title: Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain.
abstract: BACKGROUND
  A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol.
  
  
  RESULTS
  In this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 h(-1), respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH1 (81D) allele restored its growth on glucose at a maximum specific rate of 0.053 h(-1) in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h(-1).
  
  
  CONCLUSIONS
  In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. ferryboat31@hotmail.com.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. ferryboat31@hotmail.com.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. guodong@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. guodong@chalmers.se.
authors.forename: Guodong
authors.initials: G
authors.lastname: Liu
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. martin.engqvist@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. martin.engqvist@chalmers.se.
authors.forename: Martin K M
authors.initials: MK
authors.lastname: Engqvist
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. anakri@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. anakri@chalmers.se.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Krivoruchko
authors.affiliation: Science for Life Laboratory, KTH-Royal Institute of Technology, 171 21, Stockholm, Sweden. bjorn.hallstrom@scilifelab.se.
authors.forename: Björn M
authors.initials: BM
authors.lastname: Hallström
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. yunc@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. yunc@chalmers.se.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. siewers@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. siewers@chalmers.se.
authors.forename: Verena
authors.initials: V
authors.lastname: Siewers
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. nielsenj@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. nielsenj@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden. nielsenj@chalmers.se.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000235:genetics* / Q000378:metabolism; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011767:Pyruvate Decarboxylase / Q000172:deficiency* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003470:Culture Media; D051246:Glucose Transport Proteins, Facilitative; C084498:HXT2 protein, S cerevisiae; C118806:MTH1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C071665:RPD3 protein, S cerevisiae; D006655:Histone Deacetylases; D011767:Pyruvate Decarboxylase; D005947:Glucose