CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae

Abstract

The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were re-introduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation) and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. Eleven point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

“…As a result, α-amylase secretion was obviously improved with a total of seven gene deletions and 11 point mutations. 145 What is more, some genes were deleted by the CRISPR/Cas9 system to improve xylitol accumulation. As a result, the XKS1-deleted strain could produce 46.17 g/L of xylitol with a xylitol yield of 0.92 g/g during fermentation.…”

“…144 On this basis, single gene deletions were performed in parental strain AAC of S. cerevisiae by CRISPR/Cas9 to improve the secretion of αamylase effectively. 145 A total of 42 mutations identified in a S. cerevisiae mutant strain were reintroduced by CRISPR/Cas9 into the original strain to determine their individual effects on the protein function. As a result, α-amylase secretion was obviously improved with a total of seven gene deletions and 11 point mutations.…”

Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications

“…Anchor protein Yeast host Promoter Activity improve protein secretion (Wang et al, 2022). 3) Optimization of secretion pathways (Tang et al, 2017;Huang et al, 2018;Besada-Lombana & Da Silva, 2019) and stress modulation (Lamour et al, 2019).…”

“…Point mutation target genes identified from UV-mutagenized strains were analyzed to be involved in multiple intracellular biological processes. Detecting the effects of point mutations and gene deletions on the secretion of α-amylase could help to balance these biological processes and thus improve protein secretion ( Wang et al, 2022 ). 3) Optimization of secretion pathways ( Tang et al, 2017 ; Huang et al, 2018 ; Besada-Lombana & Da Silva, 2019 ) and stress modulation ( Lamour et al, 2019 ).…”

High throughput mutagenesis and screening for yeast engineering

“…In addition, more recent strategies such as CRISPR/Cas9 genome editing and CRISPR/dCas9 transcriptional control have significantly reduced the time and labor required to analyze a protein of interest in any genetic background. These tools have been implemented in a variety of applications, including increasing biochemical production (e.g., ethanol [7][8][9][10][11][12], n-butanol [10], or fatty acids [10][11][12]) and improving protein secretion (e.g., α-amylase [13]).…”