De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories

Abstract

Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L−1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L−1) and daidzin (73.2 mg L−1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.

De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts

“…According to the literature, it is essential to supply sufficient UDP-glucose for a high biosynthesis of glycosides 15 , 40 . Thus, to further boost the UDP-glucose pool, we overexpressed three genes PGM1 (phosphoglucomutase), PGM2 (phosphoglucomutase), and UGP1 (uridine triphosphate glucose-1-phosphate uridylyltransferase) in the SGN13 strain, using the plasmid pY13- PGK1p .…”

“…S. cerevisiae has generally been considered a superior host for expressing plant-derived P450s; however, in cases with low catalytic activity or expression level, their efficiency still needs to be improved 15 . Although the catalytic activity of KAH has been improved by its co-expression with CPR1 24 , the steviol titer was still limited by the transformation of EKA into steviol (Supplementary Fig.…”

“…However, synthesis efficiency was low, partially because some of the enzymes in the pathway were poorly expressed in prokaryotic cells, such as the cytochrome P450s. Compared with prokaryotic counterparts, the Generally Recognized as Safe (GRAS) chassis organism Saccharomyces cerevisiae is considered a superior host for expressing plant-derived P450s 15 . Olsson et al reported that Reb D and Reb M can be synthesized in S. cerevisiae .…”

“…According to previous reports, there are three main factors that limit the efficient production of rubusoside and rebaudiosides in yeast. The first one is the presence of rate-limiting steps in the biosynthesis pathway that must be overcome, to avoid that the precursor of diterpenes in the MVA pathway is insufficient, and that plant-derived P450s and UGTs exhibit low catalytic efficiencies 2 , 3 , 15 . The second factor is related to the need to improve the tolerance of yeast to rubusoside and rebaudiosides, as terpene accumulation often leads to cytotoxicity or growing pressure 17 .…”

Precursor Quantitation Methods for Next Generation Food Production

“…Acyl coenzyme are main precursors for natural products such as fatty acids ( Zhu et al, 2017 ) and heme (C4 pathway) ( Xin et al, 2018 ), components in meat analog. In addition, they also serve as building blocks for biosynthesis of nutritious supplements such as resveratrol ( Pannu and Bhatnagar 2019 ) and isoflavonoids ( Liu et al, 2021 ). Among them, acetyl-CoA, malonyl-CoA and methymalonyl-CoA are most widely used substrates, while other acyl derived CoAs such as ethylmalonyl-CoA, methyoxymalonyl-CoA, chloroethylmalonyl-CoA, allylmalonyl-CoA and hexylmalonyl-CoA also took part in the biosynthesis of some compounds ( Ray and Moore 2016 ).…”