Knockout and functional analysis of BSSS-related genes in Acremonium chrysogenum by novel episomal expression vector containing Cas9 and AMA1

Biotechnol Lett. 2022 Jun;44(5-6):755-766. doi: 10.1007/s10529-022-03255-w. Epub 2022 May 8.

Abstract

Objective: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum.

Result: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed.

Conclusion: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.

Keywords: Acremonium chrysogenum; CRISPR/Cas9; Cephalosporin C; Yeast bud site selection system.

MeSH terms

  • Acremonium* / genetics
  • CRISPR-Cas Systems / genetics
  • Cdc20 Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cephalosporins / metabolism
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism
  • Gene Editing
  • Genes, Fungal
  • Membrane Glycoproteins / genetics
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / metabolism

Substances

  • Ama1 protein, S cerevisiae
  • Axl2 protein, S cerevisiae
  • BUD3 protein, S cerevisiae
  • BUD4 protein, S cerevisiae
  • Cdc20 Proteins
  • Cell Cycle Proteins
  • Cephalosporins
  • Membrane Glycoproteins
  • Saccharomyces cerevisiae Proteins
  • GTP-Binding Proteins

Supplementary concepts

  • Acremonium chrysogenum