| Lignocellulose is the most abundant component of Earth's biomass,and its efficient utilization has been the focus of a worldwide research effort.Improving its utilization will help alleviate global energy crisis and reduce food security issues.Although lignocellulose-degrading enzymes have been studied for many years,there are still limitations that need to be overcome.These include previous studies being limited to single-enzyme systems,limited understanding of degradation mechanisms,and low degradation efficiencies.Therefore,the discovery of the novel high-efficiency enzymes for lignocellulose degradation is of great significance to the utilization of biomass.The Asian elephant(Elephas maximus)has been listed as a first-class protected animal in China and is characterized as an endangered species by the International Convention on Trade in Endangered Species.Their low rate of reproduction and high rate of disease in captivity along with the unusual migration of wild Asian elephants are obstacles to the conservation of this species.Traditional methods of studying the diet and living environment of Asian elephants have played an important role in improving the above problems.However,to better understand the impact of diet and the living environment on the health and reproduction of Asian elephants,it is urgent that we expand our research as well as develop new research methods in this area.The large number of microbial communities in the gastrointestinal tract of animals is closely related to their nutrition,immunity,and feeding habits.Therefore,studying the intestinal microbiome of Asian elephants will assist efforts to protect this species.Based on the survey of foraging plant species for Asian elephants,this study utilized 16 S r RNA gene sequencing,q PCR detection,metagenomic sequencing,and proteomic analysis to explore the gut microbiome of captive and wild Asian elephants.These data provide a theoretical basis to improve the protection and management of this precious species and also offer a resource for the mining of new and efficient industrial enzymes.The results of this study are summarized as follows.1.The survey results of Asian elephant foraging plant species showed that the number of foraging plant species decreased substantially(from 112 to 3),whereas the fiber content of the main plant species gradually increased(Neutral Detergent Fiber content from 49.5% to 72.8%)as the degree of captivity increased.Phylogenetic analysis of the 16 S r RNA sequencing results showed that the microbial diversity of the gut microbiome decreased as the degree of captivity increased.Thus,captivity resulted in reduced gut bacterial taxa in Asian elephants.The results of the q PCR analysis showed that the relative content of butyryl-Co A: acetate Co A-transferase was significantly decreased in pure wild Asian elephants when compared to captive,semi-captive,and semi-wild Asian elephants.These results indicate that captive Asian elephants are lacking internal resources(gut microbiota),whereas wild Asian elephants suffer from a lack of external resources(food and habitat).2.Based on metagenomic analysis of the gut microbiota from the Asian elephants,including bacteria,archaea,eukaryotes,and viruses,it was discovered that bacteria were the dominant microbial taxa.In particular,Firmicutes and Bacteroidetes were the dominant bacterial phyla in Asian elephants.Differential species analysis showed that Bacteroidetes and Proteobacteria were significantly enriched in the breastfeeding Asian elephant(BAE)group,Fibrobacteres and Spirochaetes were significantly enriched in the captive Asian elephant(CAE)group,and Firmicutes and Verrucomicrobia were significantly enriched in the wild Asian elephant(WAE)group.These differences are likely caused by differences in their diet.The functional annotation results showed that the glycoside hydrolases that were significantly enriched in the BAE group were primarily beta-galactosidase and mannosidase.Lignocellulose-degrading enzymes were primarily enriched in the CAE group,whereas the carbohydrate enzymes were not enriched but were highly diverse in the WAE group;this is likely to be related to the greater variety in diet of wild Asian elephants.3.Using a metagenomic binning technique,we assembled 139 high-quality bacterial genomes and found that the gut of Asian elephants contained many new microbial species.Metabolic reconstitution analysis revealed that 5-ribose phosphate isomerase A and glucose 6-phosphate isomerase involved in both the pentose phosphate pathway and the glycolysis pathway are abundant in the gut of Asian elephants.In contrast,these enzymes are of extremely low abundance in the rumen of cattle,suggesting Asian elephants have superior efficiency of energy metabolism.CAZy annotation of 139 metagenome assembled genomes showed that more than15,000 CAZymes were present.In terms of the ratio of CAZymes to total proteins,the Asian elephant gut CAZymes were significantly more enriched than GTDB.The low similarity to protein sequences in NCBI?NR(only 16.5% of the sequences had identity higher than 95%)and in CAZy(only 18.0% of the sequences had identity higher than 70%),indicates that the Asian elephant gut CAZymes are highly novel.Approximately 83.5% of the Asian elephant gut CAZymes have the extracellular secretion signal peptide,which may help CAZymes to secrete into the Asian elephant intestinal environment to act on plant polysaccharides.Combined with a bioinformatic analysis,we obtained detailed information on the genetic sequence of more than 15,000 new enzymes that degrade lignocellulose in the intestines of Asian elephants.This provides an impressive genetic resource for the identification of new and potentially more efficient feed enzymes as well as enzymes for the development of bioenergy.4.The analysis of Asian elephant stool samples using shotgun proteome detection revealed that the Asian elephant gut bacteria expressed 848 CAZymes,474 polysaccharide utilization loci(PUL)related proteins,and 41 cellulosome-related proteins.The bacterial species with the highest expression of these cellulosome-related proteins was Ruminococcaceae?HUN007.Thus,Ruminococcaceae?HUN007 is likely the key bacteria for the degradation of lignocellulose in the gut of Asian elephants.The expression of cellulosome-associated and PUL-associated proteins in the gut of wild Asian elephants was lower than that of captive Asian elephants.We hypothesize that this may be due to wild Asian elephants having a preference for low-fiber food such as crops,grains,and fruits. |
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