Enhancement of galactose consumption rate in Saccharomyces cerevisiae CEN.PK2-1 by CRISPR Cas9 and adaptive evolution for fermentation of Kappaphycus alvarezii hydrolysate

J Biotechnol. 2019 May 20:297:78-84. doi: 10.1016/j.jbiotec.2019.03.012. Epub 2019 Apr 5.

Abstract

Ethanol ferrmentation of Kappaphycus alvarezii hydrolysates was performed using wild-type (WT) Saccharomyces cerevisiae CEN.PK2-1, hexokinase 2 deleted (Δhxk2) and adapted strain on high galactose concentrations. The WT and Δhxk2 strains produced 8.9 and 14.67 g/L of ethanol with yield coefficient (YEtOH) of 0.20 and 0.33 (g/g), respectively. However, neither the WT nor Δhxk2strain could utilize all of the galactose, leaving 16.4 and 6.2 g/L of galactose in the fermentation broth, respectively. Therefore, fermentation with S. cerevisiae CEN.PK2-1 adapted to galactose was carried out to increase the ethanol yield coefficient (YEtOH), producing a maximum ethanol concentration of 20.0 g/L with a YEtOH of 0.44 (g/g). Ethanol concentration of adapted strain was 1.36-2.25 times higher than WT and Δhxk2 strains. The adapted yeast exhibited the highest transcript levels of GAL genes. The yeast strain via adaptive yeast strain produced ethanol with a higher titer and yield due to a modular activation of GAL genes than WT or the hxk2 deleted strains.

Keywords: Adaptive evolution; CRISPR Cas-9; Fermentation; Kappaphycus alvarezii; Pretreatment.

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Carbohydrate Metabolism
  • Directed Molecular Evolution*
  • Ethanol / metabolism
  • Fermentation*
  • Galactose / metabolism*
  • Gene Expression Regulation, Fungal
  • Genes, Fungal
  • Hydrolysis
  • Rhodophyta / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Temperature
  • Transcription, Genetic

Substances

  • Ethanol
  • CRISPR-Associated Protein 9
  • Galactose