title: The sole introduction of two single-point mutations establishes glycerol utilization inSaccharomyces cerevisiaeCEN.PK derivatives.
abstract: BACKGROUND
  Glycerol is an abundant by-product of biodiesel production and has several advantages as a substrate in biotechnological applications. Unfortunately, the popular production hostSaccharomyces cerevisiaecan barely metabolize glycerol by nature.
  
  
  RESULTS
  In this study, two evolved derivatives of the strain CEN.PK113-1A were created that were able to grow in synthetic glycerol medium (strains PW-1 and PW-2). Their growth performances on glycerol were compared with that of the previously published evolved CEN.PK113-7D derivative JL1. As JL1 showed a higher maximum specific growth rate on glycerol (0.164 h-1compared to 0.119 h-1for PW-1 and 0.127 h-1for PW-2), its genomic DNA was subjected to whole-genome resequencing. Two point mutations in the coding sequences of the genesUBR2andGUT1were identified to be crucial for growth in synthetic glycerol medium and subsequently verified by reverse engineering of the wild-type strain CEN.PK113-7D. The growth rate of the resulting reverse-engineered strain was 0.130 h-1. Sanger sequencing of theGUT1andUBR2alleles of the above-mentioned evolved strains PW-1 and PW-2 also revealed one single-point mutation in these two genes, and both mutations were demonstrated to be also crucial and sufficient for obtaining a maximum specific growth rate on glycerol of ~0.120 h-1.
  
  
  CONCLUSIONS
  The current work confirmed the importance ofUBR2andGUT1as targets for establishing glycerol utilization in strains of the CEN.PK family. In addition, it shows that a growth rate on glycerol of 0.130 h-1can be established in reverse-engineered CEN.PK strains by solely replacing a single amino acid in the coding sequences of both Ubr2 and Gut1.
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Ping-Wei
authors.identifier: 0000-0002-9301-7236
authors.initials: PW
authors.lastname: Ho
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Steve
authors.initials: S
authors.lastname: Swinnen
authors.affiliation: Systems and Computing Engineering Department, Universidad de los Andes, Cra 1 Este No 19A-40, Bogotá, Colombia.
authors.forename: Jorge
authors.initials: J
authors.lastname: Duitama
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Nevoigt
keywords: Adaptive laboratory evolution; CEN.PK; Evolutionary engineering; Glycerol; Yeast