Integrated Genomics and Phenotype Microarray Analysis of Saccharomyces cerevisiae Industrial Strains for Rice Wine Fermentation and Recombinant Protein Production

Ye Ji Son, Min-Seung Jeon, Hye Yun Moon, Jiwon Kang, Da Min Jeong, Dong Wook Lee, Jae Ho Kim, Jae Yun Lim, Jeong-Ah Seo, Seong-il Eyun, and Hyun Ah Kang

Under Review

— Figures and Tables (Colored and High resolution) —

• Fig 1. Comparative single nucleotide polymorphism (SNP) and insertion or deletion (INDEL) detection in the whole genomes of S. cerevisiae KSD-YC, 98-5, and Y2805 strains. Each structural SNP and INDEL chromosome comparison was visualized in (A) S. cerevisiae Y2805 and S288C. (B) S. cerevisiae KSD-YC and the sake yeast K7. (C) Each haplotype of S. cerevisiae 98-5. (D) Synteny analysis of the de novo assemblies of S. cerevisiae KSD-YC, 98-5, and Y2805 genomes with the reference S288C genome.






• Fig 2. Phylogenetic tree construction with maximum likelihood. The 13 concatenated genes, including YPR152C, YJL099W, YJL057C, YJL051W, YKL068W, YML080W, YML056C, YNL161W, YNL125C, YOR133W, YAR042W, YBL052C, and YBR163W, were used in the phylogenetic tree construction to classify S. cerevisiae at the strain level. The bootstrap values greater than 50% are shown at the branches. The groups of yeast strains are presented as a single model (JTT+F+I+G4) based on the tree topology. The seven region types (Europe, Australia, Africa, Middle East, Asia, Malaysia, and North America) and six source types (baking, wine, sake/ragi, huangjiu, bio-EtOH, and clinical) are represented as different colored branches and strains, respectively. The structural features with the genomic distance between PAD1 and FDC1 are indicated on the l right side. Nonfunctional genes are represented by dotted arrows.






• Fig 3. Chromosome structure analysis of S. cerevisiae KSD-YC, 98-5, and Y2805 strains. (A) Multiple comparative analysis of the whole genomes of the S. cerevisiae strains. The synteny blocks of chromosome XII of the S. cerevisiae strains were extended to compare the gene components around the rDNA cluster region. The space marked with a dotted line describes the absolute length or the interval of the gene, with the relative position of each gene block. (B) The location of the cluster of five genes conserved in the S. cerevisiae wine strains. Among the four S. cerevisiae strains, 98-5 only showed the partial cluster of those genes in chromosomes IV and XVI. The genes with a premature stop codon and intact genes are displayed in red and blue, respectively.






• Fig 4. Comparative analysis of the 4-vinylguaiacol (4-VG) bioconversion activity of S. cerevisiae strains. (A) Schematic representation of the PAD1 and FDC1 genes required for 4-VG bioconversion in S. cerevisiae S288C, CEN.PK2-1C, KSD-YC, 98-5, and Y2805 strains. The information on the accession numbers of the PAD1 and FDC1 genes with the detected SNP/deletion is provided in Table S6. (B) Heatmap of 4-VG production in S. cerevisiae strains through headspace-solid-phase microextraction with gas chromatography/mass spectrometry (HS-SPME GS/MS). To test 4-VG production capability, yeast cells were grown in YPD medium (1% yeast extract, 2% bacto peptone, and 2% glucose) in the presence of 50 ppm ferulic acid, and samples were collected after 1, 2, and 3 days of incubation.






• Fig 5. Phenotype microarray analysis of S. cerevisiae strains. Representative growth patterns of S. cerevisiae 98-5 (red), KSD-YC (blue), S288C (green), and Y2805 (yellow) strains on Biolog microplates. (A) Carbon source plate (PM01 and PM02), (B) Nitrogen source (PM03), (C) Phosphorus and sulfur source (PM04), (D) Nutrient supplements (PM05), and (E) Osmolytes (PM09), where the x- and y-axis represent time in hours and Omnilog units, respectively. The Omnilog unit is a standard representation of respiration rate.






• Fig 6. Schematic representation of structural and sequence features of S. cerevisiae Y2805 genetic markers based on deep sequencing. (A) pep4::HIS3, (B) prb1-Δ1.6R, (C) GAL2, (D) can1, (E) his3-Δ200, (F) ura3-52. The DNA fragments of the genotype marker genes were amplified by PCR from the total chromosomal DNAs, which were prepared by lysing the yeast cells with glass beads, using the gene-specific primers (Table S2). The PCR products were directly subjected to sequencing or subcloned into a T vector (T-Blunt™ PCR Cloning kit; SolGent, Daejeon, South Korea) before DNA sequencing by the dye-terminator sequencing method.






• Table 1. Saccharomyces cerevisiae strains analyzed in this study






• Table 2. Mapping of single nucleotide polymorphisms (SNPs) to the S. cerevisiae genes involved in carbon and nitrogen metabolisms based on phenotype microarray (PM) analysis data






• Table 3. Mapping of single nucleotide polymorphisms (SNPs) to the S. cerevisiae genes associated with nutrient supplements based on phenotype microarray (PM) analysis data

— Supplementary Information (Colored and High resolution) *추가 예정*—

• Figure S1. Ploidy analysis of S. cerevisiae strains using flow cytometry. Yeast cells were harvested at OD₆₀₀=3, and the cell pellets were fixed in absolute ethanol for at least 12 h. The DNA content of the sequenced strains (a measure for the ploidy level) was determined by nucleic acid staining using SYTOX Green (Thermo Fisher Scientific) as described previously (Jeong et al., 2022). The ethanol-fixed cells were resuspended in RNase solution (2 mg/mL RNaseA, 15 mM NaCl, and 50 mM Tris, pH 7.5) supplemented with proteinase K (Sigma-Aldrich, >4 units/mL), and mixed with SYTOX solution (1 µM SYTOX Green nucleic acid stain and 50 mM Tris, pH 7.5). For fluorescence-activated single-cell sorting, the Accuri C6 Plus flow cytometer (BD Biosciences) and BD Accuri C6 software (BD Biosciences) were used.






• Figure S2. Analysis of Pad1p and Fdc1p sequences and 4-vinylguaiacol (4-VG) production activity of S. cerevisiae S288C, CEN.PK2-1C, KSD-YC, 98-5, and Y2805 strains. (A) Multiple amino acid sequence alignment of Pad1p and Fdc1p from S. cerevisiae industrial strains. The red box represents a nonsense mutation site at position 98 in Pad1p in the CEN. PK2-1C strain and at position 54 in Fdc1p in the KSD-YC strain, respectively. The blue box in Fdc1p represents a deletion in the 267–298^th amino acid positions in the CEN.PK strain. (B) headspace-solid-phase microextraction with gas chromatography/mass spectrometry (HS-SPME GC/MS) analysis of 4-VG production in S. cerevisiae S288C, CEN.PK2-1C, KSD-YC, 98-5, and Y2805 strains. Yeast cells were inoculated at OD₆₀₀=0.3 in 50 mL YPD medium (1% yeast extract, 2% bacto peptone, and 2% glucose) supplemented with 50 ppm ferulic acid and cultivated in duplicate at 28°C with shaking. After 48 h incubation, the culture supernatants were subjected to HS-SPME GC/MS analysis.






• Figure S3. Multiple alignment of amino acid sequences of proteins associated with metabolic pathways, salt tolerance, and hydrophobicity of S. cerevisiae S288C, KSD-YC, 98-5, and Y2805 strains. (A) Proteins for galactose metabolism and regulation, including Gal1p, Gal2p, Gal7p, Gal10p, Gal3p, and Gal4p. (B) Proteins involved in the metabolism of maltose, sucrose, maltotriose, and turanose, including Mal31p, Suc2p, Ima3p, Ima4p, and Ima5p. (C) Proteins involved in nitrogen metabolism, including Hip1p for histidine metabolism and Oxp1p for pyroglutamic acid metabolism. (D) Proteins associated with nutrient supplements, including Cabp1p, required for conversion of pantothenic acid to coenzyme A, and Vht1p for biotin transport. Residues that are not conserved across all sequences are highlighted in grey. The red boxes show mutation sites related to the biosynthetic genes of the S. cerevisiae strains based on the phenotype microarray (PM) analysis data. (E) Proteins associated with NaCl tolerance, including Ena1p, Ena2p, and Ena5p. (F) Awa1p, a glycosylphosphatidylinositol anchor protein required for cell surface hydrophobicity.






• Figure S4. Spotting assay of S. cerevisiae strains for salt-/osmotic-tolerance (A) and for cold/high temperature resistance (B). For the spotting assay, the S. cerevisiae strains were inoculated in YPD medium (1% yeast extract, 2% bacto peptone, and 2% glucose). Serially diluted yeast cells were spotted on YPD plates under different salt-/osmotic conditions, such as the presence of NaCl, KCl, and sorbitol for 3 days at 28℃. For temperature tolerance, yeast cells were cultivated at different temperatures (18℃, 20℃, and 40℃).






• Table S1. Recombinant proteins and metabolites produced from S. cerevisiae Y2805






• Table S2. Primers used for amplification and sequencing of the selection marker genes in S. cerevisiae Y2805






• Table S3. Quality metrics of yeast assembly






• Table S4. Gene prediction and functional annotation






• Table S5. Single nucleotide polymorphisms (SNPs) in the newly reconstructed yeast genomes compared to the reference genome of S. cerevisiae S288C (Homozygous SNPs) or each haplotype (Heterozygous SNPs)






• Table S6. Structural organization of PAD1 and FDC1 in 66 S. cerevisiae strains






• Table S7. List of genes subjected to SNP analysis for integration with PM data






• Table S8. Copy number variation of fermentation process-related gene families in four S. cerevisiae strains