Reconstruction Of 4S Desulfurization Pathway In Recombinant Escherichia Coli And Desensitization Of Key Enzyme

The combustion of sulfur-containing fuels causes environmental pollution and health hazards,so the global society has gradually developed ultra-low sulfur or sulfur-free fuel oil.The biodesulfurization 4S pathway can specifically cleave the C-S bond of dibenzothiophene（DBT）while maintaining its carbon skeleton and combustion value.This advantage makes the4S pathway of biodesulfurization a low-cost and environmentally friendly technology that can be used as a supplementary strategy for mainstream hydrodesulfurization.Biological desulfurization has not yet achieved the goal of industrial application,and the inhibition of each Dsz enzyme is one of the main bottlenecks of the 4S pathway.DszC is the key starting enzyme for desulfurization.Among all Dsz enzymes,DszC is most affected by feedback inhibition from 2-hydroxyphenol（HBP）.It is also inhibited by DBT and2-hydroxybiphenyl-2-sulfinic acid（HBPS）,which severely limits the desulfurization efficiency of the 4S pathway.In this study,we aimed to attenuate the feedback inhibition and substrate inhibition of DszC by protein engineering,and combined with the kinetic modeling results to guide the overexpression of DszC to increase the desulfurization rate of the 4S pathway in recombinant E.coli strains.The specific research contents and results are as follows:（1）Functional expression of the 4S pathway in E.coli and regulation of dsz gene doseThe dszABCD gene derived from R.erythropolis IGTS8 was codon-optimized and cloned into E.coli,and the dsz expression cassette was optimized.A kinetic model was established based on the reported Dsz enzyme kinetic parameters.The predicted result was that the HBP production rate could be increased when the concentration of DszC and DszB was high.Different ratios of purified Dsz enzyme combined with BADC cell extract for in vitro desulfurization,when BADC+1A+4B+2C,the conversion rate was close to 100%,which was about 9 times higher than that of BADC cell extrace alone.The dsz gene was added to the BL21（DE3）competent cells containing pSB4A5-BADC alone or in combination.The BL21（DE3）/BADC+C strain had a HBP yield about 6 times higher than that of the BL21（DE3）/BADC strain,and its specific desulfurization rate reached 98.05μM_HBP/g_DCW/h,which was 4.3 times the highest reported value.（2）Establishment of high-throughput screening method and evaluation of DszC mutant A101VA DszC mutant library was established by error-prone PCR,and a high-throughput screening method based on resting cell module and color reaction module was established.The HBP yield of the A101V strain was increased by 27.60%compared with the wild type strain.The mutant A101V contributes to the improvement of enzyme activity,suppression of feedback inhibition,and decreasing of substrate inhibition.Through the simulation of small molecule ligands in the Discovery Studio,it was found that W327 and HBP formed aπbond in the binding pocket of A101V,which became a potential key amino acid.（3）Iterative saturation mutation and evaluation of DszCBy site-directed saturation mutation of amino acid 101,it was found that the enzyme activity of A101K mutant was the highest,and the enzyme activity of wild type DszC was increased by 57.51%.The 327th amino acid was saturatingly mutated on the basis of A101K.The AKWC and AKWY mutant enzyme activities were increased by 3.3 and 2.6 times,respectively,compared with wild type DszC,and their IC₅₀ values were increased from3.95±0.24μM to 80.38±2.52 mM and 83.00±1.12 mM respectively,indicating that mutation of amino acid 327 was critical for desensitization of DszC feedback inhibition.The substrate inhibition of AKWC was also reduced.The yield of recombinant strain HBP carrying the AKWC mutant was 4.4-fold higher than that of the wild-type strain.The yield of AKWC overexpressing recombinant strain BL21（DE3）/BADC*+C*（C*stands for AKWC）HBP was23.6 times higher than that of wild type strain,and the specific transformation rate reached214.84μmol _HBP/g_DCW/h,which is the highest reported value.It is closer to the 1-3mmolHBP/gDCW/h target required for BDS commercial applications.（4）Alanine scanning and site-directed saturation mutations alleviate DszC substrate inhibitionBy constructing 40 alanine mutants from the dimeric faces on the basis of AKWC,they were replaced with BL21（DE3）/BADC,respectively.By screening,when the DBT concentrations were 0.25 and 0.5 mM,BL21（DE3）/BAD（AKWCP413A）strain HBP production was 56.81%and 39.88%higher than BL21（DE3）/BAD（AKWC）strain,respectively.The 413th amino acid was fixedly abruptly mutated on the basis of AKWC,and the AKWCPI mutant maintained 57%of the maximum enzyme activity at a concentration of1 mM DBT.AKWCPI retains most of the advantages of resistance to HBP feedback inhibition,and it has higher tolerance to high concentration DBT,the maximum enzyme activity reaches 27.27 U/mg,which is about 43%higher than AKWC,while the single point mutant P413I has almost no Enzyme activity.When the DBT concentration was 0.25 mM,the yield of HBP obtained by BL21（DE3）/BADC**+C**was 39.89%higher than that of BL21（DE3）/BADC*+C*.The desensitization engineering of feedback inhibition and substrate inhibition combined with desensitized DszC protein overexpression strategy overcame the inhibition bottleneck of DszC enzyme in the 4S pathway,which was an effective progress in the production of sulfur-free fuel oil.At the same time,the results of this study also provided a effective strategy and a success case for optimizing the metabolic pathways with feedback inhibition and substrate inhibition.