Study On The Cleavage Mechanism Of Burkholderia Sp.HQL1813 On The C-P Bond Of Methylphosphonate

Phosphorus is one of the essential nutrients for living organisms and its availability influences the primary productivity of organisms and the structure of ecosystems.Organisms preferentially absorb and utilize soluble inorganic phosphorus,while most of the phosphorus in the environment is mainly organophosphorus,most of which can’t be directly absorbed and utilized by microorganisms.Methylphosphonate（MPn）is the simplest C-P bonded organophosphorus and is often used to study the geochemical processes of phosphorus in the environment.The decomposition of methylphosphonates in aqueous environment is affected by both UV light and microorganisms,the reaction process and mechanism of inorganic phosphate production from methylphosphonate catalyzed by UV light have been elucidated,but the mechanism of methylphosphonate conversion by microorganisms remains to be investigated.Therefore,in this study,we investigated the biological process and mechanism of microbial decomposition of methylphosphonate to produce inorganic phosphate using microbiological methods,modern molecular biology techniques combined with phosphate oxygen isotope techniques for the microbial conversion of methylphosphonate.Firstly,Burkholderia sp.HQL1813,which was screened from the cultivation base of cherry tomatoes with organophosphorus pesticides applied in Hainan,was studied by single-factor experiments to investigate the growth and phosphate release characteristics of Burkholderia sp.HQL1813under different temperature and pH conditions.The results showed that the optimum growth conditions of Burkholderia sp.HQL1813 were 28°C and pH 7.After 80 h,its OD₆₀₀ value could reach 2.0 and the concentration of released inorganic phosphate reached 100μM.Secondly,the phosphate oxygen isotope technique was used to prepare 30.64‰¹⁸O-labeled methylphosphonate solution with heavy oxygen water,the microbial transformation system of methylphosphonate was established to determine the oxygen isotope characteristics and fractionation in the inorganic phosphate produced by the transformation of methylphosphonate by Burkholderia sp.HQL1813.The results showed that the oxygen isotope ratio(δ¹⁸O_P)in the inorganic phosphate produced by the conversion of methyphosphonate by Burkholderia sp.HQL1813 was 40.88,which was higher than that by photocatalysis(δ¹⁸O_P was 9.59),and produced significant isotopic fractionation,indicating that Burkholderia sp.HQL1813 broke the C-P bond through microbial action to produce inorganic phosphate.Genome sequencing analysis of Burkholderia sp.HQL1813 revealed that the bacterium contains the Phn GHIJKLM gene cluster and ABC transporter protein genes,which are key genes involved in C-P breaking of methylphosphonate.Meanwhile,the results of transcriptome analysis in logarithmic and stable phases showed that the expression of Phn gene cluster as well as ABC transporter protein genes were maintained at high levels,indicating that Burkholderia sp.HQL1813 was active in the metabolism of methylphosphonate and continuously produced and released inorganic phosphate.In addition,the heterologous expression and purification of the seven Phn G-Phn M proteins in Burkholderia sp.HQL1813 were successfully implemented by constructing a heterologous expression system of E.coli BL21（DE3）,and in vitro degradation experiments of methylphosphonate by Phn proteins were carried out.The results showed that a single Phn protein could not effectively decompose the methylphosphonate,and a simple multi-protein mixture activity also could not decompose,indicating that the decomposition course of Phn protein on methylphosphate may be an integrated and synergistic one achieved with other proteins.Further,the knockdown experiment of Phn J gene was carried out on Burkholderia sp.HQL1813 using homologous recombination technique.The results showed that the electrotransformed strain still retained the ability to transform methylphosphonate.In summary,this study initially revealed the process of cracking the C-P bond of methylphosphonate and releasing inorganic phosphate by Burkholderia sp.HQL1813,the oxygen isotopic characteristics of phosphate and its biological mechanism,which provides an important reference for further resolving the microbial decomposition mechanism of methylphosphonate in nature and also provides a theoretical basis for elucidating the geochemical behavior of the global phosphorus cycle.