Genome sequencing and genetic breeding of a bioethanol Saccharomyces cerevisiae strain YJS329

Abstract

Background: Environmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits.

Results: We explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified.

Conclusions: Through comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.

Figure 1

Phenotypic and physiological traits of the bioethanol yeast strain YJS329. (A) Growth of BYZ1 and YJS329 on plates with and without imposed stresses. Cells were grown in YPD liquid medium at 30°C for 20 h, and 3-μL 10-fold serial dilutions of each sample were spotted onto YPD plates. The YPD plates were then subjected to the indicated stressors. Three independent experiments were conducted, and typical data from one of them are shown. (B) Relative content of physiological and biochemical factors in YJS329. Cells were cultured in YPD for 18 h and then collected. Measurement of the trehalose, glucose-6-phosphate dehydrogenase (G6PD), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), ergosterol, hydroxymethylfurfural (HMF) reductase, palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acids (C18:1), and linoleic acid (C18:2) content was then performed. The values are expressed as log2 ratios (YJS329/BYZ1) that represent the mean of three independent cultured samples (bars indicate SD). (C) Ploidy determination of YJS329 by flow cytometry. The stationary-phase cells of yeast strain BYZ1 (orange), YJS329 (green), and a triploid strain ZTW3 (violet) were fixed with 70% ethanol and stained with propidium iodide. DNA content corresponds to the intensity of red fluorescence. (D) Sporulation efficiency of YJS329. Cells were precultured in YPD and sporulated in sporulation medium. Asci were stained with fluorescein diacetate and then imaged with a confocal laser scanning microscope.

Figure 2

Genome structure analysis of YJS329. (A) Pulse-field gel electrophoresis of the BYZ1 and YJS329 chromosomes. (B) Comparison of the genome structures of BYZ1 and YJS329 by array-comparative genomic hybridization. Amplified regions and underrepresented regions in YJS329 are shown in red and green, respectively. The violet region represents the amplified regions of chromosome 4 in BYZ1. (C) Functional classification of the lost genes in YJS329. (D) Functional classification of the amplified genes in YJS329.

Figure 3

Genome variation and genetic distance revealed by whole-genome sequencing. (A) The distribution and density of SNPs in the YJSH1 genome within a sliding window of 1,000 bp. (B) A neighbor-joining tree representing the genetic distances between strains calculated from the total number of SNPs present in whole-genome alignments. The wine strains group is shown in plum, the laboratory strains in orange, and the sake strains in gray. (C) Chromosomal rearrangement events on chromosome 1 of the YJS329 genome. The full-length chromosome 1 sequences were aligned using the Artemis Comparative Tool (13). Sequences with >85% similarity are connected by red lines and sequences with <85% similarity or with no similarity are indicated by the white gaps. The green box indicates the largest indel on chromosome 1 of YJS329 and the red boxes indicate the novel ORFs EPH1 (left) and BIO6 (right). (D) From left to right, the sequence at the 5’ end of chromosome 2 in YJS329 was similar to regions of the sequences from chromosome 10 of S288c, gene MEL1, and chromosome 3 of S288c.

Figure 4

The effects of genomic variations on the transcriptional differences between BYZ1 (orange) and YJS329 (green). (A) Comparison of expression levels of HSF1 in BYZ1 and YJS329 within a sliding window of 50 bp. The N-terminal activation domain (NAD), DNA-binding domain (DBD), trimerization domain (TD), and C-terminal activation domain (CAD) of the Hsf1p [33] are highlighted by colored boxes. The orange letters represent the corresponding amino acids in BYZ1; the olive letters represent those in YJS329. (B) Comparison of the promoter and the expression levels of the SFA1 gene in BYZ1 and YJS329. The green box in the SFA1 promoter represents the Msn2/4p binding motif in YJS329. (C) The insertion of a Ty2 element into the CTR3 promoter greatly decreased the expression of the CTR3 gene in BYZ1 (sliding window of 50 bp). (D) The down-regulation of ALD6 in YJS329 might be caused by the loss of the Adr1p binding motif in the promoter (sliding window of 50 bp). (E) The relative expression level of the amplified region located on chromosome 4 of BYZ1, represented by the log2 ratio (BYZ1/YJS329), within a sliding window of 100 bp. The red dotted line indicates the mean value of the relative expression level. The up-regulated genes in the amplified region are indicated by violet boxes (P < 0.001); the genes that were not differentially expressed in this region are indicated by yellow boxes (P > 0.001).

Figure 5

Breeding strategies for YJS329. (A) After heat and ethanol treatment, the moderate up-expression of HSF1 in YJS329 improved its viability. Strains YJS329 and YJS329 + BYHSF1 (the HSF1 from BYZ1was expressed in YJS329) were pre-cultured in YPD medium and 1 mL cells (cell density was adjusted to OD₆₀₀ = 1) were then subjected to either heat (55°C, 6 min) or ethanol (15% v/v in YPD liquid medium, 10 h) treatments. The “a” indicates a significant difference between YJS329 + BYHSF1 and YJS329 (B) The impact of deletion of FPS1 and overexpression of ALD6 on the YJS329 fermentation process. The fermentation medium contained 220 g/L glucose, 10 g/L yeast extract, 20 g/L peptone. Data represent mean ± SD of three individual cultures. The “a” indicates a significant difference between YJS329ΔFPS1 and YJS329; “b” indicates a significant difference between YJS329ΔFPS1ALD6 and YJS329ΔFPS1 using the t test at the 0.05 level. (C) Deletion of FPS1 and overexpression of ALD6 improved the viability ratio of after treatment with ethanol (15%, v/v) and lignocellulosic hydrolysate (LH, containing 4 g/L acetic acid, 1 g/L furfural, and 1 g/L 5-HMF, pH4.5) for 10 h. The “a” and “b” letters have the same meaning as in Figure 5B.