A large‐scale metagenomic study for enzyme profiles using the focused identification of the NGS‐based definitive enzyme research (FINDER) strategy

Excavating the molecular details of many diverse enzymes from metagenomes remains challenging in agriculture, food, health, and environmental fields. We present a versatile method that accelerates metabolic enzyme discovery for highly selective gene capture in metagenomes using next‐generation sequencing. Culture‐independent enzyme mining of environmental DNA is based on a set of short identifying degenerate sequences specific for a wide range of enzyme superfamilies, followed by multiplexed DNA barcode sequencing. A strategy of ‘focused identification of next‐generation sequencing‐based definitive enzyme research’ enabled us to generate targeted enzyme datasets from metagenomes, resulting in minimal hands‐on obtention of high‐throughput biological diversity and potential function profiles, without being time‐consuming. This method also provided a targeted inventory of predicted proteins and molecular features of metabolic activities from several metagenomic samples. We suggest that the efficiency and sensitivity of this method will accelerate the decryption of microbial diversity and the signature of proteins and their metabolism from environmental samples.

[1]  Gavin M Douglas,et al.  PICRUSt2 for prediction of metagenome functions , 2020, Nature Biotechnology.

[2]  T. Thomas,et al.  Tax4Fun2: prediction of habitat-specific functional profiles and functional redundancy based on 16S rRNA gene sequences , 2020, Environmental Microbiome.

[3]  M. Gerstein,et al.  Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis , 2019, Nature Communications.

[4]  Sergey Koren,et al.  Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome , 2019, Nature Biotechnology.

[5]  S. Lee,et al.  antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline , 2019, Nucleic Acids Res..

[6]  H. Rehrauer,et al.  Long fragments achieve lower base quality in Illumina paired-end sequencing , 2019, Scientific Reports.

[7]  Christian Ebeling,et al.  BacDive in 2019: bacterial phenotypic data for High-throughput biodiversity analysis , 2018, Nucleic Acids Res..

[8]  M. Engqvist.  Correlating enzyme annotations with a large set of microbial growth temperatures reveals metabolic adaptations to growth at diverse temperatures , 2018, BMC Microbiology.

[9]  Yun Zhao,et al.  Metagenomic analysis of microbial community and function involved in cd-contaminated soil , 2018, BMC Microbiology.

[10]  F. Marinelli,et al.  Metagenomics: novel enzymes from non‐culturable microbes , 2017, FEMS microbiology letters.

[11]  J. Ronholm,et al.  Metagenomics: The Next Culture-Independent Game Changer , 2017, Front. Microbiol..

[12]  Sang-Jae Lee,et al.  Diversity of Extremely Halophilic Archaeal and Bacterial Communities from Commercial Salts , 2017, Front. Microbiol..

[13]  Brian L. Schmidt,et al.  Piphillin: Improved Prediction of Metagenomic Content by Direct Inference from Human Microbiomes , 2016, PloS one.

[14]  K. Pollard,et al.  Toward Accurate and Quantitative Comparative Metagenomics , 2016, Cell.

[15]  Shuichi Shoji,et al.  Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform , 2016, Scientific Reports.

[16]  C. Schadt,et al.  PanFP: pangenome-based functional profiles for microbial communities , 2015, BMC Research Notes.

[17]  Jason Stenson,et al.  Humans differ in their personal microbial cloud , 2015, PeerJ.

[18]  J. Prosser.  Dispersing misconceptions and identifying opportunities for the use of 'omics' in soil microbial ecology , 2015, Nature Reviews Microbiology.

[19]  G. Potocki-Veronese,et al.  Discovery of new protein families and functions: new challenges in functional metagenomics for biotechnologies and microbial ecology , 2015, Front. Microbiol..

[20]  S. Green,et al.  Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches , 2015, PloS one.

[21]  T. Sharpton.  An introduction to the analysis of shotgun metagenomic data , 2014, Front. Plant Sci..

[22]  Susumu Goto,et al.  Data, information, knowledge and principle: back to metabolism in KEGG , 2013, Nucleic Acids Res..

[23]  Jiajie Zhang,et al.  PEAR: a fast and accurate Illumina Paired-End reAd mergeR , 2013, Bioinform..

[24]  Na-Ri Shin,et al.  An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice , 2013, Gut.

[25]  A. Klindworth,et al.  Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies , 2012, Nucleic acids research.

[26]  William A. Walters,et al.  Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms , 2012, The ISME Journal.

[27]  J. Chun,et al.  Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. , 2012, International journal of systematic and evolutionary microbiology.

[28]  Patrick D. Schloss,et al.  Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies , 2011, PloS one.

[29]  S. Tringe,et al.  Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases , 2011, Biotechnology for biofuels.

[30]  S. Tringe,et al.  Metagenomic Discovery of Biomass-Degrading Genes and Genomes from Cow Rumen , 2011, Science.

[31]  R. Daniel,et al.  Metagenomic Analyses: Past and Future Trends , 2010, Applied and Environmental Microbiology.

[32]  Robert C. Edgar,et al.  Search and clustering orders of magnitude faster than BLAST , 2010, Bioinform..

[33]  P. Bork,et al.  A human gut microbial gene catalogue established by metagenomic sequencing , 2010, Nature.

[34]  Robert G. Beiko,et al.  Identifying biologically relevant differences between metagenomic communities , 2010, Bioinform..

[35]  John C. Wooley,et al.  A Primer on Metagenomics , 2010, PLoS Comput. Biol..

[36]  Natalia N. Ivanova,et al.  A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea , 2009, Nature.

[37]  T. Uchiyama,et al.  Functional metagenomics for enzyme discovery: challenges to efficient screening. , 2009, Current opinion in biotechnology.

[38]  James R. Cole,et al.  Gene-targeted-metagenomics reveals extensive diversity of aromatic dioxygenase genes in the environment , 2009, The ISME Journal.

[39]  Safiyh Taghavi,et al.  Bioprospecting metagenomes: glycosyl hydrolases for converting biomass , 2009, Biotechnology for biofuels.

[40]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[41]  Richard Boyce,et al.  iCODEHOP: a new interactive program for designing COnsensus-DEgenerate Hybrid Oligonucleotide Primers from multiply aligned protein sequences , 2009, Nucleic Acids Res..

[42]  Alla Lapidus,et al.  A Bioinformatician's Guide to Metagenomics , 2008, Microbiology and Molecular Biology Reviews.

[43]  Susan M. Huse,et al.  Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing , 2008, PLoS genetics.

[44]  K. Timmis,et al.  Metagenomics for Mining New Genetic Resources of Microbial Communities , 2008, Journal of Molecular Microbiology and Biotechnology.

[45]  M. Ferrer,et al.  Recent trends in industrial microbiology. , 2008, Current opinion in microbiology.

[46]  Colin J Ingham,et al.  The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms , 2007, Proceedings of the National Academy of Sciences.

[47]  Benjamin J. Raphael,et al.  The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families , 2007, PLoS biology.

[48]  Manuel Ferrer,et al.  Environmental biocatalysis: from remediation with enzymes to novel green processes. , 2006, Trends in biotechnology.

[49]  Joachim Jose,et al.  Autodisplay: efficient bacterial surface display of recombinant proteins , 2006, Applied Microbiology and Biotechnology.

[50]  Jan E Schnitzer,et al.  Screening phage display libraries for organ-specific vascular immunotargeting in vivo. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Edward M. Rubin,et al.  Metagenomics: DNA sequencing of environmental samples , 2005, Nature Reviews Genetics.

[52]  F. Arnold,et al.  Evolving strategies for enzyme engineering. , 2005, Current opinion in structural biology.

[53]  R. Daniel.  The metagenomics of soil , 2005, Nature Reviews Microbiology.

[54]  Andrew D Griffiths,et al.  High-throughput screens and selections of enzyme-encoding genes. , 2005, Current opinion in chemical biology.

[55]  J. Handelsman.  Metagenomics: Application of Genomics to Uncultured Microorganisms , 2004, Microbiology and Molecular Biology Reviews.

[56]  Andreas Plückthun,et al.  In-vitro protein evolution by ribosome display and mRNA display. , 2004, Journal of immunological methods.

[57]  O. White,et al.  Environmental Genome Shotgun Sequencing of the Sargasso Sea , 2004, Science.

[58]  S. Giovannoni,et al.  The uncultured microbial majority. , 2003, Annual review of microbiology.

[59]  P. Salamon,et al.  Metagenomic Analyses of an Uncultured Viral Community from Human Feces , 2003, Journal of bacteriology.

[60]  J. Sebat,et al.  Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library , 2003, Applied and Environmental Microbiology.

[61]  K. Katoh,et al.  MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. , 2002, Nucleic acids research.

[62]  P. Schultz,et al.  Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Eric T. Boder,et al.  Yeast surface display for screening combinatorial polypeptide libraries , 1997, Nature Biotechnology.

[64]  W. S. Rapson.  Mining, Extraction and Refining of Gold , 1992 .

[65]  Jae-Ho Shin,et al.  An improved effective microorganism (EM) soil ball-making method for water quality restoration , 2015, Environmental Science and Pollution Research.

[66]  P. Golyshin,et al.  Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes. , 2015, Advances in experimental medicine and biology.

[67]  P. Hugenholtz,et al.  Microbiology: Metagenomics , 2008, Nature.