咖啡酸是一种天然酚酸类化合物,具有抗氧化、抗肿瘤、抗炎等多种生物学活性,咖啡酸也是迷迭香酸、绿原酸和咖啡酸苯乙酯等高附加值化合物合成的重要前体,在医药、食品和化妆品等行业具有较大的应用价值。为实现咖啡酸绿色可持续生产,在前期构建的产咖啡酸酿酒酵母菌株中,敲除苯丙氨酸途径中的预苯酸脱水酶基因(PHA2),将咖啡酸产量提高42%。而敲除色氨酸途径中邻氨基苯甲酸合成酶基因(TRP2),对咖啡酸积累影响不大。之后,回补了URA3、HIS3、MET15营养标记基因,然后将工程菌株在5 L的发酵罐中进行了补料分批发酵,使得咖啡酸产量达到了9.3 g/L,是目前文献中报道微生物发酵生产咖啡酸的最高水平,该研究为咖啡酸的绿色生产以及其衍生物的高效微生物合成奠定了基础。
Caffeic acid is a natural phenolic acid with various pharmaceutical properties, including antioxidant, anti-tumor and anti-inflammatory effects.It is also an important precursor for biosynthesis of other valuable compounds such as rosmarinic acid, chlorogenic acid and phenethyl caffeate.Therefore, caffeic acid has significant value in pharmaceutical, food, cosmetic and other industries.To achieve green and sustainable production of caffeic acid, the prephenate dehydratase gene (PHA2), which is involved in the biosynthesis of phenylalanine, was knocked out in a previously constructed caffeic acid produced Saccharomyces cerevisiae, resulting in a 42% improvement in caffeic acid production.Additionally, the anthranilate synthase gene (TRP2), which is responsible for tryptophan biosynthesis, was deleted.However, this had little effect on coffee acid production.The engineered strain was further complemented with auxotrophic marker URA3, HIS3, MET15.In a subsequent fed-batch fermentation process conducted in a 5 L bioreactor, the engineered strain achieved a caffeic acid production of 9.3 g/L, this is currently the highest reported titer of caffeic acid produced by engineered microbial cells.This study provides a foundation for the green production of caffeic acid and its derivatives.
[1] CHEN J H, HO C T.Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds[J].Journal of Agricultural and Food Chemistry, 1997, 45(7):2374-2378.
[2] CHAO P C, HSU C C, YIN M C.Anti-inflammatory and anti-coagulatory activities of caffeic acid and ellagic acid in cardiac tissue of diabetic mice[J].Nutrition & Metabolism, 2009, 6:33.
[3] ESPÍNDOLA K M M, FERREIRA R G, NARVAEZ L E M, et al.Chemical and pharmacological aspects of caffeic acid and its activity in hepatocarcinoma[J].Frontiers in Oncology, 2019, 9:541.
[4] LI S Z, LIANG C N, LIU G X, et al.De novo biosynthesis of chlorogenic acid using an artificial microbial community[J].Journal of Agricultural and Food Chemistry, 2021, 69(9):2816-2825.
[5] ZHOU P P, YUE C L, ZHANG Y C, et al.Alleviation of the byproducts formation enables highly efficient biosynthesis of rosmarinic acid in Saccharomyces cerevisiae[J].Journal of Agricultural and Food Chemistry, 2022, 70(16):5077-5087.
[6] XU Y P, GENG L J, ZHANG Y W, et al.De novo biosynthesis of salvianolic acid B in Saccharomyces cerevisiae engineered with the rosmarinic acid biosynthetic pathway[J].Journal of Agricultural and Food Chemistry, 2022, 70(7):2290-2302.
[7] WANG J, MAHAJANI M, JACKSON S L, et al.Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters and amides[J].Metabolic Engineering, 2017, 44:89-99.
[8] MAGNANI C, ISAAC V L B, CORREA M A, et al.Caffeic acid:A review of its potential use in medications and cosmetics[J].Analytical Methods, 2014, 6(10):3203-3210.
[9] KIM Y H, KWON T, YANG H J, et al.Gene engineering, purification, crystallization and preliminary X-ray diffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis membrane protein[J].Protein Expression and Purification, 2011, 79(1):149-155.
[10] BERNER M, KRUG D, BIHLMAIER C, et al.Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrix espanaensis[J].Journal of Bacteriology, 2006, 188(7):2666-2673.
[11] CHOI O, WU C Z, KANG S Y, et al.Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in Escherichia coli[J].Journal of Industrial Microbiology & Biotechnology, 2011, 38(10):1657-1665.
[12] LIN Y H, YAN Y J.Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex[J].Microbial Cell Factories, 2012, 11:42.
[13] HUANG Q, LIN Y H, YAN Y J.Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain[J].Biotechnology and Bioengineering, 2013, 110(12):3188-3196.
[14] WANG L, LI N, YU S Q, et al.Enhancing caffeic acid production in Escherichia coli by engineering the biosynthesis pathway and transporter[J].Bioresource Technology, 2023, 368:128320.
[15] LIU L Q, LIU H, ZHANG W, et al.Engineering the biosynthesis of caffeic acid in Saccharomyces cerevisiae with heterologous enzyme combinations[J].Engineering, 2019, 5(2):287-295.
[16] CHEN R B, GAO J Q, YU W, et al.Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast[J].Nature Chemical Biology, 2022, 18(5):520-529.
[17] ZHOU P P, YUE C L, SHEN B, et al.Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid[J].Applied Microbiology and Biotechnology, 2021, 105(14-15):5809-5819.
[18] ZHOU P P, FANG X, XU N N, et al.Development of a highly efficient copper-inducible GAL regulation system (CuIGR) in Saccharomyces cerevisiae[J].ACS Synthetic Biology, 2021, 10(12):3435-3444.
[19] LYU X M, WANG F, ZHOU P P, et al.Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae[J].Nature Communications, 2016, 7:12851.
[20] LIU H Y, TIAN Y J, ZHOU Y, et al.Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside[J].Microbial Biotechnology, 2021, 14(6):2605-2616.
[21] CAO M F, GAO M R, SUÁSTEGUI M, et al.Building microbial factories for the production of aromatic amino acid pathway derivatives:From commodity chemicals to plant-sourced natural products[J].Metabolic Engineering, 2020, 58:94-132.
[22] GUO W, HUANG Q L, FENG Y H, et al.Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae[J].Biotechnology and Bioengineering, 2020, 117(8):2410-2419.
