Xianni Qi

Understanding the Mechanism of Thermotolerance Distinct From Heat Shock Response Through Proteomic Analysis of Industrial Strains of Saccharomyces cerevisiae

Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of ∼2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering.

High-throughput screening and characterization of xylose-utilizing, ethanol-tolerant thermophilic bacteria for bioethanol production

Aims:  To develop a high‐throughput assay for screening xylose‐utilizing and ethanol‐tolerant thermophilic bacteria owing to their abilities to be the promising ethanologens.

TALENs-Assisted Multiplex Editing for Accelerated Genome Evolution To Improve Yeast Phenotypes

Genome editing is an important tool for building novel genotypes with a desired phenotype. However, the fundamental challenge is to rapidly generate desired alterations on a genome-wide scale. Here, we report TALENs (transcription activator-like effector nucleases)-assisted multiplex editing (TAME), based on the interaction of designed TALENs with the DNA sequences between the critical TATA and GC boxes, for generating multiple targeted genomic modifications. Through iterative cycles of TAME to induce abundant semirational indels coupled with efficient screening using a reporter, the targeted fluorescent trait can be continuously and rapidly improved by accumulating multiplex beneficial genetic modifications in the evolving yeast genome. To further evaluate its efficiency, we also demonstrate the application of TAME for significantly improving ethanol tolerance of yeast in a short amount of time. Therefore, TAME is a broadly generalizable platform for accelerated genome evolution to rapidly improve yeast phenotypes.

Distinct Proteome Remodeling of Industrial Saccharomyces cerevisiae in Response to Prolonged Thermal Stress or Transient Heat Shock

To gain a deep understanding of yeast-cell response to heat stress, multiple laboratory strains have been intensively studied via genome-wide expression analysis for the mechanistic dissection of classical heat-shock response (HSR). However, robust industrial strains of Saccharomyces cerevisiae have hardly been explored in global analysis for elucidation of the mechanism of thermotolerant response (TR) during fermentation. Herein, we employed data-independent acquisition and sequential window acquisition of all theoretical mass spectra based proteomic workflows to characterize proteome remodeling of an industrial strain, ScY01, responding to prolonged thermal stress or transient heat shock. By comparing the proteomic signatures of ScY01 in TR versus HSR as well as the HSR of the industrial strain versus a laboratory strain, our study revealed disparate response mechanisms of ScY01 during thermotolerant growth or under heat shock. In addition, through proteomics data-mining for decoding transcription factor interaction networks followed by validation experiments, we uncovered the functions of two novel transcription factors, Mig1 and Srb2, in enhancing the thermotolerance of the industrial strain. This study has demonstrated that accurate and high-throughput quantitative proteomics not only provides new insights into the molecular basis for complex microbial phenotypes but also pinpoints upstream regulators that can be targeted for improving the desired traits of industrial microorganisms.

An NGS-Independent Strategy for Proteome-Wide Identification of Single Amino Acid Polymorphisms by Mass Spectrometry.

Detection of proteins containing single amino acid polymorphisms (SAPs) encoded by nonsynonymous SNPs (nsSNPs) can aid researchers in studying the functional significance of protein variants. Most proteogenomic approaches for large-scale SAPs mapping require construction of a sample-specific database containing protein variants predicted from the next-generation sequencing (NGS) data. Searching shotgun proteomic data sets against these NGS-derived databases allowed for identification of SAP peptides, thus validating the proteome-level sequence variation. Contrary to the conventional approaches, our study presents a novel strategy for proteome-wide SAP detection without relying on sample-specific NGS data. By searching a deep-coverage proteomic data set from an industrial thermotolerant yeast strain using our strategy, we identified 337 putative SAPs compared to the reference genome. Among the SAP peptides identified with stringent criteria, 85.2% of SAP sites were validated using whole-genome sequencing data obtained for this organism, which indicates high accuracy of SAP identification with our strategy. More interestingly, for certain SAP peptides that cannot be predicted by genomic sequencing, we used synthetic peptide standards to verify expression of peptide variants in the proteome. Our study has provided a unique tool for proteogenomics to enable proteome-wide direct SAP identification and capture nongenetic protein variants not linked to nsSNPs.

Metabolic and genomic characterisation of stress-tolerant industrial Saccharomyces cerevisiae strains from TALENs-assisted multiplex editing

TALENs-assisted multiplex editing (TAME) toolbox was previously established and used to successfully enhance ethanol stress tolerance of Saccharomyces cerevisiae laboratory strain. Here, the TAME toolbox was harnessed to improve and elucidate stress tolerances of S. cerevisiae industrial strain. One osmotolerant strain and one thermotolerant strain were selected from the mutant library generated by TAME at corresponding stress conditions, and exhibited 1.2-fold to 1.3-fold increases of fermentation capacities, respectively. Genome resequencing uncovered genomic alterations in the selected stress-tolerant strains, suggesting that cell wall and membrane-related proteins might be major factors behind improved tolerances of yeast to different stresses. Furthermore, amplified mitochondrial DNA might also have an important impact on increased stress tolerance. Unexpectedly, none of predesigned target potential TALENs modification sites showed any genomic variants in sequenced genomes of the selected strains, implicating that the improved stress tolerances might be due to indirect impacts of genome editing via TALENs rather than introducing genomic variants at potential target sites. Our findings not only confirmed TAME could be a useful tool to accelerate the breeding of industrial strain with multiple stress tolerance, but also supported the previous understandings of the complicated mechanisms of multiple stress tolerance in yeast.