De novo biosynthesis of quercetin in Yarrowia Lipolytica through systematic metabolic engineering for enhanced yield

Abstract

Kaempferol and quercetin possess various biological activities, making them valuable in food and medicine. However, their production via traditional methods is often inefficient. This study aims to address this gap by engineering the yeast Yarrowia lipolytica to achieve high yields of these flavonoids. We designed a kaempferol biosynthetic pathway by integrating multiple-copy fusion enzyme expression modules, F3H-(GGGGS)₂-FLS, into the genome with an optimized linker (GGGGS)₂ to enhance kaempferol production from naringenin. To synthesize quercetin de novo, we introduced the FMOCPR gene into the kaempferol-synthesizing strain using the optimized pFBAin promoter. Notably, increasing glucose concentration effectively boosted the production of both flavonoids. Our results demonstrated kaempferol and quercetin titers reaching 194.30 ± 7.69 and 278.92 ± 11.58 mg/L, respectively, in shake-flask cultures. These findings suggest that Y. lipolytica is a promising platform for the efficient production of flavonoid-derived products.

References

Al-Khayri JM, Sahana GR, Nagella P. Flavonoids as potential anti-inflammatory molecules Rev Molecules, 2022, 27(9): 2901.

Alexander C, Parsaee A, Vasefi M. Polyherbal and multimodal treatments: kaempferol- and quercetin-rich herbs alleviate symptoms of alzheimer’s disease Biology (Basel), 2023, 12(11): 1453

Bangar SP, Chaudhary V, Sharma N. Kaempferol: a flavonoid with wider biological activities and its applications Crit Rev Food Sci Nutr, 2023, 63(28): 9580-9604.

Borrás-Linares I, Fernández-Arroyo S, Arráez-Roman D. Characterization of phenolic compounds, anthocyanidin, antioxidant and antimicrobial activity of 25 varieties of Mexican Roselle (Hibiscus sabdariffa) Ind Crops Prod, 2015, 69: 385-394.

Chen X, Zaro JL, Shen W-C. Fusion protein linkers: property, design and functionality Drug Deliver Rev, 2013, 65(10): 1357-1369.

Cravens A, Payne J, Smolke CD. Synthetic biology strategies for microbial biosynthesis of plant natural products Nat Commun, 2019, 10(1): 2142.

Dabeek WM, Marra MV. Dietary quercetin and kaempferol: bioavailability and potential cardiovascular-related bioactivity in humans Nutrients, 2019, 11(10): 2288.

Duan L, Ding W, Liu X. Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae Microb Cell Fact, 2017, 16(1): 165.

He Q, Szczepańska P, Yuzbashev T. De novo production of resveratrol from glycerol by engineering different metabolic pathways in Yarrowia Lipolytica Metab Eng Commun, 2020, 11: e00146.

Jan R, Khan M, Asaf S. Bioactivity and therapeutic potential of kaempferol and quercetin: new insights for plant and human health Plants (Basel), 2022, 11(19): 2623

Liu QL, Liu Y, Li G. De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories Nat Commun, 2021, 12(1): 6085.

Liu M, Wang C, Ren X. Remodelling metabolism for high-level resveratrol production in Yarrowia Lipolytica Bioresour Technol, 2022, 365: 128178.

Marin L, Gutierrez-Del-Rio I, Entrialgo-Cadierno R. De novo biosynthesis of myricetin, kaempferol and quercetin in Streptomyces albus and Streptomyces coelicolor PLoS ONE, 2018, 13(11): e0207278.

Markham KA, Palmer CM, Chwatko M. Rewiring Yarrowia Lipolytica toward triacetic acid lactone for materials generation P Natl Acad Sci U S A, 2018, 115(9): 2096-2101.

Mckay TB, Emmitte KA, German C. Quercetin and related analogs as therapeutics to promote tissue repair Bioeng (Basel), 2023, 10(10): 1127

Miller KK, Alper HS. YarLipolyticalytica: more than an oleaginous workhorse Appl Microbiol Biot, 2019, 103(23–24): 9251-9262.

Niu T, Huang C, Wang R. Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin Microb Cell Fact, 2024, 23(1): 114.

Palmer CM, Miller KK, Nguyen A. Engineering 4-coumaroyl-CoA derived polyketide production in Yarrowia Lipolytica through a β-oxidation mediated strategy Metab Eng, 2020, 57: 174-181.

Patra P, Das M, Kundu P. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts Biotechnol Adv, 2021, 47: 107695.

Pei J, Chen A, Dong P. Modulating heterologous pathways and optimizing fermentation conditions for biosynthesis of kaempferol and astragalin from naringenin in Escherichia coli J Ind Microbiol Biotechnol, 2019, 46(2): 171-186.

Qiu Z, Liu X, Li J. Metabolic division in an Escherichia coli coculture system for efficient production of kaempferide ACS Synth Biol, 2022, 11(3): 1213-1227.

Rodriguez A, Strucko T, Stahlhut SG. Metabolic engineering of yeast for fermentative production of flavonoids BioresourTechnol, 2017, 245: 1645-1654.

Shang Y, Zhang P, Wei W. Metabolic engineering for the high-yield production of polydatin in Yarrowia Lipolytica Bioresour Technol, 2023, 381: 129129.

Smith S, Tsai S-C. The type I fatty acid and polyketide synthases: a tale of two megasynthases Nat Prod Rep, 2007, 24(5): 1041-1072.

Spagnuolo M, Hussain MS, Gambill L. Alternative substrate metabolism in Yarrowia Lipolytica Front Microbiol, 2018, 9: 1077.

Stahlhut SG, Siedler S, Malla S. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli Metab Eng, 2015, 31: 84-93.

Tartik M, Liu J, Mohedano MT. Optimizing yeast for high-level production of kaempferol and quercetin Microb Cell Fact, 2023, 22(1): 74.

Tian CL, Liu X, Chang Y. Investigation of the anti-inflammatory and antioxidant activities of luteolin, kaempferol, apigenin and quercetin S Afr J Bot, 2021, 137: 257-264.

Trantas E, Panopoulos N, Ververidis F. Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae Metab Eng, 2009, 11(6): 355-366.

Wei W, Zhang P, Shang Y. Metabolically engineering of Yarrowia Lipolytica for the biosynthesis of naringenin from a mixture of glucose and xylose Bioresource Technol, 2020, 314: 123726.

Worland AM, Czajka JJ, Li Y. Biosynthesis of terpene compounds using the non-model yeast Yarrowia Lipolytica: grand challenges and a few perspectives Curr Opin Biotech, 2020, 64: 134-140.

Yao F, Liu S-C, Wang D-N. Engineering oleaginous yeast Yarrowia Lipolytica for enhanced limonene production from xylose and lignocellulosic hydrolysate FEMS Yeast Res, 2020, 20(6): foaa046.

Funding

Shanghai Municipal Science and Technology Major Project(Shanghai Municipal Science and Technology Major Project); Natural Science Foundation of Zhejiang Province(LQ22C010005)