Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside

Abstract

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

Highly efficient production of rebaudioside D enabled by structure-guided engineering of bacterial glycosyltransferase YojK

“…To solve the solubility problem of plant-derived glycosyltransferases ( Cai et al, 2017 ; Lemmerer et al, 2019 ; Chen et al, 2020 ; Shu et al, 2020 ), YojK, a Leloir O-glycosyltransferase from B. subtilis 168, was selected to determine whether it had the catalytic ability to glycosylate Reb A to form Reb D ( Figure 1A ), as it exhibits prominent glycosylation activity to various substrates like other bacterial glycosyltransferases with a large acceptor binding pocket ( Strobel et al, 2013 ; Zhou et al, 2013 ; Pandey et al, 2014 ). Previous studies show that it can glycosylate substrates with large size like ginsenosides, crocins, flavonols and flavones ( Luo et al, 2015 ; Pandey et al, 2019 ; Wang et al, 2019 ).…”

“…EUGT11 also shows its catalytic ability to biosynthesize Reb D from Reb A at the low concentration of Reb A ( Wang et al, 2020 ). However, the heterologous expression of these plant-derived glycosyltransferases has been widely recognized as a problem ( Cai et al, 2017 ; Chen et al, 2018 ; Lemmerer et al, 2019 ; Shu et al, 2020 ). They are mainly expressed as inclusion bodies in Escherichia coli ( E. coli ) with little soluble and active protein ( Cai et al, 2017 ; Lemmerer et al, 2019 ; Shu et al, 2020 ), which greatly restricts their industrial applications for scale production of Reb D. Therefore, to identify and characterize a novel glycosyltransferase, which could be functionally expressed in E. coli with high solubility and possesses an excellent ability to catalyze the synthesis of Reb D from Reb A with great regioselectivity, is highly desired.…”

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle

“…cerevisiae. It is probably because UGT91D2 is responsible for catalyzing stevioside into Reb E and Reb A into Reb D, the high expression of UGT91D2 increased the titer of Reb E and Reb D with 50 mg/L stevioside as the substrate . UGT91D2 has a smaller active pocket than EUGT11 and is preferable when choosing stevioside as the substrate.…”

“…There are two methods to obtain Reb M by whole-cell catalysis: modifying UGT76G1 with Reb D as the substrate or modifying UGT76G1 and EUGT11 with the difficult-to-obtain Reb E or expensive Reb A as the substrate. In Escherichia coli, 1.8 g/L of Reb M was synthesized via enzymatic catalysis by promoting the solubility of UGT76G1 with 2 mM Reb D as the substrate . The UGT76G1 T284S/M88L/L200A mutant was able to produce 23.37 mg/mL Reb M from 22.58 mg/mL Reb D with a yield of 90.5% in E.…”

Rethinking Biosynthesis of Aclacinomycin A

“…In addition, heterologous bacterial hosts may generate inclusion bodies [ 89 , 90 ]. To improve the solubility of GTs, a plethora of protein fusion partners and solubility tags have been successfully utilized [ 89 , 91 , 92 ].…”