Whole-Cell Biocatalyst for Rubusoside Production in Saccharomyces cerevisiae

Abstract

Rubusoside (Rub) is a highly sweet diterpene glycoside mainly isolated from the leaves of Rubus suavissimus (Rosaceae). It has been used as a low-calorie natural sweetener for decades and was recently found to be a potential drug lead. In this study, we designed a whole-cell biocatalyst to achieve the glycosylation of steviol to Rub in Saccharomyces cerevisiae. The sucrose synthases were applied to construct a uridine diphosphate glucose regeneration system, which were coupled with optimal combinations of different uridine diphosphate (UDP) glycosyltransferases from multiple plant species. After optimization of reaction conditions, the residues in SrUGT74G1 probably influencing glycosylation efficiency were subjected to site-directed mutagenesis. Double mutations of S84A/E87A reduced the accumulation of intermediates, finally glucosylating 1.27 g/L steviol to 0.45 ± 0.06 g/ L steviolmonoside (conversion rate = 23.3%) and 1.92 ± 0.17 g/L Rub (conversion rate = 74.9%). A high efficiency of Rub biosynthesis could be achieved without supply of additional UDPG. This work provided the first example of multi-step glycosylation reactions in whole-cell biocatalysis, which laid a foundation of scalable production of the value-added diterpene sweetener in the future.

Identification and Validation of Magnolol Biosynthesis Genes in Magnolia officinalis

“…Recent studies have further indicated that Yarrowia lipolytica can supply a robust provision of acetyl-CoA precursors and ample reducing power [47,48], thus catering to the requisites of heterologous synthetic pathways. Previously, flavonoids were considered as low-yield metabolites with rate-limiting steps [49], primarily due to the limited availability of precursor acetyl-CoA and suboptimal metabolic pathway engineering, culminating in fermentative yields scarcely exceeding 1 g/L [21,50,51]. However, with the intervention of oleaginous yeast, metabolic yields for certain compounds that appeared challenging to surpass 1 g/L can now be effortlessly elevated to several grams per liter [52][53][54].…”

Rational Design for the Complete Synthesis of Stevioside in Saccharomyces cerevisiae

“…In Stevia rebaudiana, only four glycosyltransferases catalyze all glucosylation reactions, and there are more than twenty glucosylation products [32,33]. In order to obtain strains that yield high amounts of stevioside, we decided to adjust the glycosylation sequence, replacing the original SrUGT74G1 with SrUGT74G1S84A/E87A, which was directionally modified [18]; to reduce the accumulation of intermediate by-products, SrUGT85C2 and SrUGT74G1S84A/E87A were then fusion expressed using the flexible peptide "GGGS", and SrUGT91D2e_NO.5 was replaced with SrUGT91D2 [16]. This process formed the strain SST-302III-ST1, which yielded 55.6 mg/L of stevioside, but 90 mg/L of steviol remained in the fermentation so- In yeast cells, incoming glucose is phosphorylated to glucose-6-phosphate via hexokinase (HXK1), then converted to glucose-1-phosphate via phosphoglucomutase (PGM2), and, finally, condensed with uracil triphosphate-glucose (UTP) to form UDP-Glc via UDPglucose pyrophosphorylase (UGP1).…”

Progress and Prospects of Natural Glycoside Sweetener Biosynthesis: A Review

“…This study made it possible to convert simple steviol glycosides to high-value glycosides through whole-cell catalysis. In addition, under whole-cell biocatalysis, steviol can be glycosylated to generate rubusoside . The UDP-glucose regeneration system was coupled with optimal UDP, and mutations were induced at specific sites where residues could impact the glycosylation efficiency of SrUGT74G1.…”

“…In addition, under whole-cell biocatalysis, steviol can be glycosylated to generate rubusoside. 83 The UDP-glucose regeneration system was coupled with optimal UDP, and mutations were induced at specific sites where residues could impact the glycosylation efficiency of SrUGT74G1. Mutations reduced the accumulation of intermediates.…”