Rna-Seq, Phosphoproteomic Analysis And Gene Correlation Of The Xylose-fermenting Yeast Scheffersomyces Stipitis

Lignocellulose is resourceful. Its bioconversion to ethanol has attracted much attention because of the potential replacement of gasoline as a fuel. Hexose (glucose) and pentose (xylose) are the most abundant cellulosic and hemicellulosic components, respectively. Bakers’ yeast Saccharomyces cerevisiae, which is commonly used in ethanol production, cannot ferment xylose. We studied the transcriptomes and proteomes of the xylose-fermenting yeast Scheffersomyces stipitis (synonym:Pichia stipitis) cultivated in glucose or xylose as the sole carbon source, identified phosphorylation of proteins, measured metabolites in the yeast, and analyzed gene correlations through integration of those result. Indentification of crucial genes (proteins) will be an approachable route for improving xylose fermentation.RNA-Seq revealed that5,176of5,816annotated open reading frames had a uniform transcription and that214open reading frames were differentially transcribed. Differential expression analysis showed that, compared with other biological processes, carbohydrate metabolism and oxidation-reduction reactions were highly enhanced in yeast grown on xylose. We then identified4,085proteins by using label-free quantitative proteomics approach. Of them,1,551and789proteins in the xylose or glucose sample was increased in abundance, respectively. Most proteins involved in glycolysis and the TCA cycle had a uniform abundance. Of proteins involved in PPP and metabolism of fatty acid, some protein in the xylose sample was increased in abundance. The result coincided with transcriptional analysis defined by RNA-Seq. nanoLC-MS/MS analysis of TiO2-enriched phosphoprotein extracts identified982phosphopeptides, covering477,328, and177sites of Ser, Thr, and Tyr in351putative phosphoproteins. Abundance of23in the glucose sample and45phosphoproteins in the xylose sample were increased. Glucose or xylose affected phosphorylation of certain key enzymes involved in glycolysis, the TCA cycle and PPP. Protein-protein interactions (PPIs) of S. stipitis were predicted through integration of gene orthology, gene ontolgy and gene expression, which accounted for26,528PPIs involving2,306proteins. Hub proteins were clustered into class I (51proteins) and class II (176proteins). Integrated analysis of the transcriptomes, proteomes, PPIs and measurement of metabolic indicators of fermentation revealed some potential limit points in yeast grown on xylose. Gene correlations presented the variety and complexity of sugar fermentation of S. stipitis, and fluctuations of transcription, translation and post-translational modifications of those proteins involved in glycolysis, the TCA cycle, PPP, gluconeogenesis, sugar uptake, metabolism of amino acid, β-oxidation of fatty acid, the respiratory chain, oxidative phosphorylation, and transcriptional control.We presented a practical approach for the study of functional genomics in S. stipitis. The scheme was a key component for extraction and integration of data from separate source, data analysis and data diming. Some tools were developed as a solution for deficiency of bioinformatics tools in the study of S. stipitis. A Perl software package named Pscore was developed to largely determine ambiguous phosphorylation site assignment. Pscore calculation modified the algorithm for validation of PTM used in previous software, in favor of solving the obstacle of validation of PTM manually. Pscore aligned experimental m/z of ions with that of b-, y-, a-, and ammonium type ions. Pscore can distinguish ambiguous phosphorylation sites when there were equal scores, or more peptides with different phosphorylation sites were mixed.The study reveal transcriptional, proteomic, and phosphoproteomic diversity of S. stipitis cultivated in glucose or xylose, downsize the scope of potential crucial genes, and facilitate further study of genomic modifications for xylose fermentation improvement in the yeast.