*Corresponding authors
Weihong Zhong, E-mail: whzhong@zjut.edu.cn, Fax/Tel: (86)-571-88813378.
Abstract: Sakuranetin is a plant-natural product, which has increasingly been utilized in cosmetic and pharmaceutical industries for its extensive anti-inflammatory, anti-tumor, and immunomodulatory effects. Sakuranetin was mostly produced via extraction technology from plants, which is limited to natural conditions and biomass supply. In this study, a novel strategy to produce sakuranetin via de novosynthesis from glucose by engineering S. cerevisiae was introduced. After a series of heterogenous genes integration, a biosynthetic pathway of sakuranetin from glucose was successfully constructed in S. cerevisiae which sakuranetin yield reached only 4.28 mg/L. Then, a multi-module metabolic engineering strategy was applied for improving sakuranetin yield in S. cerevisiae : (1) adjusting the copy number of sakuranetin synthesis genes; (2) removing the rate-limiting factor of aromatic amino pathway and optimizing the synthetic pathway of aromatic amino acids to enhance the supply of carbon flux for sakuranetin; (3) introducing acetyl-CoA carboxylase mutantsACC1S659A, S1157A, and knocking-outYPL062W to strengthen the supply of malonyl-CoA which is another synthetic precursor of sakuranetin. The resultant mutant S. cerevisiae exhibited a more than 10-fold increase of sakuranetin titer (50.62 mg/L) in shaking flasks. Furthermore, the sakuranetin titer increased to 158.65 mg/L in a 1-L bioreactor, which is the highest sakuranetin titer among all publications reported yield of the engineered microbial cell.
Keywords: Sakuranetin; Saccharomyces cerevisiae ; De novo biosynthesis; Pathway balancing; Metabolic engineering
Introduction
Flavonoids, which are well-known plant secondary metabolites, have anti-oxidant, anti-cancer, anti-aging, and antimicrobial effects (Benkherouf et al., 2019; Berim & Gang, 2016; Li et al., 2018; Stompor et al., 2019; Stompor & Zarowska, 2016). The majority of flavonoids are produced via extraction technology from plants, which is limited to natural conditions and biomass supply and is not stable and uniform for useful flavonoid production (Newman & Cragg, 2007). Therefore, it is necessary to develop effective microbial systems for the synthesis of flavonoids via metabolic engineering (Pirie et al., 2013).
Sakuranetin (chemical name: 4 ,5-dihydroxy-7-methoxyflavanone) is a dihydroflavonoid compound originally separated from the bark of the cherry tree (Asahina, 1908). Current studies have found that sakuranetin has anti-inflammatory activity (Kim & Kang, 2016), anti-tumor (Chen et al., 2016), and antimicrobial effects (Grecco et al., 2014), especially has a therapeutic effect on asthma (Sakoda et al., 2016; Santana et al., 2019), exhibiting a wide range of medicinal application potential. Meanwhile, it can effectively resist melanin deposition and improve the dullness of the skin, and play a role in whitening and rejuvenating the skin due to its high antioxidant activity (Stompor, 2020).
So far, there have been few reports on the synthesis of sakuranetin in microorganisms. However, the synthesis of its precursor naringenin has been widely studied in S. cerevisiae and Escherichia coli . The biosynthetic pathway of naringenin begins with aromatic amino acids and is primarily mediated by four enzymes: tyrosine ammonia lyase (TAL) or phenylalanine ammonia-lyase (PAL), 4-coumaric acid-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) (Winkel-Shirley, 2001). A complete synthetic pathway of naringenin by introducing the above four genes (PAL , 4CL , CHS, and CHI ) and cinnamic acid monooxygenase (C4H) was constructed in S. cerevisiae for naringenin production via the phenylalanine pathway, the yield of naringenin increased to 54.4 mg/L in shaking flask cultures (Koopman et al., 2012). Four genes (TAL , 4CL , CHS , and CHI ) were integrated into S. cerevisiae for naringenin production via the tyrosine pathway, and the yield of naringenin reached 648.63 mg/L via fed-batch fermentation by the addition ofp -coumaric acid (Gao, Lyu, et al., 2020). The naringenin yield reached 100.64 mg/L starting with glucose in E. coli via a multi-module metabolic engineering strategy including (1) increasing the number of gene copies of the naringenin synthesis pathway, and (2) regulating the expression intensity of the promoter (Wu et al., 2014). The yield of naringenin reached 90 mg/L via metabolic engineering of precursor supply and promoter control in S. cerevisiae (Lyu et al., 2017). Furthermore, a fatty acid catabolic pathway was systematically designed for the synthesis of naringenin in S. cerevisiae , and the naringenin yield reached 1.13 g/L in a 5-L bioreactor (Zhang et al., 2021). After efficient pathway optimization via promoter engineering based on the promoter library in S. cerevisiae , a high naringenin production of 1.21 g/L fromp -coumaric acid was achieved in a 5-L bioreactor (Gao, Zhou, et al., 2020).
After the key enzyme from naringenin to sakuranetin in rice, naringenin-7-O-methyltransferase (NOMT), was identified (Shimizu et al., 2012). Several genes including NOMT were introduced for sakuranetin synthesis in E. coli , and the sakuranetin production in the shaking flask reached 40.1 mg/L (Kim et al., 2013). A two-module co-culture strategy of p -coumaric acid and sakuranetin was designed in E. coli , and the yield of sakuranetin reached 79 mg/L in a 2.5-L bioreactor (Wang et al., 2020). However, there are few reports on sakuranetin synthesis inS. cerevisiae. Given its higher ability to produce SAM than that of prokaryotes, S. cerevisiae has a natural advantage as a chassis cell to produce sakuranetin.
In this study, we proposed to construct a pathway for de novoproduction of sakuranetin from glucose in S. cerevisiae (Fig. 1) and strengthen the biosynthesis of sakuranetin via a multi-modules metabolic engineering, including (1) enhancing sakuranetin biosynthesis by adjusting the genes copy number; (2) removing the rate-limiting factor of aromatic amino pathway and optimizing the synthetic pathway of aromatic amino acids (L-Phe and L-Tyr) to enhance the supply of carbon flux of sakuranetin; and (3) introducing acetyl-CoA carboxylase mutantsACC1S659A, S1157A and knocking-outYPL062W to strengthen the supply of malonyl-CoA, another synthetic precursor of sakuranetin. The resultant mutant S. cerevisiae exhibited a more than 10-fold increase of sakuranetin at a titer of 50.62 mg/L in shake-flask cultures and 158.65 mg/L in a 1-L bioreactor, respectively.