Improved Production of a Heterologous Amylase in Saccharomyces Cerevisiae by Inverse Metabolic Engineering

Appl Environ Microbiol. 2014 Sep;80(17):5542-50. doi: 10.1128/AEM.00712-14. Epub 2014 Jun 27.

Abstract

The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amylases / genetics
  • Amylases / metabolism*
  • Metabolic Engineering / methods*
  • Mutation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae / radiation effects
  • Selection, Genetic
  • Ultraviolet Rays

Substances

  • Recombinant Proteins
  • Amylases