Metabolic Engineering of Saccharomyces cerevisiae for De Novo Production of Kaempferol

Abstract

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.

Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

“…When V461 was mutated into serine, the specificity of the downstream ketoic acid was reduced, which was conducive to the production of the precursor material amyl alcohol [32]. Kaempferol was produced by the metabolic engineering of Saccharomyces cerevisiae [33]. B-FITTER software was used to analyze the crystal structure of tryptophan synthetase to determine the temperature factor (B-factor) of each amino acid locus and to identify the adverse effects on the thermal stability of the enzyme caused by mutations of the key amino acid G395.…”

Metabolic Engineering of Microorganisms for the Production of Flavonoids

“…Biosynthesis of flavonols can be achieved by coexpressing F3H and FLS with flavanones as the precursor. Lyu et al (2019) introduced F3H and FLS into a naringenin-producing S. cerevisiae strain Y-22, which rendered the recombinant strain the ability to generate kaempferol. Further optimization strategies including screening of efficient pathway enzymes, disruption of competing pathways, enhancement of the precursors PEP and E4P supply, and mitochondrial engineering of F3H and FLS significantly improved the kaempferol titer to 86 mg/L (Lyu et al, 2019).…”

A roadmap to engineering antiviral natural products synthesis in microbes

“…A de novo biosynthetic pathway for kaempferol was reconstructed in S. cerevisiae by optimizing the expression of flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H) from a large variety of gene candidates. By blocking phenylethanol accumulation, supplementing intermediates, and overexpressing the key genes in kaempferol synthetic pathway, the final strain produced 86 mg/L kaempferol [ 40 ]. In another study, based on production of kaempferol, further introduction of an optimized flavonoid monooxygenase (FMO) and its partner CPR led the production of 20.4 mg/L quercetin in the S. cerevisiae strain.…”