Screening Of Indole-degrading Strain?functional Gene Expression And Co-culture Characteristics

Indole is a genetically toxic nitrogen-containing heterocyclic aromatic compound.At present,most of the research on indole biodegradation focuses on the identification of intermediate products and the degradation pathway.Aromatic oxygenases such as naphthalene dioxygenase and phenol hydroxylase have been reported to be capable of converting indole,but their specificity may be affected.This study takes the improvement of indole degradation efficiency as the starting point,through the optimization of environmental factors,medium components,exogenous expression of functional genes,and the construction of co-cultivation systems,aiming to develop more efficient strains and their enzyme resources as well as improve the biodegradation efficiency of indole.The main contents are as follows:An indole-degrading bacterium YBY was isolated from the soil and identified by the 16 S sequence as Alcaligens sp.The strain YBY can tolerate high concentrations of salt,and it could also use phenol,2-indolinone,and benzoic acid as the sole carbon source.The best conditions for the growth and degradation of the indole of the strain were pH 6.77 and 150 r /min.When a certain concentration of metal ions and other exogenous nutrients were added,the removal rate of indole will be reduced.LC / TOF / MS was used to analyze the indole product metabolized by the strain YBY.It is speculated that the possible metabolic pathway is indole hydroxylation to indole,and indole dimerizes to indigo,or is oxidized to istain,which is further converted into gentisic acid.This pathway is one of the three main pathways for aerobic degradation of indole.The genome of the strain YBY was analyzed using high-throughput sequencing technology.The genome of the strain was 4.3 Mb in length,with 11 contigs,and the GC content was 56.63%.The whole genome also contained genome about benzoic acid,toluene,salicylic acid,and gentisic acid metabolism.By comparing with the known indole oxygenase sequences,one indole oxygenase gene cluster was found in strain YBY,two indole oxygenase gene clusters were found in strain IDO3,and there was more than 40% similarity with the reported indole oxygenase,and it was speculated that it can catalyze the first step of indole oxidation.Then homologous recombination and enzyme digestion were used to exogenously express the two components of the indole oxygenase.DNA electrophoresis,SDS-PAGE and sequencing technology proved the successful construction of recombinant strain 6170.By optimizing the environmental factors and medium components for the indigo synthesis by recombinant strain 6170,the final indigo yield was 363 mg / L.A co-cultivation system was established between the indole-efficient degrading bacteria isolated from the laboratory earlier and the strain Alcaligenes sp.YBY,and it was found that the co-cultivation system had a higher indole degradation efficiency than the single bacteria system.As the concentration increased in 50-200 mg / L,the trend became apparent.Compared with the co-cultivation system that has been reported,the co-culture system constructed in this paper showed higher degradation efficiency.