[2S04a] 18. Biorefinery and Bioproduction - Part 2

3:00 PM - 3:15 PM JST(6:00 AM - 6:15 AM UTC)

[2BRBP-09] Multi-omics Integration and Genome-scale Screening Decodes Self-Flocculation and Stress Tolerance in Industrial Yeast

○Xue Zhang^1,2, Xin-Qing Zhao¹, Chen-Guang Liu^3,1, Zhuo Wang¹, Feng-Wu Bai¹ (1. Shanghai Jiao Tong University (China), 2. Yangtze Delta Region Institute of Tsinghua University (China), 3. Dalian University of Technology (China))

Keywords:

Saccharomyces cerevisiae,Self-flocculation,Stress tolerance,CRISPR screening,Cell factories,Genetic elements,Rational design

A unique self-flocculating yeast strain SPSC01 was developed through protoplast fusion for fuel ethanol production with high product titers. To explore the mechanism of self-flocculation and multi-stress tolerance, we conducted comparative multi-omics analyses and CRISPR-based genome-scale screening on SPSC01. Leveraging two cutting-edge third-generation sequencing technologies, we achieved a gapless, high-quality, and chromosome-level assembly for the genomes of SPSC01 and its parental strains. Through comprehensive genome analyses, we identified 25 unique genes that are absent in the parental strains, along with 13 novel genes with unknown functions. The self-flocculation of yeast cells is driven by the copy number of genetic variations and significantly upregulated transcription of FLO genes. Mutations in both cis- and trans-regulatory elements contribute to the constitutive expression of FLO1 and its derivative genes, a prerequisite for developing the self-flocculating phenotype. Notably, we discovered a novel small protein G12 that harbors a zinc finger domain, and its overexpression substantially enhanced ethanol production of engineered yeast strains. Furthermore, alterations in metabolic pathways with ergosterol, glutathione, amino acid, and glycerophospholipid are implicated for developing tolerance to ethanol and major inhibitors acetic acid and furfural that are released during the pretreatment of lignocellulosic biomass. To unbiasedly and bottom-up identify genetic determinants underlying complex traits such as flocculation and stress tolerance in a broad-spectrum, discovery-driven manner, we further performed pooled CRISPR screening in SPSC01, enabling high-throughput perturbation of candidate loci. The progress provides strategies for engineering yeast cell factories with robustness through rational design to produce biofuels and bio-based chemicals with high product titers and productivities, in particular with the biorefinery of lignocellulosic biomass for sustainable socioeconomic development.