Optimization of Pinocembrin Biosynthesis in Saccharomyces cerevisiae

Abstract

The flavonoid pinocembrin and its derivatives have gained increasing interest for their benefits on human health. While pinocembrin and its derivatives can be produced in engineered Saccharomyces cerevisiae , yields remain low. Here, we describe novel strategies for improved de novo biosynthesis of pinocembrin from glucose based on overcoming existing limitations in S. cerevisiae. First, we identified cinnamic acid as an inhibitor of pinocembrin synthesis. Second, by screening for more efficient enzymes and optimizing the expression of downstream genes, we reduced cinnamic acid accumulation. Third, we addressed other limiting factors by boosting the availability of the precursor malonyl-CoA, while eliminating the undesired byproduct 2′,4′,6′-trihydroxy dihydrochalcone. After optimizing cultivation conditions, 80 mg/L pinocembrin was obtained in a shake flask, the highest yield reported for S. cerevisiae . Finally, we demonstrated that pinocembrin-producing strains could be further engineered to generate 25 mg/L chrysin, another interesting flavone. The strains generated in this study will facilitate the production of flavonoids through the pinocembrin biosynthetic pathway.

High-Efficiency Multiplexed Cytosine Base Editors for Natural Product Synthesis in Yarrowia lipolytica

“…Tsc13 was targeted to prevent the reported promiscuous activity of Tsc13 on chalcone synthase in S. cerevisiae. − Even though the base editing efficiency was high, we could not obtain the stop codon mutations in Tsc13 with the base editor (Figure S8). Tsc13 gene encodes for very-long-chain enoyl-CoA reductase, catalyzing the final step in each cycle of long-chain fatty acid elongation This pointed to a previously unreported essentiality of the Tsc13 gene in Y.…”

Optimizing yeast for high-level production of kaempferol and quercetin

“…This beneficial effect of fed-batch-like condition has been shown before to further increase naringenin production with enhanced malonyl-CoA supply in NAG3-4 [ 31 ], which led us to consider another precursor malonyl-CoA for naringenin biosynthesis. Thus, we overexpressed a mutant version of acetyl-CoA carboxylase (Acc1 S659A, S1157A ), which demonstrated improved activity [ 41 ] and increased malonyl-CoA supply [ 43 ], in our best kaempferol producer MTK43 and quercetin producer MTQ13. Under normal batch conditions, while production of kaempferol slightly increased quercetin production was significantly reduced (Fig.…”

De Novo Synthesis of Chrysin in Saccharomyces cerevisiae

“…In S. cerevisiae, the concentration of chrysin was elevated from 3.6 mg/L to 17.3 mg/L through the manipulation of gene expression and the augmentation of malonyl-CoA availability. 11 In P. pastoris, a chrysin concentration of 20.1 mg/L was achieved by enhancing the supply of malonyl-CoA and replacing the enoyl reductase (Tsc13) with MdECR. 11 Currently reported methods to improve the concentration of chrysin mainly focus on adjusting the expression of transgenes 12 and enhancing the supply of malonyl-CoA.…”

“…11 In P. pastoris, a chrysin concentration of 20.1 mg/L was achieved by enhancing the supply of malonyl-CoA and replacing the enoyl reductase (Tsc13) with MdECR. 11 Currently reported methods to improve the concentration of chrysin mainly focus on adjusting the expression of transgenes 12 and enhancing the supply of malonyl-CoA. 11 However, these methods do not address the issue of precursor availability.…”

“…14 S. cerevisiae is capable of synthesizing a variety of flavonoids that have a similar structure to chrysin, including chlorogenic acid 15 and pinocembrin. 11 The synthesis of chrysin involves cytochrome P450 monooxygenases, which are anchored to the membrane of the endoplasmic reticulum and other organelles. 16 S. cerevisiae has a unique posttranscriptional modification mechanism, which can functionally express P450 hydroxylase to produce flavonoids, 17 such as eriodictyol 18 and daidzin.…”