title: Microbial production of next-generation stevia sweeteners.
abstract: 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 C13- and C19-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 UGT76G1Thr146Glyand 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.
authors.affiliation: Evolva A/S, Lersø Park Alle 42-44, 5th, 2100, Copenhagen OE, Denmark.|Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Kim
authors.identifier: http://orcid.org/0000-0003-0900-7519
authors.initials: K
authors.lastname: Olsson
authors.affiliation: Evolva A/S, Lersø Park Alle 42-44, 5th, 2100, Copenhagen OE, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Carlsen
authors.affiliation: Evolva A/S, Lersø Park Alle 42-44, 5th, 2100, Copenhagen OE, Denmark.
authors.forename: Angelika
authors.initials: A
authors.lastname: Semmler
authors.affiliation: Evolva A/S, Lersø Park Alle 42-44, 5th, 2100, Copenhagen OE, Denmark.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Simón
authors.affiliation: Evolva A/S, Lersø Park Alle 42-44, 5th, 2100, Copenhagen OE, Denmark. michaelm@evolva.com.
authors.forename: Michael Dalgaard
authors.initials: MD
authors.lastname: Mikkelsen
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.|Center for Synthetic Biology "bioSYNergy", University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
mesh_terms: D000595:Amino Acid Sequence; D045786:Diterpenes, Kaurane / Q000096:biosynthesis* / Q000378:metabolism; D005960:Glucosides / Q000096:biosynthesis* / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D036625:Stevia / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013549:Sweetening Agents / Q000737:chemistry / Q000378:metabolism*
chemical_list: D045786:Diterpenes, Kaurane; D005960:Glucosides; D013549:Sweetening Agents; C012043:stevioside; C012042:steviol; D016695:Glycosyltransferases