We have located links that may give you full text access.
Microbial production of next-generation stevia sweeteners.
Microbial Cell Factories 2016 December 8
BACKGROUND: The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana.
RESULTS: In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13- and C 19-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1.
CONCLUSIONS: Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1Thr146Gly and UGT76G1His155Leu, which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners.
RESULTS: In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13- and C 19-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1.
CONCLUSIONS: Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1Thr146Gly and UGT76G1His155Leu, which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners.
Full text links
Related Resources
Get seemless 1-tap access through your institution/university
For the best experience, use the Read mobile app
All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.
By using this service, you agree to our terms of use and privacy policy.
Your Privacy Choices
You can now claim free CME credits for this literature searchClaim now
Get seemless 1-tap access through your institution/university
For the best experience, use the Read mobile app