Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

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Kaempferol is a polyphenolic compound with various reported health benefits and thus harbors considerable potential for food-engineering applications. In this study, a high-yield kaempferol-producing cell factory was constructed by multiple strategies, including gene screening, elimination of the phenylethanol biosynthetic branch, optimizing the core flavonoid synthetic pathway, supplementation of precursor PEP/E4P, and mitochondrial engineering of F3H and FLS. A total of 86 mg/L of kaempferol was achieved in strain YL-4, to date the highest production titer in yeast. Furthermore, a coculture system and supplementation of surfactants were investigated, to relieve the metabolic burden as well as the low solubility/possible transport limitations of flavonoids, respectively. In the coculture system, the whole pathway was divided across two strains, resulting in 50% increased cell growth. Meanwhile, supplementation of Tween 80 in our engineered strains yielded 220 mg/L of naringenin and 200 mg/L of mixed flavonoidsamong the highest production titer reported via de novo production in yeast.

Flavonoids are plant secondary metabolites with great potential in the food industry. Metabolic engineering of Saccharomyces cerevisiae is a sustainable production technique. However, the current naringenin production yield is low because of inefficient enzymatic activity. Hence, this study uses gene source screening as a tool to identify the best gene source for enzymes such as 4-coumarate: coenzyme ligase (4CL) and chalcone synthase (CHS). For the first time, the 4CL gene from Medicago truncatula and the CHS gene from Vitis vinifera were expressed in S. cerevisiae, and this combination provided the highest yield of naringenin, which was 28-fold higher as compared to the reference strain. The combinations obtained similar performance in the Y-28 strains, where the highest production was 28.68 mg/L. Our results demonstrated that the selection and combination of enzymes from the correct gene source could greatly improve naringenin production. For the future, this could help commercialize flavonoid production, which would result in natural food preservatives and additives.