Cloning and expression of genes encoding key enzymes of a marine paraben-degrading strain

Abstract:

[Background] 4-hydroxybenzoic acid and its esters are precursors for various aromatic compounds. They are, however, difficult to be decomposed in the nature, causing environmental pollution. These pollutants migrate to the ocean along with ocean current, damaging the marine ecological environment.[Objective] To screen a 4-hydroxybenzoic acid esters-degrading strain from the marine environment, predict the metabolic pathways of 4-hydroxybenzoic acid esters by whole-genome sequencing and annotation analysis, and analyze the key enzymes in the pathways and the functions of the enzymes. [Methods] We isolated the 4-hydroxybenzoic acid esters-degrading strain through enrichment culture. We used gene cloning technology to express the genes encoding key enzymes in the metabolic pathways of 4-hydroxybenzoic acid esters in Escherichia coli and explored the activities and enzymological characteristics of the recombinant proteins. [Results] A strain (B1 strain) was screened out from the seabed sediment and identified as Citricoccus nitrophenolicus by 16S rRNA sequencing. B1 can grow with diverse 4-hydroxybenzoic acid esters, and the growth status was the best when methyl ester was the only carbon source. Genes encoding carboxylesterase and monooxygenase were successfully expressed in E. coli. The optimal reaction conditions of recombinant carboxylesterase were pH 8.0 and 30℃ (30 min). The activity of recombinant monooxygenase depended on coenzyme, and Mg²⁺, Mn²⁺, Zn²⁺, and Fe³⁺ can enhance the activity of monooxygenase. As further identified by fluorescent quantitative PCR, z13175 and z09075 were genes encoding carboxylesterase. In the absence of oxygen, B1 strain carried out anaerobic respiration and reduced nitrate to nitrite.[Conclusion] Carboxylesterase and monooxygenase are key enzymes in the conversion of 4-hydroxybenzoic acid esters to protocatechuic acid, which play an important role in the biodegradation of the esters.