Y, Yan Y. Biosynthesis of caffeic acid in Escherichiacoli using its endogenous hydroxylase complex. Microbial Cell Fact, 2012

[23] Liu J, Li H, Zhao G, Caiyin Q, Qiao J. Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions. J Ind Microbiol Biotechnol, 2018, 45(5): 313-327.

[24] Luziatelli F, Brunetti L, Ficca AG, Ruzzi M. Maximizing the efficiency of vanillin production by biocatalyst enhancement and process optimization. Front Bioeng Biotechnol, 2019, 7: 279.

[25] Ma QH, Xu Y. Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis. Biochimie, 2008, 90(3): 515-524.

[26] Martinez I, Zhu J, Lin H, Bennett GN, San KY. Replacing Escherichiacoli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridiumacetobutylicum facilitates NADPH dependent pathways. Metab Eng, 2008, 10(6): 352-359.

[27] Ng CY, Farasat I, Maranas CD, Salis HM. Rational design of a synthetic Entner-Doudoroff pathway for improved and controllable NADPH regeneration. Metab Eng, 2015, 29: 86-96.

[28] Ni J, Tao F, Du H, Xu P. Mimicking a natural pathway for denovo biosynthesis: natural vanillin production from accessible carbon sources. Sci Rep, 2015, 5: 13670.

[29] Noronha SB, Yeh HJ, Spande TF, Shiloach J. Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS. Biotechnol Bioeng, 2000, 68(3): 316-327.

[30] Rodrigues JL, Araujo RG, Prather KL, Kluskens LD, Rodrigues LR. Heterologous production of caffeic acid from tyrosine in Escherichiacoli. Enzyme Microb Technol, 2015, 71: 36-44.

[31] Rodrigues JL, Araujo RG, Prather KL, Kluskens LD, Rodrigues LR. Production of curcuminoids from tyrosine by a metabolically engineered Escherichiacoli using caffeic acid as an intermediate. Biotechnol J, 2015, 10(4): 599-609.

[32] Rodrigues JL, Gomes D, Rodrigues LR. A combinatorial approach to optimize the production of curcuminoids from tyrosine in Escherichiacoli. Front Bioeng Biotechnol, 2020, 8: 59.

[33] Sauer U, Canonaco F, Heri S, Perrenoud A, Fischer E. The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichiacoli. J Biol Chem, 2004, 279(8): 6613-6619.

[34] Serra S, Fuganti C, Brenna E. Biocatalytic preparation of natural flavours and fragrances. Trends Biotechnol, 2005, 23(4): 193-198.

[35] Sgarbossa A, Giacomazza D, di Carlo M. Ferulic acid: a hope for Alzheimer’s disease therapy from plants. Nutrients, 2015, 7(7): 5764-5782.

[36] Shiloach J, Kaufman J, Guillard AS, Fass R. Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichiacoli BL21 (DE3) and Escherichiacoli JM109. Biotechnol Bioeng, 1996, 49(4): 421-428.

[37] Wang Y, Boghigian BA, Pfeifer BA. Improving heterologous polyketide production in Escherichiacoli by overexpression of an S-adenosylmethionine synthetase gene. Appl Microbiol Biotechnol, 2007, 77(2): 367-373.

[38] Wang J-F, Xiong Z-Q, Li S-Y, Wang Y. Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichiacoli. Appl Microbiol Biotechnol, 2013, 97(18): 8057-8067.

[39] Wang J-F, Zhang S-L, Wang Y. Pathway assembly and optimization in E.coli for denovo biosynthesis of resveratrol. China Biotechnol, 2014, 34(2): 71-77.

[40] Watts KT, Lee PC, Schmidt-Dannert C. Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichiacoli. ChemBioChem, 2004, 5(4): 500-507.

[41] Wen Y, Ushio H. Ferulic acid promotes hypertrophic growth of fast skeletal muscle in zebrafish model. Nutrients, 2017

[42] Yamauchi Y, Hirasawa T, Nishii M, Furusawa C, Shimizu H. Enhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacteriumglutamicum harboring Escherichiacoli transhydrogenase gene pntAB. J Gen Appl Microbiol, 2014, 60(3): 112-118.

[43] Zhang C, Hong K. Production of Terpenoids by Synthetic Biology Approaches. Front Bioeng Biotechnol, 2020

[44] Zhao C, Zhao Q, Li Y, Zhang Y. Engineering redox homeostasis to develop efficient alcohol-producing microbial cell factories. Microb Cell Fact, 2017, 16(1): 115.

[45] Zhou JM, Gold ND, Martin VJ, Wollenweber E, Ibrahim RK. Sequential O-methylation of tricetin by a single gene product in wheat. Biochim Biophys Acta, 2006, 1760(7): 1115-1124.

[46] Zhou J-M, Seo YW, Ibrahim RK. Biochemical characterization of a putative wheat caffeic acid O-methyltransferase. Plant Physiol Biochem, 2009, 47(4): 322-326.

[47] Zocchi A, Jobe AM, Neuhaus JM, Ward TR. Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichiapastoris. Protein Expr Purif, 2003, 32(2): 167-174.

Funding

National Key Research and Development Program of China(2018YFA0900600)

the Strategic Priority Research Program ‘Molecular mechanism of Plant Growth and Development’ of CAS(XDB27020202)

National Natural Science Foundation of China(31670099)

the Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Science(No. ZSYS-016)

the International Partnership Program of Chinese Academy of Science(No. 153D31KYSB20170121)

Program of Shanghai Academic/Technology Research Leader(20XD1404400)

the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-002-15)