Cyanozymes – bioprospecting of selective biocatalyst from freshwater cyanobacteria

Abstract:

CyanoMining is dedicated to the investigation of the biology of specific cyanobacterial species thriving in identified mined-out Philippine areas or local environments with known history of mine tailings. The proposed project is aimed towards improving our knowledge on the biology of cyanobacteria with specific emphasis on strains including Synechococcus sp. and Chroococcus sp. that are ubiquitous in high-metal environments. CyanoMining deals with the elucidation of the genomes of newly identified cyanobacterial strains by deep sequencing, and an investigation of the conditions required for optimal metal accumulation.

Robert Kourist, Irene Rodriguez: Lab Tour at University of Philippines

© UP/Dolly Manic

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Implementation Period:

03/2024 – 05/2024

Project:

Project Objectives and Impact

CyanoMining is aimed towards elucidating the biology of specific cyanobacterial species thriving in identified mined-out areas or local environments with known history of mine tailings. The proposed project is aimed towards improving our knowledge on the biology of cyanobacteria with specific emphasis on strains including Synechococcus sp. and Chroococcus sp. that are ubiquitous in high-metal environments. The specific objectives of the project are:

– Knowledge transfer in the sampling and environmental context of cyanobacterial species (Philippine partner)
– Knowledge transfer in cultivation, DNA-isolation, physiological characterization and bioinformatic analysis of cyanobacteria (Austrian partner)

After the project, the partners started to plan on collaborations within the Southeast-Europe Joint Scheme on Research and Innovation (https://www.sea-europe-jfs.eu/call/2023-sti-joint-call-proposals-circular-economy-clean-accessible-and-secure-energy-supply-8th).

Results and Contributions

1. Knowledge transfer in the sampling and environmental context of cyanobacterial species (Philippine partner)

During the visit of Prof. Kourist, Dr. Schliep and Ing. Schabhüttl at the Bolinao Marine Lab, Pangasinán, The Philippines, the group of of Dr. Irene B. Rodriguez introduced the researcher to the Marine sampling sites and the cultivation opportunities at the Bolinao Marine Lab. A visit to the Campus Dilliman, UP, then introduced the visitors to the instrumental analytics available. Then, further cooperation and funding opportunities were discussed.

Prof. Kourist and Dr. Schliep then held courses at the Dilliman Campus for the group members of Dr. Rodriguez. These courses were available to other students of the UP Dilliman Campus.

Biocatalysis and Enzyme Engineering (Kourist): This course is for biologists interesting in the application of enzymes in biotechnological processes and their optimization by enzymes engineering, The course spans from examples of enzymatic reactions to the basics of protein engineering, including rational protein design, directed enzyme evolution and high-throughput assay screening. In a hands-on example, the students visualized structures of a decarboxylase and analyzed the position of amino acid mutations that changed the selectivity of the enzyme.

Bioinformatics (Schliep): This course is for biologists dealing with inference of phylogenetic trees from molecular sequences. These attendents address questions relative to the evolutionary relationships among these sequences, as well as the evolutionary forces structuring biodiversity at different scales.

The objectives of the course are: (i) to know how to choose a strategy of molecular data analysis, (ii) to be able to initiate a phylogenetic analysis starting from the files of molecular sequences using different inference methods (Maximum Parsimony, Maximum likelihood). And last, but not least (iii) interpret the results and create informative graphics.

The software we used in this workshop is centered on the R language for statistics and included particularly the specialized packages ape and phangorn.

2. Knowledge transfer in cultivation, physiological characterization and bioinformatic analysis of cyanobacteria (Austrian partner)

The two-week research visit of Dr. Irene B. Rodriguez and Dolly C. Manic from the Marine Science Institute of the University of the Philippines took place from May 6-10 and May 20-28, 2024. The Institute of Molecular Biotechnology of the Graz University of Technology provided laboratory space and supervision for training activities related to molecular biology techniques as specified in the project proposal.

During the first week of the training, the group worked on DNA extraction and purification to validate the identification of the E. coli strains used for the experiments and check if the target plasmid was present in its DNA sequence. After sequence confirmation, culture media preparation and inoculation of E. coli cells were conducted in preparation for protein production experiments. Several optical density (OD) measurements were performed during the incubation period until the target OD was achieved. Then, protein induction was carried out by adding IPTG, a molecular mimic of allolactose to trigger the transcription of the lac operon, which therefore controls the induction of protein expression.

After overnight incubation of E. coli cultures, cell-free protein analysis (BCA) was performed using a microplate reader.

In the second week, they conducted experiments using cyanobacteria Synechocystis. The expression cassette of the ene-reductase YqJM from Bacillus subtilis was integrated into the gene locus slr0168. To ensure the correct integration of the cassette into all genome copies, genomic DNA isolation, segregation check PCR, and gel electrophoresis were performed.

Next, the recombinant Synechocystis strain harboring YqjM was utilized for whole-cell cyanobacterial biotransformations. Cells previously cultivated were harvested and concentrated prior to the addition of substrate 2-methylmaleimide. During the incubation period, samples for gas chromatography as measurements were collected.

Moreover, other cyanobacterial measurements such as oxygen evolution and quantum efficiency of photosystem II were also carried out.