Precision enzyme discovery through targeted mining of metagenomic data.

Metagenomics has opened new avenues for exploring the genetic potential of uncultured microorganisms, which may serve as promising sources of enzymes and natural products for industrial applications. Identifying enzymes with improved catalytic properties from the vast amount of available metagenomic data poses a significant challenge that demands the development of novel computational and functional screening tools. The catalytic properties of all enzymes are primarily dictated by their structures, which are predominantly determined by their amino acid sequences. However, this aspect has not been fully considered in the enzyme bioprospecting processes. With the accumulating number of available enzyme sequences and the increasing demand for discovering novel biocatalysts, structural and functional modeling can be employed to identify potential enzymes with novel catalytic properties. Recent efforts to discover new polysaccharide-degrading enzymes from rumen metagenome data using homology-based searches and machine learning-based models have shown significant promise. Here, we will explore various computational approaches that can be employed to screen and shortlist metagenome-derived enzymes as potential biocatalyst candidates, in conjunction with the wet lab analytical methods traditionally used for enzyme characterization.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

Lignocellulose degradation by rumen bacterial communities: New insights from metagenome analyses.

Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment for the storage and microbial fermentation of ingested feeds dominated by plant materials. The rumen microbiota has acquired diverse and functionally overlapped enzymes for the degradation of plant cell wall polysaccharides. In rumen Bacteroidetes, enzymes involved in degradation are clustered into polysaccharide utilization loci to facilitate coordinated expression when target polysaccharides are available. Firmicutes use free enzymes and cellulosomes to degrade the polysaccharides. Fibrobacters either aggregate lignocellulose-degrading enzymes on their cell surface or release them into the extracellular medium in membrane vesicles, a mechanism that has proven extremely effective in the breakdown of recalcitrant cellulose. Based on current metagenomic analyses, rumen Bacteroidetes and Firmicutes are categorized as generalist microbes that can degrade a wide range of polysaccharides, while other members adapted toward specific polysaccharides. Particularly, there is ample evidence that Verrucomicrobia and Spirochaetes have evolved enzyme systems for the breakdown of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin. It is concluded that diversity in degradation mechanisms is required to ensure that every component in feeds is efficiently degraded, which is key to harvesting maximum energy by host animals.

Efficiency of an alkaline, thermostable, detergent compatible, and organic solvent tolerant lipase with hydrolytic potential in biotreatment of wastewater.

Discharging the tannery wastewater into the environment is a serious challenge worldwide due to the release of severe recalcitrant pollutants such as oil compounds and organic materials. The biological treatment through enzymatic hydrolysis is a cheap and eco-friendly method for eliminating fatty substances from wastewater. In this context, lipases can be utilized for bio-treatment of wastewater in multifaceted industrial applications. To overcome the limitations in removing pollutants in the effluent, we aimed to identify a novel robust stable lipase (PersiLipase1) from metagenomic data of tannery wastewater for effective bio-degradation of the oily wastewater pollution. The lipase displayed remarkable thermostability and maintained over 81 % of its activity at 60 °C.After prolonged incubation for 35 days at 60°C, the PersiLipase1 still maintained 53.9 % of its activity. The enzyme also retained over 67 % of its activity in a wide range of pH (4.0 to 9.0). In addition, PersiLipase1 demonstrated considerable tolerance toward metal ions and organic solvents (e.g., retaining >70% activity after the addition of 100 mM of chemicals). Hydrolysis of olive oil and sheep fat by this enzyme showed 100 % efficiency. Furthermore, the PersiLipase1 proved to be efficient for biotreatment of oil and grease from tannery wastewater with the hydrolysis efficiency of 90.76 % ± 0.88. These results demonstrated that the metagenome-derived PersiLipase1 from tannery wastewater has a promising potential for the biodegradation and management of oily wastewater pollution.

Chromosome-level genome assembly of the Muscovy duck provides insight into fatty liver susceptibility.

The Muscovy duck (Cairina moschata) is an economically important poultry species, which is susceptible to fatty liver. Thus, the Muscovy duck may serve as an excellent candidate animal model of non-alcoholic fatty liver disease. However, the mechanisms underlying fatty liver development in this species are poorly understood. In this study, we report a chromosome-level genome assembly of the Muscovy duck, with a contig N50 of 11.8 Mb and scaffold N50 of 83.16 Mb. The susceptibility of Muscovy duck to fatty liver was mainly attributed to weak lipid catabolism capabilities (fatty acid ß-oxidation and lipolysis). Furthermore, conserved noncoding elements (CNEs) showing accelerated evolution contributed to fatty liver formation by down-regulating the expression of genes involved in hepatic lipid catabolism. We propose that the susceptibility of Muscovy duck to fatty liver is an evolutionary by-product. In conclusion, this study revealed the potential mechanisms underlying the susceptibility of Muscovy duck to fatty liver.

Markhor-derived Introgression of a Genomic Region Encompassing PAPSS2 Confers High-altitude Adaptability in Tibetan Goats.

Understanding the genetic mechanism of how animals adapt to extreme conditions is fundamental to determine the relationship between molecular evolution and changing environments. Goat is one of the first domesticated species and has evolved rapidly to adapt to diverse environments, including harsh high-altitude conditions with low temperature and poor oxygen supply but strong ultraviolet radiation. Here, we analyzed 331 genomes of domestic goats and wild caprid species living at varying altitudes (high > 3000 m above sea level and low < 1200 m), along with a reference-guided chromosome-scale assembly (contig-N50: 90.4 Mb) of a female Tibetan goat genome based on PacBio HiFi long reads, to dissect the genetic determinants underlying their adaptation to harsh conditions on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association studies (GWAS) revealed a genomic region harboring the 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) gene showing strong association with high-altitude adaptability (PGWAS = 3.62 × 10-25) in Tibetan goats. Transcriptomic data from 13 tissues revealed that PAPSS2 was implicated in hypoxia-related pathways in Tibetan goats. We further verified potential functional role of PAPSS2 in response to hypoxia in PAPSS2-deficient cells. Introgression analyses suggested that the PAPSS2 haplotype conferring the high-altitude adaptability in Tibetan goats originated from a recent hybridization between goats and a wild caprid species, the markhor (Capra falconeri). In conclusion, our results uncover a hitherto unknown contribution of PAPSS2 to high-altitude adaptability in Tibetan goats on QTP, following interspecific introgression and natural selection.

Genomic analyses of wild argali, domestic sheep, and their hybrids provide insights into chromosome evolution, phenotypic variation, and germplasm innovation.

Understanding the genetic mechanisms of phenotypic variation in hybrids between domestic animals and their wild relatives may aid germplasm innovation. Here, we report the high-quality genome assemblies of a male Pamir argali (O ammon polii, 2n = 56), a female Tibetan sheep (O aries, 2n = 54), and a male hybrid of Pamir argali and domestic sheep, and the high-throughput sequencing of 425 ovine animals, including the hybrids of argali and domestic sheep. We detected genomic synteny between Chromosome 2 of sheep and two acrocentric chromosomes of argali. We revealed consistent satellite repeats around the chromosome breakpoints, which could have resulted in chromosome fusion. We observed many more hybrids with karyotype 2n = 54 than with 2n = 55, which could be explained by the selfish centromeres, the possible decreased rate of normal/balanced sperm, and the increased incidence of early pregnancy loss in the aneuploid ewes or rams. We identified genes and variants associated with important morphological and production traits (e.g., body weight, cannon circumference, hip height, and tail length) that show significant variations. We revealed a strong selective signature at the mutation (c.334C > A, p.G112W) in TBXT and confirmed its association with tail length among sheep populations of wide geographic and genetic origins. We produced an intercross population of 110 F2 offspring with varied number of vertebrae and validated the causal mutation by whole-genome association analysis. We verified its function using CRISPR-Cas9 genome editing. Our results provide insights into chromosomal speciation and phenotypic evolution and a foundation of genetic variants for the breeding of sheep and other animals.

Functional and phylogenetic analyses of camel rumen microbiota associated with different lignocellulosic substrates.

Rumen microbiota facilitates nutrition through digestion of recalcitrant lignocellulosic substrates into energy-accessible nutrients and essential metabolites. Despite the high similarity in rumen microbiome structure, there might be distinct functional capabilities that enable different ruminant species to thrive on various lignocellulosic substrates as feed. Here, we applied genome-centric metagenomics to explore phylogenetic diversity, lignocellulose-degrading potential and fermentation metabolism of biofilm-forming microbiota colonizing 11 different plant substrates in the camel rumen. Diversity analysis revealed significant variations in the community of rumen microbiota colonizing different substrates in accordance with their varied physicochemical properties. Metagenome reconstruction recovered genome sequences of 590 bacterial isolates and one archaeal lineage belonging to 20 microbial phyla. A comparison to publicly available reference genomes and rumen metagenome-assembled genomes revealed that most isolates belonged to new species with no well-characterized representatives. We found that certain low abundant taxa, including members of Verrucomicrobiota, Planctomycetota and Fibrobacterota, possessed a disproportionately large number of carbohydrate active enzymes per Mb of genome, implying their high metabolic potential to contribute to the rumen function. In conclusion, we provided a detailed picture of the diversity and functional significance of rumen microbiota colonizing feeds of varying lignocellulose composition in the camel rumen. A detailed analysis of 591 metagenome-assembled genomes revealed a network of interconnected microbiota and highlighted the key roles of certain taxonomic clades in rumen function, including those with minimal genomes (e.g., Patescibacteria). The existence of a diverse array of gene clusters encoding for secondary metabolites unveiled the specific functions of these biomolecules in shaping community structure of rumen microbiota.

Whole-Genome Resequencing of Worldwide Wild and Domestic Sheep Elucidates Genetic Diversity, Introgression, and Agronomically Important Loci.

Domestic sheep and their wild relatives harbor substantial genetic variants that can form the backbone of molecular breeding, but their genome landscapes remain understudied. Here, we present a comprehensive genome resource for wild ovine species, landraces and improved breeds of domestic sheep, comprising high-coverage (∼16.10×) whole genomes of 810 samples from 7 wild species and 158 diverse domestic populations. We detected, in total, ∼121.2 million single nucleotide polymorphisms, ∼61 million of which are novel. Some display significant (P < 0.001) differences in frequency between wild and domestic species, or are private to continent-wide or individual sheep populations. Retained or introgressed wild gene variants in domestic populations have contributed to local adaptation, such as the variation in the HBB associated with plateau adaptation. We identified novel and previously reported targets of selection on morphological and agronomic traits such as stature, horn, tail configuration, and wool fineness. We explored the genetic basis of wool fineness and unveiled a novel mutation (chr25: T7,068,586C) in the 3'-UTR of IRF2BP2 as plausible causal variant for fleece fiber diameter. We reconstructed prehistorical migrations from the Near Eastern domestication center to South-and-Southeast Asia and found two main waves of migrations across the Eurasian Steppe and the Iranian Plateau in the Early and Late Bronze Ages. Our findings refine our understanding of genome variation as shaped by continental migrations, introgression, adaptation, and selection of sheep.

Rich maternal and paternal genetic diversity and divergent lineage composition in wild yak (Bos mutus).

Wild yak (Bos mutus) is a vulnerable bovine species on the Qinghai-Tibetan Plateau. So far, most studies on the molecular genetic diversity of wild yak have focused on autosomal and mtDNA variations based on the small number of samples. In this study, we analyzed 84 D-loop and 24 whole mitogenome sequences of wild yak to further comprehensively explore its maternal genetic diversity and lineage composition. Meanwhile, using six yak Y-specific polymorphic markers (i.e., SRY4, USP9Y, UTY19, AMELY3, OFD1Y10 and INRA189), we assessed the paternal genetic diversity and lineage composition based on eight wild yak. Our results showed that wild yak exhibited abundant maternal genetic diversity with haplotype diversities of 0.9621 ± 0.0078 and 0.9928 ± 0.0144 in the D-loop and whole mitogenome sequences, respectively. Maternal phylogenetic analysis of wild yak uncovered three defined lineages (mt-I, mt-II and mt-III). Similarly, profuse paternal genetic diversity was observed in wild yak with Y-haplotype diversity at 0.8214 ± 0.1007. Two Y-haplogroups (Y1 and Y2) and four Y-haplotypes (yH1-yH4) were identified in paternal phylogenetic analysis, indicating wild yak to be of two paternal lineages. The present study of genetic diversity and lineage composition of wild yak would provide useful information for the genetic resource conservation and utilization of this vulnerable wild species.

Rich maternal and paternal genetic diversity and divergent lineage composition in wild yak (Bos mutus).

Wild yak (Bos mutus) is a vulnerable bovine species on the Qinghai-Tibetan Plateau (QTP). So far, most studies on molecular genetic diversity of wild yak have focused on autosomal and mtDNA variations based on small number of samples. In this study, we analyzed 84 D-loop and 24 whole mitogenome sequences of wild yak to further comprehensively explore its maternal genetic diversity and lineage composition. Meanwhile, using six yak Y-specific polymorphic markers (i.e., SRY4, USP9Y, UTY19, AMELY3, OFD1Y10 and INRA189), we assessed the paternal genetic diversity and lineage composition based on eight wild yak. Our results showed that wild yak exhibited abundant maternal genetic diversity with haplotype diversities of 0.9621 ± 0.0078 and 0.9928 ± 0.0144 in the D-loop and whole mitogenome sequences, respectively. Maternal phylogenetic analysis of wild yak uncovered three defined lineages (mt-I, mt-II and mt-III). Similarly, profuse paternal genetic diversity was observed in wild yak with Y-haplotype diversity (Hd) at 0.8214 ± 0.1007. Two Y-haplogroups (Y1 and Y2) with four Y-haplotypes (yH1-yH4) were identified in paternal phylogenetic analysis, indicating wild yak to be of two paternal lineages. This study of genetic diversity and lineage composition of wild yak would provide useful information for the genetic resource conservation and utilization of this vulnerable wild species.

Genome Divergence and Dynamics in the Thin-Tailed Desert Sheep From Sudan.

With climate change bound to affect food and feed production, emphasis will shift to resilient and adapted indigenous livestock to sustain animal production. However, indigenous livestock comprise several varieties, strains and ecotypes whose genomes are poorly characterized. Here, we investigated genomic variation in an African thin-tailed Desert Sheep sampled in Sudan, using 600K genotype data generated from 92 individuals representing five ecotypes. We included data from 18 fat-tailed and 45 thin-tailed sheep from China, to investigate shared ancestry and perform comparative genomic analysis. We observed a clear genomic differentiation between the African thin-tailed Desert Sheep and the Chinese thin-tailed and fat-tailed sheep, suggesting a broad genetic structure between the fat-tailed and thin-tailed sheep in general, and that at least two autosomal gene pools comprise the genome profile of the thin-tailed sheep. Further analysis detected two distinct genetic clusters in both the African thin-tailed Desert Sheep and the Chinese thin-tailed sheep, suggesting a fine-scale and complex genome architecture in thin-tailed sheep. Selection signature analysis suggested differences in adaptation, production, reproduction and morphology likely underly the fine-scale genetic structure in the African thin-tailed Desert Sheep. This may need to be considered in designing breeding programs and genome-wide association studies.

Population differentiated copy number variation of Bos taurus, Bos indicus and their African hybrids.

BACKGROUND: CNV comprises a large proportion in cattle genome and is associated with various traits. However, there were few population-scale comparison studies on cattle CNV. RESULTS: Here, autosome-wide CNVs were called by read depth of NGS alignment result and copy number variation regions (CNVRs) defined from 102 Eurasian taurine (EAT) of 14 breeds, 28 Asian indicine (ASI) of 6 breeds, 22 African taurine (AFT) of 2 breeds, and 184 African humped cattle (AFH) of 17 breeds. The copy number of every CNVRs were compared between populations and CNVRs with population differentiated copy numbers were sorted out using the pairwise statistics VST and Kruskal-Wallis test. Three hundred sixty-two of CNVRs were significantly differentiated in both statistics and 313 genes were located on the population differentiated CNVRs. CONCLUSION: For some of these genes, the averages of copy numbers were also different between populations and these may be candidate genes under selection. These include olfactory receptors, pathogen-resistance, parasite-resistance, heat tolerance and productivity related genes. Furthermore, breed- and individual-level comparison was performed using the presence or copy number of the autosomal CNVRs. Our findings were based on identification of CNVs from short Illumina reads of 336 individuals and 39 breeds, which to our knowledge is the largest dataset for this type of analysis and revealed important CNVs that may play a role in cattle adaption to various environments.

Genomes reveal selective sweeps in kiang and donkey for high-altitude adaptation.

Over the last several hundred years, donkeys have adapted to high-altitude conditions on the Tibetan Plateau. Interestingly, the kiang, a closely related equid species, also inhabits this region. Previous reports have demonstrated the importance of specific genes and adaptive introgression in divergent lineages for adaptation to hypoxic conditions on the Tibetan Plateau. Here, we assessed whether donkeys and kiangs adapted to the Tibetan Plateau via the same or different biological pathways and whether adaptive introgression has occurred. We assembled a de novo genome from a kiang individual and analyzed the genomes of five kiangs and 93 donkeys (including 24 from the Tibetan Plateau). Our analyses suggested the existence of a strong hard selective sweep at the EPAS1 locus in kiangs. In Tibetan donkeys, however, another gene, i.e., EGLN1, was likely involved in their adaptation to high altitude. In addition, admixture analysis found no evidence for interspecific gene flow between kiangs and Tibetan donkeys. Our findings indicate that despite the short evolutionary time scale since the arrival of donkeys on the Tibetan Plateau, as well as the existence of a closely related species already adapted to hypoxia, Tibetan donkeys did not acquire adaptation via admixture but instead evolved adaptations via a different biological pathway.

Large-scale genomic analysis reveals the genetic cost of chicken domestication.

BACKGROUND: Species domestication is generally characterized by the exploitation of high-impact mutations through processes that involve complex shifting demographics of domesticated species. These include not only inbreeding and artificial selection that may lead to the emergence of evolutionary bottlenecks, but also post-divergence gene flow and introgression. Although domestication potentially affects the occurrence of both desired and undesired mutations, the way wild relatives of domesticated species evolve and how expensive the genetic cost underlying domestication is remain poorly understood. Here, we investigated the demographic history and genetic load of chicken domestication. RESULTS: We analyzed a dataset comprising over 800 whole genomes from both indigenous chickens and wild jungle fowls. We show that despite having a higher genetic diversity than their wild counterparts (average π, 0.00326 vs. 0.00316), the red jungle fowls, the present-day domestic chickens experienced a dramatic population size decline during their early domestication. Our analyses suggest that the concomitant bottleneck induced 2.95% more deleterious mutations across chicken genomes compared with red jungle fowls, supporting the "cost of domestication" hypothesis. Particularly, we find that 62.4% of deleterious SNPs in domestic chickens are maintained in heterozygous states and masked as recessive alleles, challenging the power of modern breeding programs to effectively eliminate these genetic loads. Finally, we suggest that positive selection decreases the incidence but increases the frequency of deleterious SNPs in domestic chicken genomes. CONCLUSION: This study reveals a new landscape of demographic history and genomic changes associated with chicken domestication and provides insight into the evolutionary genomic profiles of domesticated animals managed under modern human selection.

Mitochondrial genomes from modern and ancient Turano-Mongolian cattle reveal an ancient diversity of taurine maternal lineages in East Asia.

Turano-Mongolian cattle are a group of taurine cattle from Northern and Eastern Asia with distinct morphological traits, which are known for their ability to tolerate harsh environments, such as the Asian steppe and the Tibetan plateau. Through the analysis of 170 mitogenomes from ten modern breeds, two sub-lineages within T3 (T3119 and T3055) were identified as specific of Turano-Mongolian cattle. These two T3 sub-lineages, together with the previously identified T4, were also present in six Neolithic samples, dated to ~3900 years BP, which might represent the earliest domestic taurine stocks from Southwest Asia. The rare haplogroup Q, found in three Tibetan cattle, testifies for the legacy of ancient migrations from Southwest Asia and suggests that the isolated Tibetan Plateau preserved unique prehistoric genetic resources. These findings confirm the geographic substructure of Turano-Mongolian cattle breeds, which have been shaped by ancient migrations and geographic barriers.