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Zhadyra S, Tao F, Xu P. Exploring the Microbiome and Functional Metabolism of Fermented Camel Milk (Shubat) Using Metagenomics. Foods 2025; 14:1102. [PMID: 40238218 PMCID: PMC11989172 DOI: 10.3390/foods14071102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2025] [Revised: 03/19/2025] [Accepted: 03/20/2025] [Indexed: 04/18/2025] Open
Abstract
Shubat is a traditional fermented camel milk drink that originated in Central Asia, with especially deep cultural roots in Kazakhstan. However, systematic studies on the microbial ecology and functional genes of Shubat remain scarce. As a distinctive fer-mented food, its microbial diversity and functional properties have not been fully ex-plored. This study investigates the microbial diversity and functional potential of Shubat by using advanced metagenomic techniques. Its microbial community is mainly composed of bacteria (96.6%), with Lactobacillus, Lactococcus, and Streptococcus being the dominant genera. Functional annotations through EggNOG, KEGG, and CAZy databases highlighted the metabolic versatility of Shubat's microbiota. Key pathways included amino acid and carbohydrate metabolism, vitamin biosynthesis, and central carbon metabolism, emphasizing their roles in fermentation and nutritional enhancement. The identification of various enzymes related to chemical synthesis further emphasizes the contribution of the microbiota to Shubat's unique flavor and texture. This study not only provides an important basis for the scientific understanding of Shubat but also expands the application possibilities of fermented food in the field of health and nutrition and confers modern value and significance to traditional food. This integration of science and tradition has not only facilitated the development of food microbiology but also paved new pathways for the global dissemination of traditional foods and the development of functional foods.
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Affiliation(s)
- Sagyman Zhadyra
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; (S.Z.); (P.X.)
- Laboratory of Biotechnology, Research Institute for Biotechnology and Ecology, Zhetysu University, Taldykorgan 040009, Kazakhstan
| | - Fei Tao
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; (S.Z.); (P.X.)
| | - Ping Xu
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; (S.Z.); (P.X.)
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2
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Chhiba V, Pillay P, Mtimka S, Moonsamy G, Kwezi L, Pooe OJ, Tsekoa TL. South Africa's indigenous microbial diversity for industrial applications: A review of the current status and opportunities. Heliyon 2023; 9:e16723. [PMID: 37484259 PMCID: PMC10360602 DOI: 10.1016/j.heliyon.2023.e16723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Revised: 05/05/2023] [Accepted: 05/25/2023] [Indexed: 07/25/2023] Open
Abstract
The unique metagenomic, metaviromic libraries and indigenous micro diversity within Southern Africa have the potential for global beneficiation in academia and industry. Microorganisms that flourish at high temperatures, adverse pH conditions, and high salinity are likely to have enzyme systems that function efficiently under those conditions. These attributes afford researchers and industries alternative approaches that could replace existing chemical processes. Thus, a better understanding of African microbial/genetic diversity is crucial for the development of "greener" industries. A concerted drive to exploit the potential locked in biological resources has been previously seen with companies such as Diversa Incorporated and Verenium (Badische Anilin-und SodaFabrik-BASF) both building business models that pioneered the production of high-performance specialty enzymes for a variety of different industrial applications. The market potential and accompanying industry offerings have not been fully exploited in South Africa, nor in Africa at large. Utilization of the continent's indigenous microbial repositories could create long-lasting, sustainable growth in various production sectors, providing economic growth in resource-poor regions. By bolstering local manufacture of high-value bio-based products, scientific and engineering discoveries have the potential to generate new industries which in turn would provide employment avenues for many skilled and unskilled laborers. The positive implications of this could play a role in altering the face of business markets on the continent from costly import-driven markets to income-generating export markets. This review focuses on identifying microbially diverse areas located in South Africa while providing a profile for all associated microbial/genetically derived libraries in this country. A comprehensive list of all the relevant researchers and potential key players is presented, mapping out existing research networks for the facilitation of collaboration. The overall aim of this review is to facilitate a coordinated journey of exploration, one which will hopefully realize the value that South Africa's microbial diversity has to offer.
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Affiliation(s)
- Varsha Chhiba
- Future Production: Chemicals Cluster, Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa
| | - Priyen Pillay
- Future Production: Chemicals Cluster, Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa
| | - Sibongile Mtimka
- Future Production: Chemicals Cluster, Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa
- School of Life Sciences, Discipline of Biochemistry, University of KwaZulu-Natal, Durban, South Africa
| | - Ghaneshree Moonsamy
- Future Production: Chemicals Cluster, Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa
| | - Lusisizwe Kwezi
- Future Production: Chemicals Cluster, Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa
| | - Ofentse J. Pooe
- School of Life Sciences, Discipline of Biochemistry, University of KwaZulu-Natal, Durban, South Africa
| | - Tsepo L. Tsekoa
- Future Production: Chemicals Cluster, Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa
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3
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Sartaj K, Patel A, Matsakas L, Prasad R. Unravelling Metagenomics Approach for Microbial Biofuel Production. Genes (Basel) 2022; 13:1942. [PMID: 36360179 PMCID: PMC9689425 DOI: 10.3390/genes13111942] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Revised: 10/18/2022] [Accepted: 10/21/2022] [Indexed: 09/29/2023] Open
Abstract
Renewable biofuels, such as biodiesel, bioethanol, and biobutanol, serve as long-term solutions to fossil fuel depletion. A sustainable approach feedstock for their production is plant biomass, which is degraded to sugars with the aid of microbes-derived enzymes, followed by microbial conversion of those sugars to biofuels. Considering their global demand, additional efforts have been made for their large-scale production, which is ultimately leading breakthrough research in biomass energy. Metagenomics is a powerful tool allowing for functional gene analysis and new enzyme discovery. Thus, the present article summarizes the revolutionary advances of metagenomics in the biofuel industry and enlightens the importance of unexplored habitats for novel gene or enzyme mining. Moreover, it also accentuates metagenomics potentials to explore uncultivable microbiomes as well as enzymes associated with them.
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Affiliation(s)
- Km Sartaj
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India
| | - Alok Patel
- Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental, and Natural Resources Engineering, Luleå University of Technology, SE-971 87 Luleå, Sweden
| | - Leonidas Matsakas
- Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental, and Natural Resources Engineering, Luleå University of Technology, SE-971 87 Luleå, Sweden
| | - Ramasare Prasad
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India
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4
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Tsudome M, Tachioka M, Miyazaki M, Uchimura K, Tsuda M, Takaki Y, Deguchi S. An ultrasensitive nanofiber-based assay for enzymatic hydrolysis and deep-sea microbial degradation of cellulose. iScience 2022; 25:104732. [PMID: 36039358 PMCID: PMC9418596 DOI: 10.1016/j.isci.2022.104732] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2022] [Revised: 05/10/2022] [Accepted: 07/02/2022] [Indexed: 11/18/2022] Open
Affiliation(s)
- Mikiko Tsudome
- Research Center for Bioscience and Nanoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
| | - Mikako Tachioka
- Research Center for Bioscience and Nanoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
| | - Masayuki Miyazaki
- SUGAR Program, JAMSTEC, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
| | - Kohsuke Uchimura
- Research Center for Bioscience and Nanoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
| | - Miwako Tsuda
- SUGAR Program, JAMSTEC, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
| | - Yoshihiro Takaki
- SUGAR Program, JAMSTEC, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
| | - Shigeru Deguchi
- Research Center for Bioscience and Nanoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
- Corresponding author
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Lu M, Schneider D, Daniel R. Metagenomic Screening for Lipolytic Genes Reveals an Ecology-Clustered Distribution Pattern. Front Microbiol 2022; 13:851969. [PMID: 35756004 PMCID: PMC9226776 DOI: 10.3389/fmicb.2022.851969] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2022] [Accepted: 04/28/2022] [Indexed: 12/02/2022] Open
Abstract
Lipolytic enzymes are one of the most important enzyme types for application in various industrial processes. Despite the continuously increasing demand, only a small portion of the so far encountered lipolytic enzymes exhibit adequate stability and activities for biotechnological applications. To explore novel and/or extremophilic lipolytic enzymes, microbial consortia in two composts at thermophilic stage were analyzed using function-driven and sequence-based metagenomic approaches. Analysis of community composition by amplicon-based 16S rRNA genes and transcripts, and direct metagenome sequencing revealed that the communities of the compost samples were dominated by members of the phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Chloroflexi. Function-driven screening of the metagenomic libraries constructed from the two samples yielded 115 unique lipolytic enzymes. The family assignment of these enzymes was conducted by analyzing the phylogenetic relationship and generation of a protein sequence similarity network according to an integrated classification system. The sequence-based screening was performed by using a newly developed database, containing a set of profile Hidden Markov models, highly sensitive and specific for detection of lipolytic enzymes. By comparing the lipolytic enzymes identified through both approaches, we demonstrated that the activity-directed complements sequence-based detection, and vice versa. The sequence-based comparative analysis of lipolytic genes regarding diversity, function and taxonomic origin derived from 175 metagenomes indicated significant differences between habitats. Analysis of the prevalent and distinct microbial groups providing the lipolytic genes revealed characteristic patterns and groups driven by ecological factors. The here presented data suggests that the diversity and distribution of lipolytic genes in metagenomes of various habitats are largely constrained by ecological factors.
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Affiliation(s)
| | | | - Rolf Daniel
- Department of Genomic and Applied Microbiology, Institute of Microbiology and Genetics, Georg August University of Göttingen, Göttingen, Germany
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Li J, Gu X, Zhang Q, Fu L, Tan J, Zhao L. Biochemical Characterization of a Carrageenase, Car1383, Derived From Associated Bacteria of Antarctic Macroalgae. Front Microbiol 2022; 13:851182. [PMID: 35432236 PMCID: PMC9009511 DOI: 10.3389/fmicb.2022.851182] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2022] [Accepted: 03/09/2022] [Indexed: 11/13/2022] Open
Abstract
A carrageenase gene, car1383, was obtained from the metagenome of Antarctic macroalgae-associated bacteria. The amino acid sequence of its product showed up to 33% similarity with other carrageenases and contained a GH16-family motif. The recombinant Car1383 was heterologously expressed in Eschericia coli and exhibited maximal activity at 50°C and pH 6.0, with a Km of 6.51 mg/ml and a Vmax of 55.77 U/mg. Its activity was enhanced by some cations (Na+, K+, and Fe2+), but inhibited or inactivated by others (Sr2+, Ca2+, Ni2+, Ba2+, Mn2+, Cu2+, Fe3+, and Mg2+). Car1383 degraded carrageenan into neocarrabiose and neocarratetraose. Site-directed mutagenesis indicated that putative active sites, E190 and E195, conserved sites, W183 and G255, play important roles in Car1383 activity. This study provides a new candidate for the industrial preparation of bioactive algal oligosaccharides.
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Affiliation(s)
- Jiang Li
- Key Laboratory of Ecological Environment Science and Technology, First Institute of Oceanography, Ministry of Natural Resources, Qingdao, China
- *Correspondence: Jiang Li,
| | - Xiaoqian Gu
- CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
| | - Qian Zhang
- Key Laboratory of Ecological Environment Science and Technology, First Institute of Oceanography, Ministry of Natural Resources, Qingdao, China
| | - Liping Fu
- Key Laboratory of Ecological Environment Science and Technology, First Institute of Oceanography, Ministry of Natural Resources, Qingdao, China
| | - Jiaojiao Tan
- CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
| | - Luying Zhao
- Key Laboratory of Ecological Environment Science and Technology, First Institute of Oceanography, Ministry of Natural Resources, Qingdao, China
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Singh N, Singh V, Singh MP. Microbial degradation of lignocellulosic biomass for bioenergy production: A metagenomic-based approach. BIOCATAL BIOTRANSFOR 2022. [DOI: 10.1080/10242422.2022.2056451] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- Nidhi Singh
- Centre of Bioinformatics, University of Allahabad, Allahabad, India
- School of Biochemical Engineering, IIT (BHU), Varanasi, India
| | - Veer Singh
- Centre of Biotechnology, University of Allahabad, Allahabad, India
| | - Mohan P. Singh
- Centre of Biotechnology, University of Allahabad, Allahabad, India
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Ajeje SB, Hu Y, Song G, Peter SB, Afful RG, Sun F, Asadollahi MA, Amiri H, Abdulkhani A, Sun H. Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective. Front Bioeng Biotechnol 2021; 9:794304. [PMID: 34976981 PMCID: PMC8715034 DOI: 10.3389/fbioe.2021.794304] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 11/02/2021] [Indexed: 12/13/2022] Open
Abstract
The bioconversion of lignocellulose into monosaccharides is critical for ensuring the continual manufacturing of biofuels and value-added bioproducts. Enzymatic degradation, which has a high yield, low energy consumption, and enhanced selectivity, could be the most efficient and environmentally friendly technique for converting complex lignocellulose polymers to fermentable monosaccharides, and it is expected to make cellulases and xylanases the most demanded industrial enzymes. The widespread nature of thermophilic microorganisms allows them to proliferate on a variety of substrates and release substantial quantities of cellulases and xylanases, which makes them a great source of thermostable enzymes. The most significant breakthrough of lignocellulolytic enzymes lies in lignocellulose-deconstruction by enzymatic depolymerization of holocellulose into simple monosaccharides. However, commercially valuable thermostable cellulases and xylanases are challenging to produce in high enough quantities. Thus, the present review aims at giving an overview of the most recent thermostable cellulases and xylanases isolated from thermophilic and hyperthermophilic microbes. The emphasis is on recent advancements in manufacturing these enzymes in other mesophilic host and enhancement of catalytic activity as well as thermostability of thermophilic cellulases and xylanases, using genetic engineering as a promising and efficient technology for its economic production. Additionally, the biotechnological applications of thermostable cellulases and xylanases of thermophiles were also discussed.
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Affiliation(s)
- Samaila Boyi Ajeje
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Yun Hu
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Guojie Song
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Sunday Bulus Peter
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Richmond Godwin Afful
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Fubao Sun
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Mohammad Ali Asadollahi
- Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
| | - Hamid Amiri
- Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
| | - Ali Abdulkhani
- Department of Wood and Paper Science and Technology, Faculty of Natural Resources, University of Tehran, Karaj, Iran
| | - Haiyan Sun
- Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China
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Degradation activity of fungal communities on avocado peel (Persea americana Mill.) in a solid-state process: mycobiota successions and trophic guild shifts. Arch Microbiol 2021; 204:2. [PMID: 34870719 DOI: 10.1007/s00203-021-02600-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 11/19/2021] [Accepted: 11/21/2021] [Indexed: 10/19/2022]
Abstract
To explore the capability of soil mycobiota to degrade avocado peel waste and identify relevant successions and trophic guild shifts, fungal communities from three environments with different land uses were evaluated in a solid-state process. Soil samples used as inoculum were collected from a pristine mature tropical forest, a traditionally managed Mayan land, and an intensively managed monospecific avocado plantation. Soil-substrate mixes were evaluated for 52 weeks to evaluate organic matter decay and the carbon-to-nitrogen ratio. Amplicon-based high-throughput sequencing from internally transcribed spacer (ITS) analysis revealed significant differences in fungal communities widely dominated by Fusarium sp. and Clonostachys sp.; however, less represented taxa showed relevant shifts concomitantly with organic matter content drops. Trophic guild assignment revealed different behaviors in fungal communities between treatments over the 52 weeks, suggesting distinct preconditioning of fungal communities in these environments. Overall, the results lead to the identification of promising degradation moments and inoculum sources for further consortia enrichment or bioprospecting efforts.
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10
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Thermostable cellulose saccharifying microbial enzymes: Characteristics, recent advances and biotechnological applications. Int J Biol Macromol 2021; 188:226-244. [PMID: 34371052 DOI: 10.1016/j.ijbiomac.2021.08.024] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 07/19/2021] [Accepted: 08/03/2021] [Indexed: 12/12/2022]
Abstract
Cellulases play a promising role in the bioconversion of renewable lignocellulosic biomass into fermentable sugars which are subsequently fermented to biofuels and other value-added chemicals. Besides biofuel industries, they are also in huge demand in textile, detergent, and paper and pulp industries. Low titres of cellulase production and processing are the main issues that contribute to high enzyme cost. The success of ethanol-based biorefinery depends on high production titres and the catalytic efficiency of cellulases functional at elevated temperatures with acid/alkali tolerance and the low cost. In view of their wider application in various industrial processes, stable cellulases that are active at elevated temperatures in the acidic-alkaline pH ranges, and organic solvents and salt tolerance would be useful. This review provides a recent update on the advances made in thermostable cellulases. Developments in their sources, characteristics and mechanisms are updated. Various methods such as rational design, directed evolution, synthetic & system biology and immobilization techniques adopted in evolving cellulases with ameliorated thermostability and characteristics are also discussed. The wide range of applications of thermostable cellulases in various industrial sectors is described.
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11
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Sung JY, Lee YJ, Cho YJ, Shin MN, Lee SJ, Lee HS, Koh H, Bae JW, Shin JH, Kim HJ, Lee DW. A large-scale metagenomic study for enzyme profiles using the focused identification of the NGS-based definitive enzyme research (FINDER) strategy. Biotechnol Bioeng 2021; 118:4360-4374. [PMID: 34309016 DOI: 10.1002/bit.27904] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2021] [Revised: 04/23/2021] [Accepted: 07/23/2021] [Indexed: 11/09/2022]
Abstract
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.
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Affiliation(s)
- Jae-Yoon Sung
- Department of Biotechnology, Yonsei University, Seoul, South Korea
| | - Yong-Jik Lee
- Department of Bio-Cosmetics, Seowon University, Chung-Ju, South Korea
| | - Yong-Joon Cho
- Department of Biological Sciences and Research Institute of Basic Sciences, Seoul National University, Seoul, South Korea
| | - Myeong-Na Shin
- Department of Central Area Crop Science, NICS, RDA, Suwon, South Korea
| | - Sang-Jae Lee
- Major in Food Biotechnology, Silla University, Busan, South Korea
| | - Han-Seung Lee
- Major in Food Biotechnology, Silla University, Busan, South Korea
| | - Hong Koh
- Department of Pediatrics, Yonsei University, Seoul, South Korea
| | - Jin-Woo Bae
- Department of Biology, Kyung Hee University, Seoul, South Korea
| | - Jae-Ho Shin
- Department of Applied Biosciences, Kyungpook National University, Daegu, South Korea
| | - Hyun Jung Kim
- Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas, USA
| | - Dong-Woo Lee
- Department of Biotechnology, Yonsei University, Seoul, South Korea
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12
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New Family of Carbohydrate-Binding Modules Defined by a Galactosyl-Binding Protein Module from a Cellvibrio japonicus Endo-Xyloglucanase. Appl Environ Microbiol 2021; 87:e0263420. [PMID: 33355108 DOI: 10.1128/aem.02634-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, for example, by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is a valuable source for CAZyme and CBM discovery and characterization due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (X181), and a glycoside hydrolase family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase (M. A. Attia, C. E. Nelson, W. A. Offen, N. Jain, et al., Biotechnol Biofuels 11:45, 2018, https://doi.org/10.1186/s13068-018-1039-6). In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest products of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes. IMPORTANCE This study reveals carbohydrate-binding module family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the carbohydrate-active enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.
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Pabbathi NPP, Velidandi A, Tavarna T, Gupta S, Raj RS, Gandam PK, Baadhe RR. Role of metagenomics in prospecting novel endoglucanases, accentuating functional metagenomics approach in second-generation biofuel production: a review. BIOMASS CONVERSION AND BIOREFINERY 2021; 13:1371-1398. [PMID: 33437563 PMCID: PMC7790359 DOI: 10.1007/s13399-020-01186-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 10/30/2020] [Accepted: 12/01/2020] [Indexed: 05/02/2023]
Abstract
As the fossil fuel reserves are depleting rapidly, there is a need for alternate fuels to meet the day to day mounting energy demands. As fossil fuel started depleting, a quest for alternate forms of fuel was initiated and biofuel is one of its promising outcomes. First-generation biofuels are made from edible sources like vegetable oils, starch, and sugars. Second-generation biofuels (SGB) are derived from lignocellulosic crops and the third-generation involves algae for biofuel production. Technical challenges in the production of SGB are hampering its commercialization. Advanced molecular technologies like metagenomics can help in the discovery of novel lignocellulosic biomass-degrading enzymes for commercialization and industrial production of SGB. This review discusses the metagenomic outcomes to enlighten the importance of unexplored habitats for novel cellulolytic gene mining. It also emphasizes the potential of different metagenomic approaches to explore the uncultivable cellulose-degrading microbiome as well as cellulolytic enzymes associated with them. This review also includes effective pre-treatment technology and consolidated bioprocessing for efficient biofuel production.
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Affiliation(s)
- Ninian Prem Prashanth Pabbathi
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
| | - Aditya Velidandi
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
| | - Tanvi Tavarna
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
| | - Shreyash Gupta
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
| | - Ram Sarvesh Raj
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
| | - Pradeep Kumar Gandam
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
| | - Rama Raju Baadhe
- Integrated Biorefinery Research Lab, Department of Biotechnology, National Institute of Technology, Warangal, Telangana 506004 India
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Chettri D, Verma AK, Verma AK. Innovations in CAZyme gene diversity and its modification for biorefinery applications. BIOTECHNOLOGY REPORTS (AMSTERDAM, NETHERLANDS) 2020; 28:e00525. [PMID: 32963975 PMCID: PMC7490808 DOI: 10.1016/j.btre.2020.e00525] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 08/04/2020] [Accepted: 08/30/2020] [Indexed: 02/07/2023]
Abstract
For sustainable growth, concept of biorefineries as recourse to the "fossil derived" energy source is important. Here, the Carbohydrate Active enZymes (CAZymes) play decisive role in generation of biofuels and related sugar-based products utilizing lignocellulose as a carbon source. Given their industrial significance, extensive studies on the evolution of CAZymes have been carried out. Various bacterial and fungal organisms have been scrutinized for the development of CAZymes, where advance techniques for strain enhancement such as CRISPR and analysis of specific expression systems have been deployed. Specific Omic-based techniques along with protein engineering have been adopted to unearth novel CAZymes and improve applicability of existing enzymes. In-Silico computational research and functional annotation of new CAZymes to synergy experiments are being carried out to devise cocktails of enzymes for use in biorefineries. Thus, with the establishment of these technologies, increased diversity of CAZymes with broad span of functions and applications is seen.
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15
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Hinsu AT, Patel AB, Pandit RJ, Thakkar JR, Shah RK, Jakhesara SJ, Koringa PG, Joshi CG. MetaRNAseq analysis of surti buffalo rumen content reveals that transcriptionally active microorganisms need not be abundant. Mol Biol Rep 2020; 47:5101-5114. [PMID: 32557173 DOI: 10.1007/s11033-020-05581-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Accepted: 06/10/2020] [Indexed: 11/26/2022]
Abstract
The present study describes rumen microbiota composition and their functional profiles in Indian Surti buffaloes by metagenomic (MG) and metatranscriptomic (MT) approaches. The study compares samples from buffaloes fed three different proportion of roughages; green and dry type of roughage; and different rumen liquor fractions. Irrespective of sample, Bacteroidetes and Firmicutes were the most predominant bacterial phyla, followed by Proteobacteria, Fibrobacteres and Actinobacteria while, Prevotella, Bacteroides, Ruminococcus and Clostridium were the most abundant genera. Different proportions of taxa were observed in both MG and MT approaches indicating the differences in organisms present and organisms active in the rumen. Higher proportions of fungal taxa were observed in MT while important organisms like Fibrobacter and Butyrivibrio and abundant organisms like Bacteroides and Prevotella were underrepresented in MT data. Functionally, higher proportions of genes involved in Carbohydrate metabolism, Amino acid metabolism and Translation were observed in both data. Genes involved in Metabolism were observed to be underrepresented in MT data while, those involved in Genetic information processing were overrepresented in MT data. Further, genes involved in Carbohydrate metabolism were overexpressed compared to genes involved in Amino acid metabolism in MT data compared to MG data which had higher proportion of genes involved in Amino acid metabolism than Carbohydrate metabolism. In all significant differences were observed between both approaches, different fractions of rumen liquor (liquid and solid) and different proportions of roughage in diet.
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Affiliation(s)
- Ankit T Hinsu
- Department of Animal Genetics & Breeding, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Avani B Patel
- Department of Animal Genetics & Breeding, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Ramesh J Pandit
- Department of Animal Biotechnology, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Jalpa R Thakkar
- Department of Animal Biotechnology, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Ravi K Shah
- Department of Animal Biotechnology, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Subhash J Jakhesara
- Department of Animal Biotechnology, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Prakash G Koringa
- Department of Animal Biotechnology, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India
| | - Chaitanya G Joshi
- Department of Animal Biotechnology, College of Veterinary Sciences & A.H, Anand Agricultural University, Anand, Gujarat, India.
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16
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Peng Z, Zhu X, Wang Z, Yan X, Wang G, Tang M, Jiang A, Kristiansen K. Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics. Bioinform Biol Insights 2020; 14:1177932220915459. [PMID: 32546984 PMCID: PMC7271268 DOI: 10.1177/1177932220915459] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2019] [Accepted: 02/25/2020] [Indexed: 01/12/2023] Open
Abstract
Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.
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Affiliation(s)
- Zonghui Peng
- BGI Americas Corporation, Cambridge, MA, USA.,Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | | | | | | | | | | | | | - Karsten Kristiansen
- Department of Biology, University of Copenhagen, Copenhagen, Denmark.,BGI-Shenzhen, Shenzhen, China.,China National GeneBank, Shenzhen, China
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17
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Haidas D, Napiorkowska M, Schmitt S, Dittrich PS. Parallel Sampling of Nanoliter Droplet Arrays for Noninvasive Protein Analysis in Discrete Yeast Cultivations by MALDI-MS. Anal Chem 2020; 92:3810-3818. [DOI: 10.1021/acs.analchem.9b05235] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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18
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Liu J, Lian Q, Chen Y, Qi J. Amino acid based de Bruijn graph algorithm for identifying complete coding genes from metagenomic and metatranscriptomic short reads. Nucleic Acids Res 2019; 47:e30. [PMID: 30657979 PMCID: PMC6412133 DOI: 10.1093/nar/gkz017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Revised: 12/19/2018] [Accepted: 01/08/2019] [Indexed: 11/12/2022] Open
Abstract
Metagenomic studies, greatly promoted by the fast development of next-generation sequencing (NGS) technologies, uncover complex structures of microbial communities and their interactions with environment. As the majority of microbes lack information of genome sequences, it is essential to assemble prokaryotic genomes ab initio aiming to retrieve complete coding genes from various metabolic pathways. The complex nature of microbial composition and the burden of handling a vast amount of metagenomic data, bring great challenges to the development of effective and efficient bioinformatic tools. Here we present a protein assembler (MetaPA), based on de Bruijn graph searching on oligopeptide spaces and can be applied on both metagenomic and metatranscriptomic sequencing data. When public homologous protein sequences are involved to guide the assembling procedures, MetaPA assembles 85% of total proteins in complete sequences with high precision of 83% on real high-throughput sequencing datasets. Application of MetaPA on metatranscriptomic data successfully identifies the majority of actively transcribed genes validated in related studies. The results suggest that MetaPA has a good potential in both metagenomic and metatranscriptomic studies to characterize the composition and abundance of microbiota.
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Affiliation(s)
- Jiemeng Liu
- State key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China.,The T-Life Research Center, Fudan University, Shanghai 200433, China
| | - Qichao Lian
- State key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
| | - Yamao Chen
- State key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
| | - Ji Qi
- State key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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19
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Wierzbicka-Woś A, Henneberger R, Batista-García RA, Martínez-Ávila L, Jackson SA, Kennedy J, Dobson ADW. Biochemical Characterization of a Novel Monospecific Endo-β-1,4-Glucanase Belonging to GH Family 5 From a Rhizosphere Metagenomic Library. Front Microbiol 2019; 10:1342. [PMID: 31258522 PMCID: PMC6587912 DOI: 10.3389/fmicb.2019.01342] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Accepted: 05/29/2019] [Indexed: 11/16/2022] Open
Abstract
Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-β-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modeling analysis indicated that CelRH5 is related to endo-β-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum, and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC), and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5–8.5. Highest activity was observed at 40°C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30°C. The presence of the final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+, and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulfate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications.
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Affiliation(s)
- Anna Wierzbicka-Woś
- Environmental Research Institute, University College Cork, Cork, Ireland.,Department of Microbiology, Faculty of Biology, University of Szczecin, Szczecin, Poland
| | - Ruth Henneberger
- Environmental Research Institute, University College Cork, Cork, Ireland.,Institute for Molecular Health Sciences, ETH Zürich, Zurich, Switzerland
| | - Ramón Alberto Batista-García
- Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Mexico
| | - Liliana Martínez-Ávila
- Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Mexico
| | - Stephen A Jackson
- Environmental Research Institute, University College Cork, Cork, Ireland.,School of Microbiology, University College Cork, Cork, Ireland
| | | | - Alan D W Dobson
- Environmental Research Institute, University College Cork, Cork, Ireland.,School of Microbiology, University College Cork, Cork, Ireland
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20
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Fortune B, Mhlongo S, van Zyl LJ, Huddy R, Smart M, Trindade M. Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome. BMC Biotechnol 2019; 19:22. [PMID: 30999885 PMCID: PMC6472066 DOI: 10.1186/s12896-019-0510-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2018] [Accepted: 03/13/2019] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. RESULTS Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0-6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s- 1 and 219 s- 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside. CONCLUSION All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.
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Affiliation(s)
- Brent Fortune
- Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa
| | - Sizwe Mhlongo
- Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa
| | - Leonardo Joaquim van Zyl
- Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa
| | - Robert Huddy
- Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa.,Centre for Bioprocess Engineering Research, University of Cape Town, Cape Town, Western Cape, South Africa
| | - Mariette Smart
- Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa.,Centre for Bioprocess Engineering Research, University of Cape Town, Cape Town, Western Cape, South Africa
| | - Marla Trindade
- Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa.
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21
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Meta-Omics Tools in the World of Insect-Microorganism Interactions. BIOLOGY 2018; 7:biology7040050. [PMID: 30486337 PMCID: PMC6316257 DOI: 10.3390/biology7040050] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 10/25/2018] [Revised: 11/16/2018] [Accepted: 11/22/2018] [Indexed: 02/07/2023]
Abstract
Microorganisms are able to influence several aspects of insects’ life, and this statement is gaining increasing strength, as research demonstrates it daily. At the same time, new sequencing technologies are now available at a lower cost per base, and bioinformatic procedures are becoming more user-friendly. This is triggering a huge effort in studying the microbial diversity associated to insects, and especially to economically important insect pests. The importance of the microbiome has been widely acknowledged for a wide range of animals, and also for insects this topic is gaining considerable importance. In addition to bacterial-associates, the insect-associated fungal communities are also gaining attention, especially those including plant pathogens. The use of meta-omics tools is not restricted to the description of the microbial world, but it can be also used in bio-surveillance, food safety assessment, or even to bring novelties to the industry. This mini-review aims to give a wide overview of how meta-omics tools are fostering advances in research on insect-microorganism interactions.
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22
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Jardine JL, Stoychev S, Mavumengwana V, Ubomba-Jaswa E. Screening of potential bioremediation enzymes from hot spring bacteria using conventional plate assays and liquid chromatography - Tandem mass spectrometry (Lc-Ms/Ms). JOURNAL OF ENVIRONMENTAL MANAGEMENT 2018; 223:787-796. [PMID: 29986326 DOI: 10.1016/j.jenvman.2018.06.089] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/26/2017] [Revised: 06/15/2018] [Accepted: 06/28/2018] [Indexed: 06/08/2023]
Abstract
The search for an eco-friendly, non-toxic, economical and efficient means of cleaning water through bioremediation is not only more favourable but critical to maintaining water quality globally especially in water-scarce countries. Thermophilic bacteria including Bacillus species are an important source of novel enzymes for biotechnology applications. In this study, 56 bacterial isolates which were cultured from five hot springs in South Africa were identified predominantly as Bacillus sp. or Bacillus-related spp by 16S rDNA gene sequencing. These isolates were screened for potentially useful enzymes for water bioremediation. Using conventional agar plate assays, 56% (n = 43), 68% (n = 38) and 16% (n = 31) were positive for amylase, protease and bromothymol blue decolorisation respectively. In liquid starch culture, three amylase-positive isolates differentially degraded starch by 34% (isolate 20S) to 98% (isolate 9T). Phenol degradation revealed that five out of thirty reduced phenol up to 42% by colorimetric assay. A thermophilic strain of Anoxybacillus rupiensis 19S (optimal growth temperature of 50 °C), which degraded starch, protein and phenol, was selected for further analysis by tandem LC-MS/MS. This newer technique identified potential enzymes for water bioremediation relating to pollutants from the food industry (amylase, proteases), polyaromatic hydrocarbons and dye pollutants (catalase peroxidase, superoxide dismutase, azoreductase, quinone oxidoreductase), antibiotic residues (ribonucleases), solubilisation of phosphates (inorganic pyrophosphatase) and reduction of chromate and lead. In addition, potential enzymes for biomonitoring of environmental pollutants were also identified. Specifically, dehydrogenases were found to decrease as the level of inorganic heavy metals and petroleum increased in soil samples. This study concludes that bacteria found in South African hot springs are a potential source of novel enzymes with tandem LC-MS/MS revealing substantially more information compared with conventional assays, which can be used for various applications of water bioremediation.
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Affiliation(s)
- J L Jardine
- Department of Biotechnology, University of Johannesburg, 37 Nind Street, Doornfontein, Gauteng, South Africa
| | - S Stoychev
- Council for Scientific and Industrial Research, Biosciences, Box 395, Pretoria 0001, South Africa
| | - V Mavumengwana
- Department of Biotechnology, University of Johannesburg, 37 Nind Street, Doornfontein, Gauteng, South Africa
| | - E Ubomba-Jaswa
- Department of Biotechnology, University of Johannesburg, 37 Nind Street, Doornfontein, Gauteng, South Africa; Water Research Commission, Lynnwood Bridge Office Park, Bloukrans Building, 4 Daventry Street, Lynnwood Manor, Pretoria, South Africa.
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23
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Cheng P, Wang Y, Liang J, Wu Y, Wright A, Liao X. Exploratory Analysis of the Microbiological Potential for Efficient Utilization of Fiber Between Lantang and Duroc Pigs. Front Microbiol 2018; 9:1342. [PMID: 29988353 PMCID: PMC6023970 DOI: 10.3389/fmicb.2018.01342] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2018] [Accepted: 05/31/2018] [Indexed: 01/08/2023] Open
Abstract
There is growing interest in the use of unconventional feed ingredients containing higher dietary fiber for pig production due to increasing prices of cereal grains and the potential health benefits of dietary fiber on host animals. This study aimed to gain insight into the community-wide microbiome population between the Chinese native Lantang pigs and the commercial Duroc pigs to uncover the microbiological mechanisms for the degradation capacity of fiber in pigs. Utilizing the metagenomics approach, we compared the phylogeny and functional capacity of the fecal microbiome from approximately 150-day-old female Lantang and Duroc pigs fed a similar diet. The structure of the fecal microbial community from the two pig breeds was different at the genus level; the number of genes associated with fiber degradation was higher in Lantang pigs. Further analysis and prediction of their functions from the fecal microbiomes of the two pig breeds revealed that the degradation capacities of fiber, branched chain fatty acids, and oligosaccharides were higher in Lantang pigs. The ability of lignocellulose bonding modules and the transport capacities of xylose, L-arabinose, ribose and methyl galactose were also higher in Lantang pigs. Similarly, the metabolic capacities of xylose, ribose, and fucose and the potential effectiveness of the tricarboxylic acid cycle (TCA) and gene abundance in the hydrogen sink pathway were higher in the fecal microbiome from Lantang pigs. Lantang pigs have a higher capacity to utilize dietary fiber than Duroc pigs, and the differences in the capability to utilize dietary fiber between the indigenous and commercial pigs could be differences in the composition and biological function of the gut microbiota.
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Affiliation(s)
- Penghui Cheng
- College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Yan Wang
- College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Juanboo Liang
- Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Seri Kembangan, Malaysia
| | - Yinbao Wu
- College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Andredenis Wright
- School of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ, United States
| | - Xindi Liao
- College of Animal Science, South China Agricultural University, Guangzhou, China
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24
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Attia MA, Nelson CE, Offen WA, Jain N, Davies GJ, Gardner JG, Brumer H. In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions. BIOTECHNOLOGY FOR BIOFUELS 2018; 11:45. [PMID: 29467823 PMCID: PMC5816542 DOI: 10.1186/s13068-018-1039-6] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/01/2017] [Accepted: 02/01/2018] [Indexed: 05/15/2023]
Abstract
BACKGROUND Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. RESULTS Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. CONCLUSIONS Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural-functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.
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Affiliation(s)
- Mohamed A. Attia
- Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4 Canada
- Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 Canada
| | - Cassandra E. Nelson
- Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250 USA
| | - Wendy A. Offen
- Department of Chemistry, University of York, Heslington, York, YO10 5DD UK
| | - Namrata Jain
- Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4 Canada
- Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 Canada
| | - Gideon J. Davies
- Department of Chemistry, University of York, Heslington, York, YO10 5DD UK
| | - Jeffrey G. Gardner
- Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250 USA
| | - Harry Brumer
- Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4 Canada
- Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 Canada
- Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3 Canada
- Department of Botany, University of British Columbia, 6270 University Blvd., Vancouver, BC V6T 1Z4 Canada
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25
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Abstract
Saturation mutagenesis is conveniently located between the two extremes of protein engineering, namely random mutagenesis, and rational design. It involves mutating a confined number of target residues to other amino acids, and hence requires knowledge regarding the sites for mutagenesis, but not their final identity. There are many different strategies for performing and designing such experiments, ranging from simple single degenerate codons to codon collections that code for distinct sets of amino acids. Here, we provide detailed information on the Dynamic Management for Codon Compression (DYNAMCC) approaches that allow us to precisely define the desired amino acid composition to be introduced to a specific target site. DYNAMCC allows us to set usage thresholds and to eliminate undesirable stop and wild-type codons, thus allowing us to control library size and subsequently downstream screening efforts. The DYNAMCC algorithms are free of charge and are implemented in a website for easy access and usage: www.dynamcc.com .
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Affiliation(s)
- Gur Pines
- Renewable and Sustainable Energy Institute (RASEI), University of Colorado Boulder, Boulder, CO, USA. .,Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA.
| | - Ryan T Gill
- Renewable and Sustainable Energy Institute (RASEI), University of Colorado Boulder, Boulder, CO, USA.,Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA
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Andrade AC, Fróes A, Lopes FÁC, Thompson FL, Krüger RH, Dinsdale E, Bruce T. Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome. MICROBIAL ECOLOGY 2017; 74:89-105. [PMID: 28070679 DOI: 10.1007/s00248-016-0911-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 12/05/2016] [Indexed: 06/06/2023]
Abstract
Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and associated biotechnological applications.
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Affiliation(s)
- Ana Camila Andrade
- Faculdade de Tecnologia e Ciências, Grupo de Biotecnologia Ambiental, Department of Bioenergy, Salvador, Brazil
| | - Adriana Fróes
- Laboratory of Microbiology, Institute of Biology, and SAGE-COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
| | | | - Fabiano L Thompson
- Laboratory of Microbiology, Institute of Biology, and SAGE-COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
| | | | | | - Thiago Bruce
- Faculdade de Tecnologia e Ciências, Grupo de Biotecnologia Ambiental, Department of Bioenergy, Salvador, Brazil.
- Department of Biology, San Diego State University, San Diego, CA, USA.
- Institute of Biology, Microbiology department, Universidade Federal da Bahia (UFBA), Rio de Janeiro, Brazil.
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Tiwari R, Nain L, Labrou NE, Shukla P. Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review. Crit Rev Microbiol 2017; 44:244-257. [DOI: 10.1080/1040841x.2017.1337713] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Rameshwar Tiwari
- Department of Microbiology, Laboratory of Enzyme Technology and Protein Bioinformatics, Maharshi Dayanand University, Rohtak, India
- Division of Microbiology, Indian Agricultural Research Institute, New Delhi, India
| | - Lata Nain
- Division of Microbiology, Indian Agricultural Research Institute, New Delhi, India
| | - Nikolaos E. Labrou
- Department of Biotechnology, School of Food, Biotechnology and Development, Laboratory of Enzyme Technology, Agricultural University of Athens, Athens, Greece
| | - Pratyoosh Shukla
- Department of Microbiology, Laboratory of Enzyme Technology and Protein Bioinformatics, Maharshi Dayanand University, Rohtak, India
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1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life. Nat Biotechnol 2017; 35:676-683. [DOI: 10.1038/nbt.3886] [Citation(s) in RCA: 179] [Impact Index Per Article: 22.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2016] [Accepted: 04/21/2017] [Indexed: 12/16/2022]
Abstract
Abstract
We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster with potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.
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29
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Mining lipolytic enzymes in community DNA from high Andean soils using a targeted approach. Antonie van Leeuwenhoek 2017; 110:1035-1051. [DOI: 10.1007/s10482-017-0877-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/02/2016] [Accepted: 04/18/2017] [Indexed: 12/23/2022]
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Montella S, Ventorino V, Lombard V, Henrissat B, Pepe O, Faraco V. Discovery of genes coding for carbohydrate-active enzyme by metagenomic analysis of lignocellulosic biomasses. Sci Rep 2017; 7:42623. [PMID: 28198423 PMCID: PMC5309792 DOI: 10.1038/srep42623] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2016] [Accepted: 01/13/2017] [Indexed: 12/03/2022] Open
Abstract
In this study, a high-throughput sequencing approach was applied to discover novel biocatalysts for lignocellulose hydrolysis from three dedicated energy crops, Arundo donax, Eucalyptus camaldulensis and Populus nigra, after natural biodegradation. The microbiomes of the three lignocellulosic biomasses were dominated by bacterial species (approximately 90%) with the highest representation by the Streptomyces genus both in the total microbial community composition and in the microbial diversity related to GH families of predicted ORFs. Moreover, the functional clustering of the predicted ORFs showed a prevalence of poorly characterized genes, suggesting these lignocellulosic biomasses are potential sources of as yet unknown genes. 1.2%, 0.6% and 3.4% of the total ORFs detected in A. donax, E. camaldulensis and P. nigra, respectively, were putative Carbohydrate-Active Enzymes (CAZymes). Interestingly, the glycoside hydrolases abundance in P. nigra (1.8%) was higher than that detected in the other biomasses investigated in this study. Moreover, a high percentage of (hemi)cellulases with different activities and accessory enzymes (mannanases, polygalacturonases and feruloyl esterases) was detected, confirming that the three analyzed samples were a reservoir of diversified biocatalysts required for an effective lignocellulose saccharification.
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Affiliation(s)
- Salvatore Montella
- Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cintia, 4 80126 Naples, Italy
| | - Valeria Ventorino
- Department of Agricultural Sciences, University of Naples "Federico II", Portici (Napoli), Italy
| | - Vincent Lombard
- CNRS UMR 7257, Aix-Marseille University, 13288 Marseille, France.,INRA, USC 1408 AFMB, 13288 Marseille, France
| | - Bernard Henrissat
- CNRS UMR 7257, Aix-Marseille University, 13288 Marseille, France.,INRA, USC 1408 AFMB, 13288 Marseille, France.,Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Olimpia Pepe
- Department of Agricultural Sciences, University of Naples "Federico II", Portici (Napoli), Italy
| | - Vincenza Faraco
- Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cintia, 4 80126 Naples, Italy
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31
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Ábrego U, Chen Z, Wan C. Consolidated Bioprocessing Systems for Cellulosic Biofuel Production. ADVANCES IN BIOENERGY 2017. [DOI: 10.1016/bs.aibe.2017.01.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
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32
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Lacerda Júnior GV, Noronha MF, de Sousa STP, Cabral L, Domingos DF, Sáber ML, de Melo IS, Oliveira VM. Potential of semiarid soil from Caatinga biome as a novel source for mining lignocellulose-degrading enzymes. FEMS Microbiol Ecol 2016; 93:fiw248. [PMID: 27986827 DOI: 10.1093/femsec/fiw248] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 08/11/2016] [Accepted: 12/13/2016] [Indexed: 11/14/2022] Open
Abstract
The litterfall is the major organic material deposited in soil of Brazilian Caatinga biome, thus providing the ideal conditions for plant biomass-degrading microorganisms to thrive. Herein, the phylogenetic composition and lignocellulose-degrading capacity have been explored for the first time from a fosmid library dataset of Caatinga soil by sequence-based screening. A complex bacterial community dominated by Proteobacteria and Actinobacteria was unraveled. SEED subsystems-based annotations revealed a broad range of genes assigned to carbohydrate and aromatic compounds metabolism, indicating microbial ability to utilize plant-derived material. CAZy-based annotation identified 7275 genes encoding 37 glycoside hydrolases (GHs) families related to hydrolysis of cellulose, hemicellulose, oligosaccharides and other lignin-modifying enzymes. Taxonomic affiliation of genes showed high genetic potential of the phylum Acidobacteria for hemicellulose degradation, whereas Actinobacteria members appear to play an important role in celullose hydrolysis. Additionally, comparative analyses revealed greater GHs profile similarity among soils as compared to the digestive tract of animals capable of digesting plant biomass, particularly in the hemicellulases content. Combined results suggest a complex synergistic interaction of community members required for biomass degradation into fermentable sugars. This large repertoire of lignocellulolytic enzymes opens perspectives for mining potential candidates of biochemical catalysts for biofuels production from renewable resources and other environmental applications.
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Affiliation(s)
- Gileno V Lacerda Júnior
- Research Center for Chemistry, Biology and Agriculture (CPQBA), UNICAMP, Division of Microbial Resources, Zip code 13148-218, Paulínia, São Paulo, Brazil
| | - Melline F Noronha
- Research Center for Chemistry, Biology and Agriculture (CPQBA), UNICAMP, Division of Microbial Resources, Zip code 13148-218, Paulínia, São Paulo, Brazil
| | - Sanderson Tarciso P de Sousa
- Research Center for Chemistry, Biology and Agriculture (CPQBA), UNICAMP, Division of Microbial Resources, Zip code 13148-218, Paulínia, São Paulo, Brazil
| | - Lucélia Cabral
- Research Center for Chemistry, Biology and Agriculture (CPQBA), UNICAMP, Division of Microbial Resources, Zip code 13148-218, Paulínia, São Paulo, Brazil
| | - Daniela F Domingos
- Department of Bioengineering, University of California San Diego, La Jolla, CA 92093-0412, USA
| | - Mírian L Sáber
- Laboratory of Environmental Microbiology, Brazilian Agricultural Research Corporation, EMBRAPA Environment, Jaguariúna, Zip code 13820-000, Brazil
| | - Itamar S de Melo
- Laboratory of Environmental Microbiology, Brazilian Agricultural Research Corporation, EMBRAPA Environment, Jaguariúna, Zip code 13820-000, Brazil
| | - Valéria M Oliveira
- Research Center for Chemistry, Biology and Agriculture (CPQBA), UNICAMP, Division of Microbial Resources, Zip code 13148-218, Paulínia, São Paulo, Brazil
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Adesioye FA, Makhalanyane TP, Biely P, Cowan DA. Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases. Enzyme Microb Technol 2016; 93-94:79-91. [DOI: 10.1016/j.enzmictec.2016.07.001] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2016] [Revised: 06/18/2016] [Accepted: 07/01/2016] [Indexed: 02/06/2023]
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34
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Kim DR, Lim HK, Lee KI, Hwang IT. Identification of a novel cellulose-binding domain within the endo -β-1,4-xylanase KRICT PX-3 from Paenibacillus terrae HPL-003. Enzyme Microb Technol 2016; 93-94:166-173. [DOI: 10.1016/j.enzmictec.2016.07.014] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Revised: 04/19/2016] [Accepted: 07/25/2016] [Indexed: 10/21/2022]
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35
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Bioprospecting metagenomics of a microbial community on cotton degradation: Mining for new glycoside hydrolases. J Biotechnol 2016; 234:35-42. [DOI: 10.1016/j.jbiotec.2016.07.017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Revised: 07/20/2016] [Accepted: 07/22/2016] [Indexed: 01/30/2023]
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36
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Kohl KD, Connelly JW, Dearing MD, Forbey JS. Microbial detoxification in the gut of a specialist avian herbivore, the Greater Sage-Grouse. FEMS Microbiol Lett 2016; 363:fnw144. [DOI: 10.1093/femsle/fnw144] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/24/2016] [Indexed: 12/25/2022] Open
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37
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Gherbovet O, Fauré R, Ferreira F, Durand J, Ragon M, Hostyn G, Record E, Bozonnet S, O’Donohue MJ. Design of chromogenic probes for efficient screening and evaluation of feruloyl esterase-like activities. ACTA ACUST UNITED AC 2016. [DOI: 10.1016/j.molcatb.2016.01.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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38
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Attia M, Stepper J, Davies GJ, Brumer H. Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation. FEBS J 2016; 283:1701-19. [PMID: 26929175 DOI: 10.1111/febs.13696] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2015] [Revised: 02/09/2016] [Accepted: 02/25/2016] [Indexed: 11/27/2022]
Abstract
UNLABELLED The heteropolysaccharide xyloglucan (XyG) comprises up to one-quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone-cleaving endo-xyloglucanases and side-chain cleaving exo-glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram-negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side-chain cleaving exo-glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N-terminal glycoside hydrolase family 74 (GH74) endo-xyloglucanase module in train with two carbohydrate-binding modules (CBMs) from families 10 and 2 (CBM10 and CBM2). The GH74 catalytic domain generates Glc4 -based xylogluco-oligosaccharide (XyGO) substrates for downstream enzymes through an endo-dissociative mode of action. X-ray crystallography of the GH74 module, alone and in complex with XyGO products spanning the entire active site, revealed a broad substrate-binding cleft specifically adapted to XyG recognition, which is composed of two seven-bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose-binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization. DATABASE Structural data have been deposited in the RCSB protein database under the Protein Data Bank codes: 5FKR, 5FKS, 5FKT and 5FKQ.
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Affiliation(s)
- Mohamed Attia
- Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, Canada
| | | | | | - Harry Brumer
- Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, Canada
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39
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Ufarté L, Bozonnet S, Laville E, Cecchini DA, Pizzut-Serin S, Jacquiod S, Demanèche S, Simonet P, Franqueville L, Veronese GP. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes. Methods Mol Biol 2016; 1399:257-71. [PMID: 26791508 DOI: 10.1007/978-1-4939-3369-3_15] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.
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Affiliation(s)
- Lisa Ufarté
- INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse, France
- UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400, Toulouse, France
- UMR5504, CNRS, 31400, Toulouse, France
| | - Sophie Bozonnet
- INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse, France
- UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400, Toulouse, France
- UMR5504, CNRS, 31400, Toulouse, France
| | - Elisabeth Laville
- INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse, France
- UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400, Toulouse, France
- UMR5504, CNRS, 31400, Toulouse, France
| | - Davide A Cecchini
- INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse, France
- UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400, Toulouse, France
- UMR5504, CNRS, 31400, Toulouse, France
| | - Sandra Pizzut-Serin
- INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse, France
- UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400, Toulouse, France
- UMR5504, CNRS, 31400, Toulouse, France
| | - Samuel Jacquiod
- Laboratoire Ampère, CNRS UMR5005, Ecole Centrale de Lyon, Université de Lyon, 69134, Ecully, France
| | - Sandrine Demanèche
- Laboratoire Ampère, CNRS UMR5005, Ecole Centrale de Lyon, Université de Lyon, 69134, Ecully, France
| | - Pascal Simonet
- Laboratoire Ampère, CNRS UMR5005, Ecole Centrale de Lyon, Université de Lyon, 69134, Ecully, France
| | - Laure Franqueville
- Laboratoire Ampère, CNRS UMR5005, Ecole Centrale de Lyon, Université de Lyon, 69134, Ecully, France
| | - Gabrielle Potocki Veronese
- INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse, France.
- UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400, Toulouse, France.
- UMR5504, CNRS, 31400, Toulouse, France.
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40
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Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters. Appl Environ Microbiol 2015; 82:1519-1529. [PMID: 26712547 DOI: 10.1128/aem.03360-15] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Accepted: 12/18/2015] [Indexed: 01/03/2023] Open
Abstract
Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions.
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41
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Montella S, Amore A, Faraco V. Metagenomics for the development of new biocatalysts to advance lignocellulose saccharification for bioeconomic development. Crit Rev Biotechnol 2015; 36:998-1009. [PMID: 26381035 DOI: 10.3109/07388551.2015.1083939] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification.
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Affiliation(s)
- Salvatore Montella
- a Department of Chemical Sciences , University of Naples "Federico II", Complesso Universitario Monte S. Angelo , Naples , Italy
| | - Antonella Amore
- a Department of Chemical Sciences , University of Naples "Federico II", Complesso Universitario Monte S. Angelo , Naples , Italy
| | - Vincenza Faraco
- a Department of Chemical Sciences , University of Naples "Federico II", Complesso Universitario Monte S. Angelo , Naples , Italy
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White biotechnology: State of the art strategies for the development of biocatalysts for biorefining. Biotechnol Adv 2015; 33:1653-70. [PMID: 26303096 DOI: 10.1016/j.biotechadv.2015.08.004] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2015] [Revised: 07/31/2015] [Accepted: 08/17/2015] [Indexed: 12/31/2022]
Abstract
White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose.
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Lobb B, Kurtz DA, Moreno-Hagelsieb G, Doxey AC. Remote homology and the functions of metagenomic dark matter. Front Genet 2015; 6:234. [PMID: 26257768 PMCID: PMC4508852 DOI: 10.3389/fgene.2015.00234] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2015] [Accepted: 06/22/2015] [Indexed: 01/26/2023] Open
Abstract
Predicted open reading frames (ORFs) that lack detectable homology to known proteins are termed ORFans. Despite their prevalence in metagenomes, the extent to which ORFans encode real proteins, the degree to which they can be annotated, and their functional contributions, remain unclear. To gain insights into these questions, we applied sensitive remote-homology detection methods to functionally analyze ORFans from soil, marine, and human gut metagenome collections. ORFans were identified, clustered into sequence families, and annotated through profile-profile comparison to proteins of known structure. We found that a considerable number of metagenomic ORFans (73,896 of 484,121, 15.3%) exhibit significant remote homology to structurally characterized proteins, providing a means for ORFan functional profiling. The extent of detected remote homology far exceeds that obtained for artificial protein families (1.4%). As expected for real genes, the predicted functions of ORFans are significantly similar to the functions of their gene neighbors (p < 0.001). Compared to the functional profiles predicted through standard homology searches, ORFans show biologically intriguing differences. Many ORFan-enriched functions are virus-related and tend to reflect biological processes associated with extreme sequence diversity. Each environment also possesses a large number of unique ORFan families and functions, including some known to play important community roles such as gut microbial polysaccharide digestion. Lastly, ORFans are a valuable resource for finding novel enzymes of interest, as we demonstrate through the identification of hundreds of novel ORFan metalloproteases that all possess a signature catalytic motif despite a general lack of similarity to known proteins. Our ORFan functional predictions are a valuable resource for discovering novel protein families and exploring the boundaries of protein sequence space. All remote homology predictions are available at http://doxey.uwaterloo.ca/ORFans.
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Affiliation(s)
- Briallen Lobb
- Department of Biology, University of Waterloo Waterloo, ON, Canada
| | - Daniel A Kurtz
- Department of Biology, University of Waterloo Waterloo, ON, Canada
| | | | - Andrew C Doxey
- Department of Biology, University of Waterloo Waterloo, ON, Canada
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Wei Y, Zhou H, Zhang J, Zhang L, Geng A, Liu F, Zhao G, Wang S, Zhou Z, Yan X. Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes. PLoS One 2015; 10:e0129921. [PMID: 26070087 PMCID: PMC4466528 DOI: 10.1371/journal.pone.0129921] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Accepted: 05/13/2015] [Indexed: 12/30/2022] Open
Abstract
Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.
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Affiliation(s)
- Yongjun Wei
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Haokui Zhou
- Department of Microbiology, the Chinese University of Hong Kong, the Prince of Wales Hospital, Hong Kong, China
| | - Jun Zhang
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Lei Zhang
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Alei Geng
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Fanghua Liu
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Guoping Zhao
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
- Department of Microbiology, the Chinese University of Hong Kong, the Prince of Wales Hospital, Hong Kong, China
- Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai, China
| | - Shengyue Wang
- Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai, China
| | - Zhihua Zhou
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
- * E-mail: (XY); (ZZ)
| | - Xing Yan
- CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
- * E-mail: (XY); (ZZ)
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Zhong C, Yang Y, Yooseph S. GRASP: guided reference-based assembly of short peptides. Nucleic Acids Res 2015; 43:e18. [PMID: 25414351 PMCID: PMC4330339 DOI: 10.1093/nar/gku1210] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2014] [Revised: 11/01/2014] [Accepted: 11/05/2014] [Indexed: 12/22/2022] Open
Abstract
Protein sequences predicted from metagenomic datasets are annotated by identifying their homologs via sequence comparisons with reference or curated proteins. However, a majority of metagenomic protein sequences are partial-length, arising as a result of identifying genes on sequencing reads or on assembled nucleotide contigs, which themselves are often very fragmented. The fragmented nature of metagenomic protein predictions adversely impacts homology detection and, therefore, the quality of the overall annotation of the dataset. Here we present a novel algorithm called GRASP that accurately identifies the homologs of a given reference protein sequence from a database consisting of partial-length metagenomic proteins. Our homology detection strategy is guided by the reference sequence, and involves the simultaneous search and assembly of overlapping database sequences. GRASP was compared to three commonly used protein sequence search programs (BLASTP, PSI-BLAST and FASTM). Our evaluations using several simulated and real datasets show that GRASP has a significantly higher sensitivity than these programs while maintaining a very high specificity. GRASP can be a very useful program for detecting and quantifying taxonomic and protein family abundances in metagenomic datasets. GRASP is implemented in GNU C++, and is freely available at http://sourceforge.net/projects/grasp-release.
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Affiliation(s)
- Cuncong Zhong
- Informatics Department, J. Craig Venter Institute, La Jolla, CA 92037, USA
| | - Youngik Yang
- Informatics Department, J. Craig Venter Institute, La Jolla, CA 92037, USA
| | - Shibu Yooseph
- Informatics Department, J. Craig Venter Institute, La Jolla, CA 92037, USA
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Wang WL, Xu SY, Ren ZG, Tao L, Jiang JW, Zheng SS. Application of metagenomics in the human gut microbiome. World J Gastroenterol 2015; 21:803-814. [PMID: 25624713 PMCID: PMC4299332 DOI: 10.3748/wjg.v21.i3.803] [Citation(s) in RCA: 222] [Impact Index Per Article: 22.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Revised: 09/30/2014] [Accepted: 11/07/2014] [Indexed: 02/06/2023] Open
Abstract
There are more than 1000 microbial species living in the complex human intestine. The gut microbial community plays an important role in protecting the host against pathogenic microbes, modulating immunity, regulating metabolic processes, and is even regarded as an endocrine organ. However, traditional culture methods are very limited for identifying microbes. With the application of molecular biologic technology in the field of the intestinal microbiome, especially metagenomic sequencing of the next-generation sequencing technology, progress has been made in the study of the human intestinal microbiome. Metagenomics can be used to study intestinal microbiome diversity and dysbiosis, as well as its relationship to health and disease. Moreover, functional metagenomics can identify novel functional genes, microbial pathways, antibiotic resistance genes, functional dysbiosis of the intestinal microbiome, and determine interactions and co-evolution between microbiota and host, though there are still some limitations. Metatranscriptomics, metaproteomics and metabolomics represent enormous complements to the understanding of the human gut microbiome. This review aims to demonstrate that metagenomics can be a powerful tool in studying the human gut microbiome with encouraging prospects. The limitations of metagenomics to be overcome are also discussed. Metatranscriptomics, metaproteomics and metabolomics in relation to the study of the human gut microbiome are also briefly discussed.
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Mhuantong W, Charoensawan V, Kanokratana P, Tangphatsornruang S, Champreda V. Comparative analysis of sugarcane bagasse metagenome reveals unique and conserved biomass-degrading enzymes among lignocellulolytic microbial communities. BIOTECHNOLOGY FOR BIOFUELS 2015; 8:16. [PMID: 25709713 PMCID: PMC4337096 DOI: 10.1186/s13068-015-0200-8] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/27/2014] [Accepted: 01/08/2015] [Indexed: 05/18/2023]
Abstract
BACKGROUND As one of the most abundant agricultural wastes, sugarcane bagasse is largely under-exploited, but it possesses a great potential for the biofuel, fermentation, and cellulosic biorefinery industries. It also provides a unique ecological niche, as the microbes in this lignocellulose-rich environment thrive in relatively high temperatures (50°C) with varying microenvironments of aerobic surface to anoxic interior. The microbial community in bagasse thus presents a good resource for the discovery and characterization of new biomass-degrading enzymes; however, it remains largely unexplored. RESULTS We have constructed a fosmid library of sugarcane bagasse and obtained the largest bagasse metagenome to date. A taxonomic classification of the bagasse metagenome reviews the predominance of Proteobacteria, which are also found in high abundance in other aerobic environments. Based on the functional characterization of biomass-degrading enzymes, we have demonstrated that the bagasse microbial community benefits from a large repertoire of lignocellulolytic enzymes, which allows them to digest different components of lignocelluoses into single molecule sugars. Comparative genomic analyses with other lignocellulolytic and non-lignocellulolytic metagenomes show that microbial communities are taxonomically separable by their aerobic "open" or anoxic "closed" environments. Importantly, a functional analysis of lignocellulose-active genes (based on the CAZy classifications) reveals core enzymes highly conserved within the lignocellulolytic group, regardless of their taxonomic compositions. Cellulases, in particular, are markedly more pronounced compared to the non-lignocellulolytic group. In addition to the core enzymes, the bagasse fosmid library also contains some uniquely enriched glycoside hydrolases, as well as a large repertoire of the newly defined auxiliary activity proteins. CONCLUSIONS Our study demonstrates a conservation and diversification of carbohydrate-active genes among diverse microbial species in different biomass-degrading niches, and signifies the importance of taking a global approach to functionally investigate a microbial community as a whole, as compared to focusing on individual organisms.
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Affiliation(s)
- Wuttichai Mhuantong
- />Enzyme Technology Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Pathumthani, 12120 Thailand
| | - Varodom Charoensawan
- />Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand
- />Integrative Computational BioScience (ICBS) Center, Mahidol University, Nakhon Pathom, 73170 Thailand
| | - Pattanop Kanokratana
- />Enzyme Technology Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Pathumthani, 12120 Thailand
| | - Sithichoke Tangphatsornruang
- />Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Pathumthani, 12120 Thailand
| | - Verawat Champreda
- />Enzyme Technology Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Pathumthani, 12120 Thailand
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Kračun SK, Schückel J, Westereng B, Thygesen LG, Monrad RN, Eijsink VGH, Willats WGT. A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. BIOTECHNOLOGY FOR BIOFUELS 2015; 8:70. [PMID: 25969695 PMCID: PMC4428106 DOI: 10.1186/s13068-015-0250-y] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/27/2015] [Accepted: 03/30/2015] [Indexed: 05/02/2023]
Abstract
BACKGROUND Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. RESULTS We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained. CONCLUSIONS We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems.
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Affiliation(s)
- Stjepan Krešimir Kračun
- />Department of Plant and Environmental Sciences, Thorvaldsensvej 40, Frederiksberg, C 1871 Denmark
| | - Julia Schückel
- />Department of Plant and Environmental Sciences, Thorvaldsensvej 40, Frederiksberg, C 1871 Denmark
| | - Bjørge Westereng
- />Department of Plant and Environmental Sciences, Thorvaldsensvej 40, Frederiksberg, C 1871 Denmark
- />Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Chr. M. Falsens vei 1., Aas, 1432 Norway
- />University of Copenhagen, Faculty of Science, Rolighedsvej 23, Frederiksberg, C 1958 Denmark
| | | | | | - Vincent G H Eijsink
- />Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Chr. M. Falsens vei 1., Aas, 1432 Norway
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Isolation and characterization of Achromobacter sp. CX2 from symbiotic Cytophagales, a non-cellulolytic bacterium showing synergism with cellulolytic microbes by producing β-glucosidase. ANN MICROBIOL 2014. [DOI: 10.1007/s13213-014-1009-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
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Shelomi M, Jasper WC, Atallah J, Kimsey LS, Johnson BR. Differential expression of endogenous plant cell wall degrading enzyme genes in the stick insect (Phasmatodea) midgut. BMC Genomics 2014; 15:917. [PMID: 25331961 PMCID: PMC4221708 DOI: 10.1186/1471-2164-15-917] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2014] [Accepted: 10/01/2014] [Indexed: 12/04/2022] Open
Abstract
BACKGROUND Stick and leaf insects (Phasmatodea) are an exclusively leaf-feeding order of insects with no record of omnivory, unlike other "herbivorous" Polyneoptera. They represent an ideal system for investigating the adaptations necessary for obligate folivory, including plant cell wall degrading enzymes (PCWDEs). However, their physiology and internal anatomy is poorly understood, with limited genomic resources available. RESULTS We de novo assembled transcriptomes for the anterior and posterior midguts of six diverse Phasmatodea species, with RNA-Seq on one exemplar species, Peruphasma schultei. The latter's assembly yielded >100,000 transcripts, with over 4000 transcripts uniquely or more highly expressed in specific midgut sections. Two to three dozen PCWDE encoding gene families, including cellulases and pectinases, were differentially expressed in the anterior midgut. These genes were also found in genomic DNA from phasmid brain tissue, suggesting endogenous production. Sequence alignments revealed catalytic sites on most PCWDE transcripts. While most phasmid PCWDE genes showed homology with those of other insects, the pectinases were homologous to bacterial genes. CONCLUSIONS We identified a large and diverse PCWDE repertoire endogenous to the phasmids. If these expressed genes are translated into active enzymes, then phasmids can theoretically break plant cell walls into their monomer components independently of microbial symbionts. The differential gene expression between the two midgut sections provides the first molecular hints as to their function in living phasmids. Our work expands the resources available for industrial applications of animal-derived PCWDEs, and facilitates evolutionary analysis of lower Polyneopteran digestive enzymes, including the pectinases whose origin in Phasmatodea may have been a horizontal transfer event from bacteria.
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Affiliation(s)
- Matan Shelomi
- />Department of Entomology and Nematology, University of California-Davis, Davis, CA 95616 USA
- />Department of Entomology, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
| | - W Cameron Jasper
- />Department of Entomology and Nematology, University of California-Davis, Davis, CA 95616 USA
| | - Joel Atallah
- />Department of Entomology and Nematology, University of California-Davis, Davis, CA 95616 USA
| | - Lynn S Kimsey
- />Department of Entomology and Nematology, University of California-Davis, Davis, CA 95616 USA
| | - Brian R Johnson
- />Department of Entomology and Nematology, University of California-Davis, Davis, CA 95616 USA
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