| Hydrogen sulfide,normally found in oil and gas and their down streaming processes,represents a highly toxic environmental threat by forming acid rain.Thioalkalivibrio versutus D306,an autotrophic;high GC content and gram-negative polyextremophile,can strongly desulfurize natural gas and produce sulfur and the less favorable,sulfate.To strengthen the power of T.versutus D306 as a natural gas desulfurizing bacterium,this thesis aimed to solve three different obstacles for this objective,which are lack of genome editing method,unclear sulfur metabolism and unfavorable sulfate production which is both;harmful to industrial equipment and toxic to the cells.To develop efficient genome editing strategy for T.versutus D306 using CRISPR-cas9 system based on known strain information,cell death induced(CDI)strategy was followed.The strategy used cell death induction,calculated as colony forming units,as an indicator for screening the best knockout plasmid without the need to check colonies using PCR.Firstly,a CRISPR-cas9 system with editing efficiency of 8.2%of the screened colonies was first developed.Further optimization of the system,using different expression levels of Cas9 protein and sgRNA,was screened again by the CDI strategy.The editing efficiency was increased to 41.2%using touchdown PCR technique developed especially for T.versutus D306.Therefore,CDI strategy showed a success in simplifying workflow for CRISPR system development in T.versutus D306 and can be applied to other autotrophs or polyextremophiles.Secondly,50 sulfur related proteins were identified in T.versutus D306 and their localization were assumed,which gave us a clear picture for different sulfur metabolism steps in the strain.The CRISPR system was used to knockout four sulfate producing key genes to enhance the desulfurization process.HdrB protein knockout enabled us to decrease sulfate production by 20.8%and 55.1%for thiosulfate and sulfide grown T.versutus,respectively,as compared to the native strain,which means more sulfur,was produced.It also prevented the strain to consume sulfur as the strain energy substrate.These results were also confirmed by electron microscope and strain growth.The desulfurization process profitability was further improved by proving the ability of T.versutus D306 to produce nano sulfur(less than 50 nm)from sulfide as a substrate using XRD,electron microscope and grain size measurements.Thirdly,a tightly inducible expression system was built using fed-batch and Design,build,test and validate approach for expressing toxic proteins by nature like Cas9 protein,Class 2 CRISPR main effector protein.The fed batch strategy improved the growth of T.versutus D306 to facilitate expression system measurements.The final constructed system,using ferric uptake regulator,enabled the expression to be repressed near the control strain values and increased again to 27 times the control.The system successfully expressed a highly toxic protein as a validation step.Fourthly,a multi gene editing CRISPR-cas9 system was constructed.The universal tRNA was selected as the target of gene editing,and 13 different tRNAs,which were 1 native T.versutus D306,1 rice native one and 11 E.coli ones,were studied in E.coli.The results showed that the editing efficiency of the constructed system for LeuW,ValU and LysV was 100%,66.7%and 33.3%respectively.In conclusion,a first knockout system,based on CRISPR-cas9 system and cell-death induced strategy,was built in T.versutus D306.The system was used to improve productivity of desulfurization process by knocking out unfavorable sulfate production pathway genes.The nanometric characteristics of T.versutus sulfur was proved for more profitable bio desulfurization process.Another system for inducible protein expression was developed using ferric repression protein depending upon design,build,test and validate approach.Finally,multiplexing CRISPR was studied in E.coli for future application in T.versutus D306. |
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