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

Abstract

cThe 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.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

“…The best clone B184 had a nearly sixfold improvement in α-amylase production. In our previous work the mutant that was selected manually, M715 (12), had only about a 1.5-fold improvement in SD-2×SCAA medium (an optimized medium for protein production) compared with the wild-type strain AAC. This shows a clear potential of high-throughput screening from large libraries for selecting cell factories with improved desired phenotype.…”

Rational strain engineering interventions to enhance cellulase secretion by Saccharomyces cerevisiae

“…To fully exploit strain engineering as a means to enhance the secretion of relevant proteins, numerous studies have focused on generating ‘supersecreting’ strains and identifying secretion enhancing genetic elements . Early studies used random mutagenesis and plate assays to identify strains with an enhanced secretion phenotype for a particular heterologous protein.…”

“…Recent advances in high throughput DNA sequencing technologies allowed researchers to overcome the limitations of SNP identification in supersecreting strains generated by random mutagenesis . Liu et al . obtained an S. cerevisiae strain, through UV‐random mutagenesis, that was capable of producing 5‐fold more heterologous secreted amylase than the original strain.…”

“…Allele alterations responsible for improved secretion may imply a range of up‐ and downregulation or even deletion of the allele in question. The synergistic effect of these genetic changes and the partial secretion enhancing effect of the VTI1 allele (involved in cis‐Golgi trafficking) compared to the original mutant strain did not conform to simple Mendelian characteristics …”

Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains

“…This result suggests that the cell-specific secreted activities of these strains are improved under oxygen-limited conditions. Recent research has demonstrated that α-amylase enzyme production from S. cerevisiae in aerobic conditions is limited by the protein folding capacity and that higher productivity can be obtained under anaerobic conditions (80). The amylase yield increased 2-fold in anaerobic cultivation compared to aerobic conditions suggesting that amylase tends to gain higher production and reduced rates of ER misfolding in these conditions.…”