Microbial Communities And Functional Genes Of Indole Aerobic Biotransformation

Indole,which can be produced by various microorganisms,plants and animals,is widespread in nature.Indole is a typical N-heterocyclic aromatic pollutant in coking and livestock wastewater,posing a threat to human health and natural environment.The study of indole biotransformation processes is the key to realize the biological treatment and reclamation of related wastewater.The researches of indole biotransformation could be dated back to 1920s,while the microbial community information for indole biotransformation is lacking,microbial resources are limited,and degradation mechanism has not yet been revealed.Herein,the microbial transformation of indole was systematically investigated from microbial community,pure strain and functional gene levels.Main contents and conclusions are as follows:The microbial community compositions and structures of coking sludge and indole wastewater reactor sludge were analyzed using Illumina high-throughput sequencing technique.The sludge systems from different actual coking wastewater treatment plants exhibited a similar community composition,and dominant genera were Thiobacillus,Comamonas,Gp4,Azoarcus,Thauera,Kineosporia,Ensifer and Rhodoplanes.Subsequently,two types of indole-containing wastewater treatment bioreactors,i.e.municipal sludge system(A system)and coking sludge system(B system),were successfully constructed and operated.Both systems could efficiently remove indole and produce indigoids by product analyses.Microbial community analysis showed that Azoarcus and Thauera were the core bacteria when indole and glucose were co-metabolized,and their relative abundances in system A and B were 37.94%and 24.34%,respectively.When indole was used as the the sole carbon source,Alcaligenes and Comamonas emerged as the core genera,and the percentage of Alcaligenes in the system B reached as high as 49.41%.Other major genera in the systems included Burkholderia and Pseudomonas.The indole-degrading bacteria were obtained by microbial pure culture technique from indole domesticated mircrobial communities and their degradation characteristics were investigated.Six strains capable of growing with indole as sole carbon source were successfully obtained from activated sludge and other environmental samples.Strains Comomonas sp.IDO1,Comomonas sp.ID02 and Xenophilus sp.ID04 could remove 100 mg/L indole within one week,and strains Burkholderia sp.ID03,Cupriavidus sp.IDO and Cupriavidus sp.SHE presented higher degradation efficiency,which could remove 100 mg/L indole within 24 h.Further studies on strain Cupriavidus sp.SHE were conducted.High performance liquid chromatography and mass spectrometry analyses showed that isatin,isatic acid and anthranilate were the intermediate products when strain SHE was grown in indole mineral salt medium.Genomic and comparative proteomic sequencing were performed,and a total of 6581 protein coding sequences were identified in strain SHE.Proteomic assay detected 1117 proteins,among which 118 proteins were significantly up-regulated and 59 proteins were down-regulated.There were two gene clusters(Cluster ? and ?)in the up-regulated proteins.Sequence analysis showed that Cluster I might be involved in the process of indole oxidation,and Cluster ? might be involved in the anthranilate metabolic process.The metabolic mechanism of indole degradation in strain SHE was revealed by molecular biological techniques based on the genomic and proteomic information.RT-qPCR result showed that she5651-she5654 in Cluster I were all significantly up-regulated in the indole mineral salt medium.The gene knockout strains SHE-A5652 and SHE-?5653?565 were successfully obtained by homologous recombination method.Results showed that the indole degradation capacities of mutant strains were notably reduced,and they could not utilize indole as the sole carbon source for growth.Gene cloning experiments proved that Cluster I could catalyze indole conversion,and gene she5652,termed as indA,was the functional gene responsible for indole oxidation.RT-qPCR result showed that she1808-she1814 in Cluster II were significantly up-regulated in indole or anthranilate mineral salt medium.Indole degradation performance of mutant strain SHE-?1814 was similar with that of wild strain SHE,but the mutant strain could not grow with indole or anthranilate as the sole carbon source.Heterologous expression and enzymatic studies of the gene she1810 were carried out.Product analysis showed that enzyme She1810 could convert anthranilate to 2-aminobenzoyl-CoA.The indole degradation pathway in strain SHE was proposed based on above results.Indole was firstly oxidized by Cluster I,the oxidation products were further oxidized to isatin and anthranilate,and anthranlilate was finally degraded via the unusual 2-aminobenzoyl-CoA pathway catalyzed by Cluster II.To in-depth investigate the functional proteins in indole degradation process,the characteristics and application of indole oxygenase were explored by bioinformatics and gene recombination technique.IndA exhibited around 30%similarity with the conventional styrene monooxygenase StyA in Pseudomonas strains,ands 40-60%similarities with the novel styrene monooxygenases StyAl and StyA2B in Rhodococcus opacus.IndA belonged to an independent branch in the phylogenetic tree,and it was widely distributed in the genera of Cupriavidus,Acientobacter and Burkholderia.IndA could catalyze indole transformation reaction in the presence of FAD and NADH,which showed highest catalytic activity at 37? and pH 8.0 in potassium phosphate buffer.The Km and kcat values for indole were 0.36±0.10 mmol/L and 1.53±0.16 min-1,and IndA could also catalyze the transformation of indole derivatives.The recombinant strain IND_AB was constructed by expressing gene indAB in Escherichia coli BL21(DE3).Strain IND_AB was able to produce indigo from tryptophan.The optimal conditions for indigo production were temperature 30?,rotation speed 150 r/min and 1.0 mmol/L IPTG as the inducer,and strain was cultured in modified tryptophan medium containing 1 g/L tryptophan,3.55 g/L NaCl and 5.12 g/L yeast extract.After 48 h incubation,307 mg/L indigo was produced,increased by 339%compared with the original yield(70 mg/L).