Optimizing Oleaginous Yeast Cell Factories for Flavonoids and Hydroxylated Flavonoids Biosynthesis

Abstract

Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. To date, plant extraction is still the primary route to produce high-value natural products which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces a large amount of lipid bodies, the abundant membrane structure and the lipophilic environment provide the ideal environment for the regioselectivity and stereoselectivity of many plant-derived P450 enzymes. In this work, we used modular method to construct, characterize, and optimize the flavonoid pathways in Yarrowia lipolytica. We also evaluated various precursor biosynthetic routes and unleashed the metabolic potential of Y. lipolytica to produce flavonoids and hydroxylated flavonoids. Specifically, we have identified that chalcone synthase (CHS) and cytochrome P450 reductases (CPR) were the bottlenecks of hydroxylated flavonoid production. We determined the optimal gene copy number of CHS and CPR to be 5 and 2, respectively. We further removed precursor pathway limitations by expressing genes associated with chorismate and malonyl-CoA supply. With pH and carbon−nitrogen ratio (C/N) optimization, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol, and 110.5 mg/L taxifolin from glucose in shake flasks. Flavonoid and its hydroxylated derivatives are most prominently known as antioxidant and antiaging agents. These findings demonstrate our ability to harness the oleaginous yeast as the microbial workhorse to expand nature's biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

“…Aromatic compounds have wide applications ranging from health care to food industry, nutraceutical supplements and pharmaceutical intermediates, which collectively represent a multi-billion-dollar global market 32–34 . Although metabolically-engineered E. coli has achieved gram-per-liter levels of aromatic compounds, primarily phenylpropanoids 35, 36 , yeast proves to be a more attractive host, due to its GRAS status, robust cell growth, tolerance of harsh conditions (such as low pH and high osmolarity), and the spatially-organized subcellular compartment for regio- or stereo-activity of cytochrome P450 enzymes 7,32,37 . Up to date, various metabolic engineering strategies have been implemented in E. coli and S. cerevisiae to improve aromatics production, including expression of the feedback-insensitive DAHP synthases 38 and chorismite synthase, enhancing the shikimate pathway 39 and modular microbial coculture 40–42 .…”

Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization

“…Y. lipolytica is considered to be a promising microbial workhorse for production of high value-added products [39,40]. In this study, we reported the development of quantitative measurement of violacein, based on both HPLC and microplate reader.…”

“…pH has played a major role in regulating cell physiology and metabolic activity. Low pH has been routinely observed in Y. lipolytica culture due to the accumulation of organic acids [39]. To evaluate the effects of pH on cell growth and violacein production, the cultivation pH was adjusted by adding 10 g/L CaCO 3 , or by buffering with NaH 2 PO 4 and Na 2 HPO 4 to pH 6.0, 6.5, 7.0, and 7.5, with initial unbuffered media as control.…”

Characterization of Met25 as a Color Associated Genetic Marker inYarrowia lipolytica

“…The recent development of genome-editing tools have made Y. lipolytica an ideal host for various applications, ranging from biofuel production [24][25][26] , natural product biosynthesis [27][28][29] and commodity chemical manufacturing 30,31 . To further expand the genetic toolbox and understand the sulfur metabolism, we hypothesized that MET25 could be utilized as a counter selectable color-associated genetic marker in Y. lipolytica.…”