Metabolic engineering of a stable haploid strain derived from lignocellulosic inhibitor tolerant Saccharomyces cerevisiae natural isolate YB-2625

Authors: Hector, Ronald - Ron; Mertens, Jeffrey; Nichols, Nancy

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2023
Publication Date: 12/6/2023
Citation: Hector, R.E., Mertens, J.A., Nichols, N.N. 2023. Metabolic engineering of a stable haploid strain derived from lignocellulosic inhibitor tolerant Saccharomyces cerevisiae natural isolate YB-2625. Biotechnology for Biofuels and Bioproducts. https://doi.org/10.1186/s13068-023-02442-9.
DOI: https://doi.org/10.1186/s13068-023-02442-9

Interpretive Summary: Production of advanced fuels and chemicals is a national priority. Generally, they are manufactured using agricultural residues (e.g., corn stover) where the biomass sugars are extracted and fermented by microbes. The sugars generated are unrefined and inhibit the growth of currently available commercial distillers’ yeast strains. These yeast are also unable to ferment the sugar xylose, which accounts for 30-40% of the sugars present in biomass syrups. ARS and other researchers have developed tools needed to genetically engineer yeast to ferment xylose. However, yeast strains naturally available that ferment unrefined biomass sugars are undomesticated and not amendable to genetic engineering. The reason is that these yeast strains are “polyploid” and molecular biologist prefer “haploid” yeast. The difference is that haploid yeast have only one copy of their genes and polyploid ones have multiple copies. It was expected that the yeast needed to be polyploid to be hardy. Here we refute this hypothesis. Earlier we screened 160 yeast strains from the ARS culture collection (Peoria, IL) and identified three strains with exceptional hardiness; all were polyploid. Herein, we were able to convert some of these special yeast to haploid yeast, screened the haploids for fermentation of biomass sugars, and identified some that were as hardy as their diploid parents. One haploid was chosen and successfully engineered to ferment xylose. Therefore, a new yeast strain was developed that is of commercial interest because it is hardy and convenient to genetically engineer. Notably, this work also dispels the false belief that haploids cannot be hardy. This work will be of specific interest to agriculture processors interested in expanding into advanced biofuels. It is of general interest to farmers looking to develop markets for their crop residues and to governmental officials setting policies to achieve climate change targets for reduced greenhouse gas emissions.

Technical Abstract: Background: Significant genetic diversity exists across Saccharomyces strains. Natural isolates and domesticated brewery and industrial strains are typically more robust than laboratory strains when challenged with inhibitory lignocellulosic hydrolysates. These strains also contain genes that are not present in lab strains and likely contribute to their superior inhibitor tolerance. However, many of these strains have poor sporulation efficiencies and low spore viability making subsequent gene analysis, further metabolic engineering, and genomic analyses of the strains challenging. This work aimed to develop an inhibitor tolerant haploid with stable mating type from S. cerevisiae YB-2625, which was originally isolated from bagasse . Results: Haploid spores isolated from four tetrads from strain YB-2625 were tested for tolerance to furfural and HMF. Due to natural mutations present in the HO-endonuclease, all haploid strains maintained a stable mating type. One of the haploids, YRH1946, did not flocculate and showed tolerance to furfural and HMF at levels comparable to the parent diploid. The tolerant haploid strain was further engineered for xylose fermentation by integration of the genes for xylose metabolism at two separate genomic locations (ho' and pho13'). In fermentations supplemented with inhibitors from acid hydrolyzed corn stover, the engineered haploid strain derived from YB-2625 was able to ferment all of the glucose and 19% of the xylose, whereas the engineered lab strains performed poorly in fermentations. Conclusions: Understanding the molecular mechanisms of inhibitor tolerance will aid in developing strains with improved growth and fermentation performance using biomass-derived sugars. The inhibitor tolerant, xylose fermenting, haploid strain described in this work has potential to serve as a platform strain for identifying pathways required for inhibitor tolerance, and for metabolic engineering to produce fuels and chemical from undiluted lignocellulosic hydrolysates.