代谢工程强化脱氮副球菌DYTN-1去除氮素污染物

Metabolic engineering enhances the nitrogen removal by Paracoccus denitrificans DYTN-1

摘要:

【目的】脱氮副球菌(Paracoccus denitrificans)是一种环境友好的α-变形菌纲菌株，在有氧条件下也可进行反硝化过程，具有较好的脱氮能力。本研究以脱氮副球菌DYTN-1为底盘细胞，筛选氮素诱导型启动子用于强化硝化和反硝化途径，进而达到代谢工程强化脱氮副球菌DYTN-1去除氮素污染物的目的。【方法】通过接合转移的方法分别将过表达amoA、amoB、hao和nirS基因的重组质粒导入脱氮副球菌DYTN-1细胞中。经过荧光定量检测和氮素定量检测对脱氮副球菌DYTN-1的基因元件和氮去除能力进行表征。【结果】从基因组中挖掘了6个受NO₂^‒、NO₃^‒和NH₄⁺诱导的启动子，诱导差异为2‒26倍；且过表达nirS的菌株用2 g/L KNO₃处理24 h后培养基中NO₃^‒的残余量为野生型菌株的67%。同时过表达hao和nirS基因的菌株在用1 g/L NH₄Cl和2 g/L KNO₃处理12 h后，其NO₃^‒的剩余量仅为野生型菌株的50%，且最终总氮的降解效率达79.5%，剩余总氮仅为野生型菌株的一半。【结论】上述研究表明，利用筛选获得的启动子工具在P. denitrificans DYTN-1中进行代谢工程改造强化氮素污染物的去除具有可行性。

关键词:诱导型启动子;硝化途径;反硝化途径;脱氮副球菌DYTN-1

Abstract:

[Objective] Paracoccus denitrificans is an environment-friendly strain of α-Proteobacteria.It can also perform denitrification under aerobic conditions and has good denitrification ability.This study intended to use P.denitrificans DYTN-1 as the chassis cell to screen nitrogen inducible promoters for strengthening the nitrification and denitrification pathways,thus achieving the purpose of enhancing the removal of nitrogen pollutants by metabolic engineering.[Methods] Recombinant plasmids overexpressing amoA,amoB,hao and nirS genes were introduced into P.denitrificans DYTN-1 cells by conjugation.The gene elements and nitrogen removal ability of P.denitrificans DYTN-1 were characterized by fluorescence quantitative detection and nitrogen quantitative detection.[Results] Six promoters induced by NO₂^‒,NO₃^‒ and NH₄⁺ were obtained at 2 to 26-fold induction.The residual amount of NO₃⁻ in the medium of the nirS overexpressed strain was 67% of that of the wild-type strain after treatment with 2 g/L KNO₃for 24 h.The strains overexpressing both hao and nirS had only 50% of the residual NO₃⁻ of the wild-type strain after treatment with 1 g/L NH₄Cl and 2 g/L KNO₃ for 12 h.Furthermore,the final degradation efficiency of total nitrogen reached 79.5% and the residual total nitrogen was only half of that of the wild-type strain.[Conclusion] It is feasible to carry out metabolic engineering with the aboved screened promoters to enhance the removal of nitrogen pollutants in P.denitrificans DYTN-1.

Key words:inducible promoter;nitrification pathway;denitrification pathway;Paracoccus denitrificans DYTN-1