The Pathway Study Of Sulfur Metabolism In Rhodococcus Erythropolis XP Based On Proteomics

The removal of sulfur-containing organic compounds in fossil fuels has been a major challenge in zero-sulfur fuel processes.Biodesulfurization（BDS）is a green desulfurization strategy that can remove refractory organosulfur compounds from fossil fuels.Rhodococcus erythropolis XP is considered one of the BDS potential strains due to its high tolerance to extreme environments and organic solvents,as well as its sulfur-specific 4S pathway with the ability to effectively degrade the sulfur-containing model compound dibenzothiophene（DBT）.Recently,there is growing interested in the effect of altered sulfur metabolism in Rhodococcus on the DBT desulfurization efficiency.Exploring the sulfur metabolism of Rhodococcus has vital research implications for improving BDS efficiency and constructing industrial BDS strains.In this paper,the growth model under inorganic sulfur source（Na₂SO₄）and organosulfur source（DBT）was established.And then,TMT relative quantitative proteomics assay was tested to analyze the protein expression differences of logarithmic period XP strains under the above sulfur sources.Proteomic identification and quantification results showed the total number of quantified proteins reached 4075.Hierarchical clustering algorithms were used to categorize the differentially expressed proteins,illustrating the significant effect of different sulfur sources on the samples between groups.Based on the standard of expression ploidy>1.2-fold and p value<0.05,523 up-regulated proteins and 549 down-regulated proteins were obtained.These differential proteins were annotated and analyzed for metabolic pathways by GO（Gene Ontology）analysis and KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway analysis,respectively,predicting the sulfur metabolic pathway in R.erythropolis XP and screening for seven genes that might affect sulfur metabolism.These results provide a theoretical basis for further exploration of sulfur metabolism of R.erythropolis XP.Due to no effective gene regulation system having been constructed in R.erythropolis XP,we have attempted to construct different gene editing systems,including heterologous recombinases system and CRISPR/Cas9 system,to validate predicted sulfur metabolism genes.Experimental results showed that the above two systems failed to achieve gene editing R.erythropolis XP from the DNA level.Using the sfgfp gene（encoding green fluorescent protein）as the reporter gene,small regulatory RNAs（sRNA）system with different scaffolds were able to influence the expression of target proteins from the mRNA level.The constructed RhlS-sRNA system effectively inhibited sfGFP protein expression with the highest fluorescence suppression efficiency reached 69.17%.The ubiquity of RhlS-sRNA system was further validated with mCherry gene（encoding red fluorescent protein）.In addition,the mechanism of sRNA systems with another scaffold was explored.To investigate the influence of proteomics predicted genes on sulfur metabolism and BDS efficiency,the predicted genes were overexpressed,and assayed the amount of2-hydroxybiphenyl（2-HBP,the DBT metabolite）by shake flask fermentation experiment to analyze whether these genes influenced the BDS process.Based on experimental results,the RhlS-sRNA systems were constructed to validate different target genes,including dszA and dszC genes from the 4S pathway and cbs,egtA,and metY genes that markedly affect DBT degradation.Experimental results showed that the RhlS-sRNA system was able to regulate the endogenous gene expression,and that the suppression of cbs,egt A,and metY genes in part reduced the ability of R.erythropolis XP to degrade DBT under sulfur starvation.Through the comprehensive results analysis of quantitative proteomics,overexpression of predicted genes,and the sRNA repression system,we suggest the close relationship between sulfur metabolism and BDS processes.The expression levels of L-cysteine,L-homocysteine,and L-methionine in sulfur metabolism may be important factors in determining whether R.erythropolis XP is in a sulfur-starved state.This study analyzed the protein expression differences under different sulfur sources based on TMT relative quantitative proteomics results,and speculated the effects of sulfur metabolism on BDS under sulfur starvation,providing a theoretical basis for the research of sulfur metabolism and BDS in R.erythropolis XP.Moreover,the effective application of the RhlS-sRNA system which breaks through the limitation of negative regulation in R.erythropolis XP will support targeted protein regulation in engineered strains and establishing ideal BDS strains.Following the continued exploration of key sulfur metabolism genes affecting the BDS process,reasonable control of sulfur metabolism will hopefully enhance the DBT desulfurization efficiency to a new level and accelerate the industrial process of zero-sulfur fuel production.