Projects / Programmes source: ARRS

Community level transcriptomic de-novo assembly reveals microbial enzymes that effectively contribute to complex plant polymere degradation

Research activity

Code Science Field Subfield
1.03.00  Natural sciences and mathematics  Biology   

Code Science Field
B110  Biomedical sciences  Bioinformatics, medical informatics, biomathematics biometrics 

Code Science Field
1.06  Natural Sciences  Biological sciences 
transcriptomics, de-novo assembly, enzyme, plant polymere degradation, microbial communities, industrial use
Evaluation (rules)
source: COBISS
Researchers (5)
no. Code Name and surname Research area Role Period No. of publications
1.  24975  PhD Damjan Makuc  Chemistry  Researcher  2014 - 2017  117 
2.  20183  PhD Boštjan Murovec  Systems and cybernetics  Researcher  2014 - 2017  194 
3.  10082  PhD Janez Plavec  Chemistry  Researcher  2014 - 2017  1,182 
4.  27547  PhD Zala Prevoršek  Animal production  Researcher  2014 - 2017  40 
5.  19104  PhD Blaž Stres  Animal production  Principal Researcher  2014 - 2017  348 
Organisations (3)
no. Code Research organisation City Registration number No. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,824 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  64,524 
3.  1538  University of Ljubljana, Faculty of Electrical Engineering  Ljubljana  1626965  26,846 
The plant material rich in complex polysaccharides is an important component of the diet of herbivorous and omnivorous mammals. Mammals do not possess their own digestive enzymes for the break down of plant cell wall polysaccharides. Polymer hydrolysis is provided by microbial communities in digestive tract. The conditions in gastrointestinal tract favor the development of dense microbial communities with high abundance completely dominated by strict and obligatory anaerobic microorganisms. The main products of anaerobic fermentation are short chain fatty acids, which contribute up to 70% of the energy required for growth and development in the case of large ruminants. Microbial degradation of the gastrointestinal tract of herbivorous mammals, particularly in the rumen, is much faster than the rest, indicating that the extremely high, rapid and efficient degradation capacity of lignocellulosic feedstock of the gut microbiota. As a result, there is outstanding interest in the industrial exploitation of enzymes involved in the conversion of plant polysaccharides. Within the framework of the international project " Global Census of Yellow Microbial Diversity Project" AgResearch , New Zealand, (www.globalrumencensus.org.nz ) samples of the contents of ruminants from different locations around the world, of distinct species, breeding, sex and feed where collected, and samples of wild ruminants, camels and macropods represented a special case of microbial ecosystem with maximum degradation efficiency of plant material. Bioinformatic analyses of deep sequencing data from phylogenetic markers targeting bacteria, archaea , protozoa, fungi, and of selected functional genes, were used to determine phylogenetic composition of microbial communities, define the core communities, identify diversity of microbial groups and their dependence on the type of occurrence, nutrition, breeding, feed conversion efficiency and other measured parameters . Below we intend to perform deep sequencing of prokaryotic transcriptome to determine the extent and type of microbial transcripts with denovo assembly. This will serve as a basis for the identification of transcripts of functional genes in the mammalian gastrointestinal tract involved in the degradation of the plant cell wall polysaccharides, their nature and dynamics of the expression of key genes for the degradation of complex polysaccharides. Microbial community samples collected from the digestive tracts of wild ruminants with high efficiency degradation of plant polymers will serve as the baseline. The analysis of datasets produced in this study, their time-line and co-occurrence assembly and correlation to metabolites is going to provide an approach to identification of yet undescribed or putative enzyme coding sequences amenable for subcloning. Direct cell-independent expression system is going to enable us the production of sufficient amounts of targeted enzymes that are going to be available for tests of specific activities on plant substrates and for expression in live host organisms (E.coli, Prevotella ruminicola, Bacteroides sp).
Significance for science
Results contributed to understanding of how the number of different transcripts of polysaccharidase genes, their relative distribution and temporal dynamics of expression reflected the potential for microbial degradation of complex plant polymers of natural origin, as for the successful decommissioning of polymeric materials the presence, relative distribution and dynamics of change are important. Due to the direct sequencing and denovo reconstruction of transcripts, the analysis is database independent (independent of databases containing largely only cultivated microbial representatives of the genomic and transcriptomic data). This is the first time that transcripts were physically mapped to operons highlighting the importance of coordinated gene expression as a novel paradigm important for understanding the function of microbial systems, for the understanding of the microbial ecology of the gastrointestinal tract, increasing the productivity of metabolic processes, and capturing the resulting degradation products into other industrial processes such as use of enzymes in industrial decommissioning of plant polymers (e.g. wood industry in conjunction with paper and cleaning products, biogas, industrial fermentations, biofuels). Last but not least, the detailed mapping of transcripts of the microbial system of the digestive tract based on the large amount of data, it was possible to connect findings with other microbial systems where similar studies have not yet been carried out, but there are partial data where recent molecular studies have shown the presence of related microorganisms or individual enzymes or their partial transcripts, such as decomposition of residues from paper production. Due to the large amount of symmetrically structured data and factorial design studies, time slices and the metabolism of metatranscriptom with respect to the metabolomes we acquired large amounts of data that require substantial in-depth period of analysis and are thus the basis for preparation of final publications in scientific publications. We were already able to realize some of them while the vast majority are in the pipeline. Two patent applications were submitted to the UK-IPO, two prototypes of measurement machines with associated software and engine control were prepared, improved and standardized techniques were developed for TSOC analysis, reducing sugars, SCFA and aromatic compounds through spectroscopy, that represent a significant methodological boost for future projects and hands-on industrial tasks. A specific spin-out of the methodological development in this project is the introduction into routine use of NMR technology in liquid and solid for analysis of the metabolomes of anaerobic processes at the level of systems biology of anaerobic environments: rumen, anaerobic reactors of biogas plants, gastrointestinal tract of the human, fluid of the human body (urine, blood). In the present, NMR technique was used as a flow-through technique for mapping the state of biochemical reactions without prior target substance extractions in all of the abovementioned anaerobic systems as well as aerobic (human metabolomics). By testing on samples of various types of ruminants, we developed analytical platform for early diagnostic tools of industrial reactor inhibition and the detection of (sub)acute rumen acidosis in ruminants, which, among the first, could systematically extend the use of NMR techniques to other industrial anaerobic systems (biogas power plants, veterinary, animal production). 1H-NMR substituted other more time-consuming and complex analyses of the small number of organic compounds by gas chromatography, which is generally carried out through the extraction of organic substances in organic solvents, and is currently the main analytical technique in these fields. NMR technique was used to determine enzymatic reactions in the degradation of complex plant polymers with yeast cell plants or in vitro expressed proteins
Significance for the country
Due to the large amount of symmetrically structured data and factorial design studies, time slices, the identification of new individual enzymes, identification of previously unspecified enzymes mixtures that participate at certain time intervals in decomposition complex organic polymers, represents the way forward to the preparation of industrial preparations for use in various types of industry ( paper, detergents, fermentation, feed preparation, food processing, biogas). From the analysis of time slices and the decomposition of plant substrates, new mRNA sequences were obtained, and for their functional protein products proved their industrial value. The preparation of materials for the production of target enzymes was performed at the level of in vitro protein synthesis and in the form of yeast cell factories and using 1H-NMR confirmed their activity for the degradation of lignin gratings in real lignocellulosic substrates. We have not yet decided to protect intellectual property and transfer to production on a larger scale in existing or newly created companies (spin-off companies from public research organizations), because of the time-consuming process of implementing these procedures at the UL, misunderstanding at the home institution, systemically unregulated application process and the protection of intellectual property, hindering the installation of a laboratory for the production of these products, as well as because the data for the reasons listed have not yet been fully publicly released until all these parameters are regulated. The application potential is therefore conserved for industrial use. We were first to obtain information on the genetic potential of microbial gastrointestinal associations of Slovenian autochthonous species of wild ruminants, which until now have not yet been described, information for degradation of plant polymers, thus significantly enhancing knowledge of their structures, as part of the natural heritage, especially in comparison with the already known data on domesticated ruminants. Last but not least, the extensions of knowledge about the setup of wild ruminants are undoubtedly welcome also for all nature lovers and managers of forests and hunters. On the basis of the collected data, we created a local database of collected information on the metagenomes, metatranscriptomes and metabolomes of the animal species under consideration, all in conjunction with NMR analyzes of plant substrates and a review of identified plant species and types of plant polymers in ruminant feeds that already represents an excellent database of data on intestine of wild ruminants in slovenian montane region. At the work we also prepared a library of genetic sequences of dominant plants in the investigated area and their identification and diversity in the digestive tract of ruminants. Within the project, two master's students of microbiology successfully completed their research and obtained their master's degrees. Introducing the NMR technique in routine monitoring of microbiological processes in the anaerobic systems has great potential for effective management on an industrial scale, reduction in inhibition events due to relative simplicity of analyses without pretreatment, high speed and sensitivity, repeatability, wide detection of organic molecules and new statistical analyses. Due to coincidence between processes taking place within gastrointestinal tracts, on industrial scale of biogas reactors and in rice fields, we have proved the importance of NMR for areas related to veterinary, nutrition and animal health (early detection of the occurrence of subacute rumen acidosis) and human food consumption (environment). Finally, this development neabled the use of research facilities for industrial tasksk as well.
Most important scientific results Annual report 2015, final report
Most important socioeconomically and culturally relevant results Annual report 2015, final report
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