Synthesis of rebaudioside D, using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration

Chen, Liangliang; Sun, Ping; Zhou, Fangfang; Li, Yan; Chen, Kequan; Jia, Honghua; Yan, Ming; Gong, De; Ouyang, Pingkai

doi:10.1016/j.foodchem.2018.03.126

Cited by 56 publications

(53 citation statements)

References 27 publications

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“…In the proposed system, UDP and inositol were reused and regenerated to push sucrose to form the desired product. To decrease production costs, cell extracts containing expressed enzymes and a small amount of UDP were used in the reaction medium to avoid enzyme purification and additional supplementation with UDP (31). Stachyose was produced by in vitro enzymatic catalysis, a process that has not been reported in previous studies.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Tian

Yang

Zeng

et al. 2019

Appl Environ Microbiol

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Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained. IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Tian

Yang

Zeng

et al. 2019

Appl Environ Microbiol

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“…Moreover, it has shown that plant‐derived glucosyltransferases such as UGT76G1 (Wang et al., ) can insert and produce rebaudioside A from stevioside in Saccharomyces cerevisiae (Olsson et al., ; Li et al., ) and Escherichia coli system (Wang et al., ). Production of rebaudioside I, rebaudioside D, and glucosyl rebaudioside A from rebaudioside A by enzymatic biotransformation has also been reported (Gerwig, Te Poele, Dijkhuizen & Kamerling, ; Te Poele et al., ; Chen et al., ). Lactobacillus reuteri 180 glucansucrase can transferred several glucosyl residues from one moiety to nine moieties into rebaudioside A from sucrose (Gerwig et al., ).…”

Section: Introductionmentioning

confidence: 89%

Enzymatic Synthesis of Glucosyl Rebaudioside A and its Characterization as a Sweetener

Lee

Kim

et al. 2019

Journal of Food Science

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Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O‐α‐D‐glucosyl‐(1″→6′) rebaudioside A with yield of 86%. O‐α‐D‐glucosyl‐(1″→6′) rebaudioside A was purified using HPLC and Diaion HP‐20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O‐α‐D‐glucosyl‐(1″→6′) rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O‐α‐D‐glucosyl‐(1″→6′) rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O‐α‐D‐glucosyl‐(1″→6′) rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. Practical Application The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value‐added bioactive compounds by enzymatic modification.

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“…The optimized genes encoding a fusion protein of 3′‐phosphoadenosine‐5′‐phosphatase (NCBI Reference Sequence: 948728) and UGT76G1 (NCBI Reference Sequence: LC037193) in pET‐C76G1 (Chen et al , ) and StSUS1 (NCBI Reference Sequence: LC430937) in pRSF‐SL2‐SUS1 (Chen et al , ) were subcloned into the NdeI/XhoI and NcoI/EcoRI sites of pCDFDuet‐1 (Novagen, Madison, WI, USA), respectively, resulting the plasmid pCDF‐C76G1‐SUS1. Plasmid pRSF‐SL2‐SUS1 (Chen et al , ) served as a template for site‐directed mutagenesis of UGTSL2 (NCBI Reference Sequence: XP_004250485.1) to mutate Asn358 to other 19 amino acid residues. The resulting plasmids carrying wild‐type or mutant UGTSL2 were transformed into the Escherichia coli BL21 (DE3) strain (TransGen Biotech, Beijing, China), with or without pCDF‐C76G1‐SUS1.…”

Section: Methodsmentioning

confidence: 99%

Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi‐enzyme system

Chen

Cai

Weng

et al. 2020

Microbial Biotechnology

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Rebaudioside D is a sweetener from Stevia rebaudiana with superior sweetness and organoleptic properties, but its production is limited by its minute abundance in S. rebaudiana leaves. In this study, we established a multi-enzyme reaction system with S. rebaudiana UDP-glycosyltransferases UGT76G1, Solanum lycopersicum UGTSL2 and Solanum tuberosum sucrose synthase StSUS1, achieving a two-step glycosylation of stevioside to produce rebaudioside D. However, an increase in the accumulation of rebaudioside D required the optimization of UGTSL2 catalytic activity towards glucosylation of rebaudioside A and reducing the formation of the side-product rebaudioside M2. On the basis of homology modelling and structural analysis, Asn358 in UGTSL2 was subjected to saturating mutagenesis, and the Asn358Phe mutant was used instead of wildtype UGTSL2 for bioconversion. The established multi-enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l À1 (1.6 times of wild-type UGTSL2) rebaudioside D from 20 g l À1 stevioside after reaction for 24 h. This system is useful for large-scale rebaudioside D production and expands our understanding of the pathways involved in its synthesis.

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Synthesis of rebaudioside D, using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration

Cited by 56 publications

References 27 publications

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Enzymatic Synthesis of Glucosyl Rebaudioside A and its Characterization as a Sweetener

Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi‐enzyme system

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