Stress tolerance enhancement via SPT15 base editing in Saccharomyces cerevisiae

Abstract

Background Saccharomyces cerevisiae is widely used in traditional brewing and modern fermentation industries to produce biofuels, chemicals and other bioproducts, but challenged by various harsh industrial conditions, such as hyperosmotic, thermal and ethanol stresses. Thus, its stress tolerance enhancement has been attracting broad interests. Recently, CRISPR/Cas-based genome editing technology offers unprecedented tools to explore genetic modifications and performance improvement of S. cerevisiae. Results Here, we presented that the Target-AID (activation-induced cytidine deaminase) base editor of enabling C-to-T substitutions could be harnessed to generate in situ nucleotide changes on the S. cerevisiae genome, thereby introducing protein point mutations in cells. The general transcription factor gene SPT15 was targeted, and total 36 mutants with diversified stress tolerances were obtained. Among them, the 18 tolerant mutants against hyperosmotic, thermal and ethanol stresses showed more than 1.5-fold increases of fermentation capacities. These mutations were mainly enriched at the N-terminal region and the convex surface of the saddle-shaped structure of Spt15. Comparative transcriptome analysis of three most stress-tolerant (A140G, P169A and R238K) and two most stress-sensitive (S118L and L214V) mutants revealed common and distinctive impacted global transcription reprogramming and transcriptional regulatory hubs in response to stresses, and these five amino acid changes had different effects on the interactions of Spt15 with DNA and other proteins in the RNA Polymerase II transcription machinery according to protein structure alignment analysis. Conclusions Taken together, our results demonstrated that the Target-AID base editor provided a powerful tool for targeted in situ mutagenesis in S. cerevisiae and more potential targets of Spt15 residues for enhancing yeast stress tolerance.

Enhancing the thermotolerance and erythritol production of Yarrowia lipolytica by introducing heat-resistant devices

“…Improving the heat resistance of yeast strains is of great significance for reducing the energy cost required for cooling bioreactors in industrial production processes. However, thermotolerance is a complex phenotype involving various aspects of cellular physiology and metabolism ( Alper et al, 2006 ; Liu et al, 2021 ). Large amounts of reactive oxygen species (ROS) are generated under higher temperatures, causing severe oxidative damage to cells, such as lipid peroxidation in cellular membranes, nucleic acid damage and increased toxicity by oxidation of proteins ( Ahn et al, 2003 ).…”

Recent Advances in Directed Yeast Genome Evolution

“…Ref. [ 68 ] leveraged Target-AID (activation-induced cytidine deaminase) base editor to enable C-to-T substitutions of SPT15 and obtain 36 mutants with various stress tolerances [ 68 ]. By screening the expression of these STP15 mutants, tolerance to a number of stresses including against hyperosmotic, thermal and ethanol stresses were shown, and at the same time, 1.5-fold increases in fermentation capacities were generated.…”

Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts

“…Apart from gene disruption, this base editor was further employed for situ mutagenesis, thus enabling it to obtain the desired phenotype. Recently, the general transcription factor gene SPT15 in S. cerevisiae was mutated by Target-AID base editor to enhance the stress tolerances ( Liu et al, 2021 ). Furthermore, this strategy has also been applied in mammals ( Ma et al, 2016 ) and plant ( Li et al, 2020 ) but has not been widely reported in non-conventional yeasts.…”