Comparative Proteomic Analysis Of Inhitory Effect Of Furfural And Phenol On Saccaromyces Serevisiae And Engineering Of Echesrichia Coli For Fatty Acid Production

Study of inhibitory effect of lignocellulosic inhibotors on S.cerevisiae is important for the construction of inhibitor-tolerant ethanologenic yeast and will benifit that S.cerevisiae produces ethanol directly from lignocellusic hydrolysates. In this study, comparative proteome was utilized to study the mechnism of two major inhibitor furfural and phenol on S.cerevisiae at the protein level.¹⁸O labeling comparative proteomic analysis was carried out to study the adaptation and tolerance of ethanologenic S.cerevisiae （both the wild type and furfural-tolerant strains, individually） to 17 g/L furfural at 20 min and 2 h. Also, the response of the wild type strain to 0.04 mol/L phenol at 20 min and the phenol-tolerant strain to 0.04 mol/L phenol at 20 min and 40 min were analyzed. A total of 76, 66, 53,48,15,46 and 23 differentially expressed proteins were indentified individually under each condition. Functional categories of the differentially expressed proteins obtained by ¹⁸O labeling comparative proteome under different inbitory conditions were carried out by FuncatDB in MIPs. Protein synthesis is the major functional group for all of the tested conditions, and most of protein synthesis related proteins showed an upregualated comparative expression. Energy, c-compound and carbohydrate metabolism, and puring nucleotide metabolism are the overpresented funtional categories of the proteins affected by furfural, while homeostasis of protons is the overpresented funtional categorie specific for phenol. Both furfural and phenol affect transcription in yest in 20 min and metabolism in 2 h. Amino acid metabolism is the overpresented funtional categories of the proteins in both the phenol-tolerant strain and the furfural-tolerant strain, which may be respossible for the tolerance acquistion in these strains. The furfural-related biomarkers alcohol dedydronase Adh1p and glucokinase Glk1p were found. Ribosomal protein Rpl32p、peripheral membrane protein Zeo1p and subunit A of the yeast V-ATPase V1 domain Tfp1p are the potential biomarkers for both phenol and furfural.The temporal proteomic response of S. cerevisiae to furfural under aerobic conditions was profiled utilizing an iTRAQ mediated shotgun proteome strategy. In the presence of furfural, the expression levels of proteins involved in protein synthesis showed a retarded downregulation, while the expression levels of enzymes catalyzing glucose fermentation and the TCA cycle displayed a delayed upregulation. Proteins catalyzing the upper part of the super pathway of sulfur amino acid biosynthesis were repressed by furfural at all time points. These insights into the response of yeast to the presence of furfural and phenol will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology.A bi-level metabolic and regulatory network optimization model for microbial strain design was developed and utilized to suggest gene manipulation for improved fatty acid production. According to the suggestion by the modeling, knockout strainâ–³cyoAâ–³AdhEâ–³nuoAâ–³ndhâ–³pta was obtained with a 45% increase in the total amount of fatty acid. The triacylglycerol pathway was introduced into Bl21 Star DE3 by knockout of dgkA and overexpression of WS/DGAT enzyme. This demonstrated the possiblity of engineering E.coli to behave like an oily bacterial and provide high-quality precusor for bio-hydrocarbon production.