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Advanced bioinformatics for genome and metagenome analyses and discovery of novel biocatalists from extremophiles: implications for improving industrial bioprocesses

Periodic Reporting for period 2 - MeTABLE (Advanced bioinformatics for genome and metagenome analyses and discovery of novel biocatalists from extremophiles: implications for improving industrial bioprocesses)

Reporting period: 2016-12-01 to 2018-11-30

"The purpose of this project is to discover biomolecules that may be of use for everyday life and that make human activities environmentally sustainable. The discovery of molecular tools from cold-loving organisms may help in making ordinary activities, such as laundry and food storage, with low energy costs and more efficient. This is very important for all European citizens since it may result in money savings.

We started our project by isolating microorganism from cold extreme environments. We isolated ciliates from the Passu and Hisper glaciers. Furthermore, we isolated five bacterial strains from a consortium associated with the Antarctic ciliate Euplotes focardii, an organism that survives only in the cold. They were isolated by using selective temperatures, such as 4°C, and media such as diesel and heavy metals. Therefore, we can consider these bacteria as extremophiles (that love extreme environments). The genomes of these bacterial strains have been sequenced and analyzed.

The innovation raised from the characterization of the bacterial Marinomonas strain has been patented. The patent file is yet under the secret period (18 months from the patent filing).

By using bioinformatic tools, we characterized enzymes and metabolic pathways that may be used for industrial applications. We focused mainly on alpha amylases since these have potential application in a wide number of industrial processes such as food, fermentation, textile, paper, detergent, and pharmaceutical industries. The enzymes that we characterized were studied at biochemical levels. We discovered the molecular characteristics that make these enzymes active at low temperatures.
Furthermore, we also focused on antifreeze proteins, which can be used for food or biological material preservation. We biochemically characterized antifreeze proteins from an Antarctic bacterium and we found that it possesses a high ice crystallization inhibition property (i.e. inhibit the formation of ice crystals that are known to damage cell membrane), an important property for food preservation in the cold.



We also analyzed the transcriptome from Euplotes focardii. By this analysis, we wanted to verify which genes are transcribed to live in cold conditions. We found that most of the genes that are expressed by these organisms are involved in the defense against oxidative stress or in the maintenance of protein homeostasis (e.g. chaperones). Oxidative stress is one of the major problems of Antarctic marine organisms, as oxygen is more soluble at low temperatures, causing an increase of reactive oxygen species concentration. Quantitative analysis showed that the expression of Hsp70 genes was induced when E. focardii cells were subjected to oxidative stress. By contrast, thermal stress did not cause Hsp70 induction. These results argue that E. focardii in its current environment is well protected against reactive oxygen species and are consistent with prior reports of constitutive Hsp70 expression as a defense against cold-induced protein denaturation. To better understand the mechanisms responsible for cold-adaptation, we also compared the E. focardii transcriptome with those from the mesophilic Euplotes species, E. crassus and E. harpa. This analysis shows that the main difference is due to the differential expression of rRNA.

With our results we produced 10 papers and 1 patent.

We organized the following workshops and seminars:
1- “Training School/Workshop: ""Bioinformatics approaches to adaptations, genome evolution and biological associations"", Computer Laboratory, University of Cambridge, 23th-27th March 2015” (http://www.metable.eu/project/training-school-persons). During this workshop, we performed the kick-off meeting and we discussed future plans.
2- “Dialogue on methods for ecology” November 15 2016, Cambridge (UK).
3- Prof. Pietro Liò seminar at BMR Genomics: “Introduction to Neural Networks in Bioinformatics”. September 4th, 2017
4- Prof. Pietro Liò seminar at BMR Genomics: “Machine learning analysis of gene families”. September 6th 2017.

We organized the following summer school:
1- “Toward a bio-based economy: science, innovation, economics, education” 4-8 of September 2017.

Dissemination activity: During the national congress of Italian Society of protistology (14-16 of September 2016, Torre del Cerrano, Pineto, Italy), Metable organized an open day where Researchers showed to ordinary people how protist may be an excellent source for biotechnology and for study adaptation and evolution.


Main challenges: Bureaucracy was a real unexpected challenge, in particular for VISA application to perform the secondment. An unexpected problem was the earthquake that struck the region that hosts UNICAM. This delayed the research activities in UNICAM for at least two months.

To have more information about our research you can visit our web page (http://www.metable.eu/metable/). You can leave comment and follow our everyday activities by following us on Twitter or Facebook.
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We characterized biocatalysts of interest.
a- Lipases : Thanks to the secondments from UNIMIB to EPI, new lipases sequences have been characterized from the genome and transcriptome of the psychrophilic ciliate Euplotes focardii. These sequences were analysed by bioinformatics tools (molecular modelling, molecular dynamics). The comparison with homologous from Euplotes crassus, a non cold-adapted congeneric species, allowed to characterize the unique residues from E. focardii lipases that may be responsible for cold-adaptation (manuscript in preparation). UNIMIB performed the biochemical characterization of these lipases.
b- Superoxide dismutases: At Unicam, Superoxide Dismutases (SODs) have been identified. These proteins have been biochemically characterized by UNIMIB (Mangiagalli et al., 2018).
c- alpha amylases: we characterized and also optimized alpha amylases for industrial purposes (Yang et al, 2017).
d- we characterized a new antifreeze protein that can be exploited for industrial application (Mangiagalli et al., 2017).

We have discovered bacterial secondary metabolites that have activity against nosocomial bacteria.
We deposited a patent and created a new spin-off named IriDEs.
We have discovered bacterial secondary metabolites that have activity against nosocomial bacteria. The secondary metabolites are now the topic of a patent that has been deposited. The impact of this new discovery is very important in this period in which nosocomial pathogens are becoming more and more resistant to traditional antibiotics.