Metabolic Engineering Of Paracoccus Denitrificans For Enhancing The Degradation Of Sulfamethoxazole

Sulfamethoxazole（SMX）is a synthetic broad-spectrum antibiotic with bacteriostatic and anti-inflammatory effects,which is widely applied in clinical and veterinary medicine.However,the overuse of SMX destroyed the community structure of microorganisms in the environment,and then break the ecological balance among the environment,animals and human beings,which seriously endangered the health of human beings and animals.Therefore,efficient degradation of SMX in the environment is urgent to ensure ecological safety.Biological removal of SMX is currently the most widely used,efficient,economical and practical method,which relied on naturally selected SMX-degrading bacteria to achieve efficient degradation of SMX.However,there were issues of naturally selected SMX-degrading microorganisms,such as low degradation efficiency and poor environmental adaptability.To further improve the biodegradation of SMX,using metabolic engineering to enhance the efficiency of environmental microbial degradation of SMX is a new strategy with great potential.However,the lack of tools and methods for metabolic engineering modification in environmental microorganisms hindered metabolic engineering modification.In order to solve this problem,this study firstly used a superior nitrogen removal environment strain—Paracoccus denitrificans DYTN-1as chassis cells,then excavated and characterized the gradient intensity promoters,which were used in metabolic engineering system to regulate the expression level of SMX degradation gene clusters（sad A,sad B and sad C）.At the same time,the source of flavin mononucleotide（FMN）reductase was optimized.Finally,the degradation ability of SMX by P.denitrificans DYTN-1 was significantly improved.This was the first study of enhanceing degradation of SMX by synthetic biotechnology at home and abroad,and the constructed SMX-degrading strain owned the functions of removing SMX and nitrogen pollutants simultaneously,which provided a technical reference for environmental remediation of multiple pollution.The details of their research are as follows.（1）Mining and characterization of endogenous gradient strength promoters of P.denitrificans DYTN-1.The evaluation and characterization of the endogenous promoters of P.denitrificans DYTN-1 was beneficial to optimize the expression of SMX degradation genes and improve the degradation efficiency.The first was to clone promoters P_{gln A},P_nir,P_{rh O} and P_cs from the P.denitrificans DYTN-1 genome,respectively.The four amplified promoters showed a gradient intensity for sf GFP,and the expression level ratio of GFP/OD₆₀₀ was 10:12:14:17.Using the four endogenous promoters to control the expression levels of sad A,sad B and sad C,respectively,the m RNA expression differences of them reached 2.1-25.3 fold,providing tools for further optimization of the SMX degradation pathway subsequently.（2）Optimization of expression levels of SMX degradation pathway genes to enhance SMX degradation.Overexpression of heterologous genes can be burdensome to both the growth and metabolism of the host bacteria,so systematically optimizing the expression levels of SMX degradation genes is the key to improve the degradation efficiency and cell growth.First,the expression levels of sad A were optimized using the screened gradient-strength promoters P_{gln A},P_nir,and P_cs.The degradation efficiency of all three promoter-regulated sad A strains was increased to about18.0%for 100 mg·L^-1 SMX,demonstrating the degradation of SMX by Sad A.The accumulation of intermediate metabolites affected the expression of sad A due to the metabolic limitation caused by the unexpressed downstream gene sad B.Using the above gradient strength promoter to optimize the co-expression of sad A-sad B through a pseudo-manipulator structure,the co-expression of sad B gene promoted the degradation of SMX.It was also found that the combination of sad A and sad B expression was optimized best at lower levels of promoter P_{gln A} regulation,and continuing to increase the transcriptional level of the genes only resulted in excessive protein misfolding,which ultimately led to a decrease in the amount of active enzyme and degradation efficiency.Finally,the highest degradation efficiency of 27.7%was obtained for strain P_{gln A}-sad A-P_{gln A}-sad B.（3）Optimization of FMN reductase source and expression level to enhance SMX degradation.Insufficient supply of FMNH₂ may be the main factor limiting the degradation of SMX by Sad A and Sad B.Therefore,screening for highly active FMN reductase and optimizing their expression level by gradient intensity promoters could help to further enhance the degradation effect.Consequently,overexpression of Sad C and other five FMN reductases from different sources further improved the degradation of SMX.Except for the sad C strain,the degradation efficiency and the degradation capacity per unit biomass of strains increased with the expression level of fmn R,among other five FMN reductase overexpressing strains.The comparative analysis of the enzymatic activity and degradation efficiency of FMN reductase revealed that the best degradation efficiency of Cog A,Sad C,Psu K and Sut R overexpressing strains in each group increased with the increase of enzymatic activity.While Ssu E and Rut F own the highest enzymatic activity,their overexpression strains led to a decrease in the degradation efficiency of SMX instead due to the high expression level.Therefore,the metabolic regulation not only relies on the high enzymatic activity of the acting enzyme,but also the adaptability of the regulatory elements is a key factor in the regulation strategy.Finally,the degradation of SMX by the constructed optimal strain P.d-p IAB₄-P_cs-sut R was increased to 44.0%(17.0 mg·(L·OD₆₀₀)^-1).（4）Performance study of P.denitrificans DYTN-1 for simultaneous degradation of SMX and nitrogen.To investigate the effects of SMX and SMX degradation genes on nitrification and denitrification of P.dentirificans DYTN-1,the ability to co-degrade 100 mg·L^-1 SMX and 1 g·L^-1NO₃^-（or NH₄⁺）was tested.It was found that denitrification of wild-type strains was inhibited by SMX in the early growth phase,while the recombinant strains expressing SMX degradation genes alleviated the"SMX stress".In contrast,the nitrification process of P.dentirificans DYTN-1 was not affected by SMX or sad AB-fmn R expression at all.The removal rates of NH₄⁺in wild-type strains with or without SMX addition were 69.5%and 67.8%,respectively,while the removal rates of NH₄⁺for strains expressing SMX degradation genes were also in the range of 67.9%to 71.2%.Finally,the constructed new strain was suitable for dual degradation of SMX and ammonia-nitrogen pollutants,and the removal efficiencies reached 71.2%and 44.0%,respectively.