Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain

Microbial Cell Factories

Research

Anastasia Krivoruchko¹  Guodong Liu¹  Yun Chen¹  Martin K M Engqvist¹  Yiming Zhang¹  Verena Siewers¹  Jens Nielsen²  Björn M Hallström³ 

[1] Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden;Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden;Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden;Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark;Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden;Science for Life Laboratory, KTH-Royal Institute of Technology, 171 21, Stockholm, Sweden;

关键词: Pyruvate decarboxylase;    Genomic DNA sequencing;    Yeast;    Reverse engineering;    MTH1;    Hexose transporter;    Citrate synthase;    Histone deacetylase;   

DOI  :  10.1186/s12934-015-0305-6

 received in 2015-02-25, accepted in 2015-07-25,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundA 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.ResultsIn 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 MTH181D 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.ConclusionsIn 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.

【 授权许可】

CC BY   
© Zhang et al. 2015