Applied Basic Research Of Gut Microbiome Of Black Bears Based On Metagenomic Sequences

Traditional Chinese medicine（TCM）plays a decisive role in the status of China’s pharmaceutical industry,"Chinese medicine innovation and development plan（2006-2020）" clearly put forward to support the rare and endangered varieties of Chinese herbal medicine protection,breeding and alternative research.For the development of artificial substitutes of expensive TCM bear bile,after repeated investigation and demonstration,the National Science and Technology Major Projects for “Major New Drugs Innovation and Development”（2014ZX09301306）was approved.The composition of the bear bile is complex,and the main medicinal value component of bear bile is ursodeoxycholic acid.Ursodeoxycholic acid is biotransformed by 7α-hydroxysteroid dehydrogenase（7α-HSDH）and 7β-hydroxysteroid dehydrogenase（7β-HSDH）from gut microbiome,use chenodeoxycholic acid as a substrate.Black bear as a special species that can produce a large amount of ursodeoxycholic acid,it is of great scientific significance and industrial value to study the structure and function of their gut microbiota,and to excavate the 7α-/7β-HSDHs with good enzymological properties in their gut microbiome.However,most of the microbes in nature are non-culturable,and the animal’s intestines are complex anaerobic environments,which limits the development of microbial resources.Sequencing the metagenome of the environmental samples allows us to understand the structure and function of the microbial population faster and better,and to excavate the functional genes.Therefore,based on the research strategy of metagenomics,this paper used the second generation sequencing technology,compared the structural characteristics of gut microbiota of black bears in different regions,reveals the common microbial composition of microbes in black bear,analyzed the gene distribution of the gut microbiome of the black bear,so that provide the basis for understanding the structure and function of the gut microbiome of the black bear;On this basis,the 7α-HSDHs and 7β-HSDHs encoding genes in the metagenomic sequences of the black bear gut microbiome were excavated and characterized.A series of enzymatic properties were studied to screen 7α-/7β-HSDH with high activity,strong catalytic efficiency and good thermal stability,to provide high quality candidate enzymes for in vitro biotransformation of ursodeoxycholic acid,and to provide material basis for the industrialization of cultivation artificial bear bile powder in vitro.The main research contents and results are as follows:First,the composition and structure of gut microbiota in captive black bears from Sichuan,Yunnan and Heilongjiang were studied based on sequence information of V3-V4 region in 16 S rRNA.A total of 12 phyla,192 genera of microorganisms were identified classification.Among them,Firmicutes and Proteobateria are the dominant phylum of Asiatic black bear intestinal flora,which are widely found in all fecal samples of the three experimental groups;Clostridium_sensu_stricto₁,Turicibacter,Escherichia-Shigella,（uncultured）Peptostreptococcaceae Incertae_Sedis,Peptostreptococcaceae Incertae_Sedis,Clostridium_glycolicum and Streptococcus are the shared microbes of the intestinal microflora of the three captive black bears;The genus LefSe analysis showed that the three genera of the genomes were Escherichia-Shigella,Streptococcus and Pseudomonas.Alpha diversity analysis and Beta diversity analysis showed that there were no significant differences in composition and structure between the gut microbiota of captive black bears in Sichuan and Yunnan,while the gut microbiota of captive black bears in Heilongjiang had a greater diversity of flora and the structure with the previous two is significant different,which indicated that that biogeographical and climatic factors may contributed to the community structure of the gut microbiota of the black bears.The above results provide the basis for understanding the structure and characteristics of gut microbiota of black bears in China.Secondly,the metagenomic sequencing of three black bear stool samples from Sichuan,Yunnan and Heilongjiang were sequenced by Hiseq sequencing platform,which revealing the functional characteristics and metabolic pathways in the gut microbiome of black bear.After splicing,assembling and predicting the sequences,a total of 400118 ORFs were obtained,which indicated that was a huge treasure trove of microbial gene resources.Based on the function index of COG database,it is shown that the genome of black bear is rich in functional genes involved in Metabolism,among which carbohydrate transport is the highest with Carbohydrate metabolic transport and metabolic genes,and amino acid transport and metabolic genes.According to NR gene classification,three samples In the sequencing of the ORF gene derived from Firmicutes;KEGG metabolic network shows that the gut microbiome of black bears are wealth in carbon metabolism,nucleic acid metabolism and amino acid metabolism pathway.The above study provides some theoretical reference for understanding the function of gut microbiome of black bear,and also provides the data base for the next step to explore the functional genes.Then,the putative 7α-/7β-HSDH genes were detected and cloned from the metagenomic sequences of the gut microbiome of black bears by local BLAST contrast strategy,and the catalytic function was characterized by in vitro expression,and the protein sequence was analyzed by bioinformatics.A total of 38 putative 7α-HSDH coding genes and 10 putative 7β-HSDH genes were found,and five 7α-HSDH coding genes with one full length sequence and one 7β-HSDH coding gene were cloned and expressed.Compared with 7α-HSDH（Clo.sa-a）and 7β-HSDH（Clo.sa-b）enzymes from Clostridium absonum ATCC27555,which were used for the biotransformation of ursodeoxycholic acid,7α-HSDH protein S1-a-1 and S1-a-2 showed superior catalytic activity,which was significantly higher than that of Clo.sa-a,and the 7β-HSDH protein Y1-b-1 from the gut microbiome of black bear also showed an equal catalytic activity with Clo.sa-b.The results showed that metagenomic strategy sequencing has successfully discovered 7α-HSDHs and 7β-HSDHs from the gut microbiome.The results of homology analysis and amino acid sequence showed high homology of six HSDH proteins and conserved SDR superfamily protein sequence characteristics.Finally,the enzymatic properties of 7α-/7β-HSDHs derived from the gut microbiome of black bears were studied systematically,and compared those enzymes with Clo.sa-a and Clo.sa-b in the enzymatic kinetic parameters and thermal stability.The catalytic efficiency of protein S1-a-1 was significantly higher than that of other 7α-HSDHs,10 times of Clo.sa-a,and Y1-b-1 was comparable to that of Clo.sa-b.Under the experimental conditions,the optimum pH of this six HSDH proteins was between 7.5 and 9.0,and the optimum temperature was between 30-50 °C.The thermal stability of the five 7α-HSDH was similar to that of Clo.sa-a,and the thermal stability of Y1-b-1 is better than that of Clo.sa-b,especially at 37 °C,the thermal stability is better than that of Clo.sa-b.The above experiment further screened 7α-/7β-HSDH with industrial application prospect,which not only provided a meaningful data reference for its follow-up application,but also provided a strong reference for the development of other functional genes in the gut microbiome.