Antonio García-Moyano (Uni Research, Norway) - Discovery of proteases for bioconversion of marine biomass
Gro Elin Kjæreng Bjerga1, Runar Stokke2, Øivind Larsen3, Hasan Arsin2, Pål Puntervoll4, Ingar Leiros5, Arne O. Smalås5 and Ida Steen2
1Norzymed, Uni Research Miljø, Bergen, NOR
2Department Of Biology, University Of Bergen, Bergen, NOR
3Uni Research, Bergen, NOR
4Center For Applied Biotechnology, Uni Research, Bergen, NOR
5Department Of Chemistry, University Of Tromsø, Tromsø, NOR
New enzymes will be key to future developments and value creation in the emerging marine biobased industry. The Norwegian fishing industry generates 890 000 tons by-products, such as heads and frames annually. This is mainly used in animal feed, oil and fish silage, or discarded. The by-products contain high-quality proteins and have a great potential as high-value products for human consumption. The few commercial enzymes used for fish hydrolysis are, however, not optimal. Together with the Norwegian ingredient industry and academic partners we utilized various strategies to identify new proteases for the marine bio-based industry. Using (meta)genomic mining new proteolytic enzymes were identified on a panel of substrates. The genes originate from in situ enrichments at deep-sea hot vent systems and from Arctic, marine microbial species. In-depth characterization and structural studies of enzymes are ongoing and their performance in hydrolysis of marine biomass at pilot scale is currently evaluated.
Anja Martinez (Thermo Fisher Scientific, Sweden) - Enhanced CRISPR/Cas9-mediated precise genome editing
Jason Potter1, Xiquan Liang1, Shantanu Kumar1, Namritha Ravinder1, Anja Martinez2, Julie Wood3 and Jonathan D Chesnut1
1Synthetic Biology R&D Thermo Fisher Scientific, Carlsbad, USA
2Synthetic Biology, Thermo Fisher Scientific, Stockholm, SWE
3Synthetic Biology, Thermo Fisher Scientific, London, GBR
While CRISPR-based gene knockout in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. We now present up to 56% targeted integration efficiency of up to a six-nucleotide insertion in HEK293 cells. In iPSCs we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. Also, the use of a dsDNA oligonucleotide with 3’ overhangs allowed integration of a longer tag at rates of up to 50%. We propose a model favoring donor DNAs with the change as close to the cleavage site as possible. For small changes, asymmetric single-stranded donors seem to be favored. For larger insertions (e.g. FLAG tag), a dsDNA donor with protruding 3’ homology arms of 30 bases is favored. Modifying the donor DNA ends with phosphorothioate further improves the editing efficiency.
Jenny M. Söderberg (UiT The Arctic University of Norway) - Development of a novel expression host for challenging enzymes
Jenny Söderberg1, Miriam Grgic1 and Peik Haugen1
1Department Of Chemistry , The Artic University Of Norway Uit, Tromsø, NOR
Successful expression and purification of enzymes represents a major challenge, in particular psychrophilic (cold adapted) enzymes are difficult to express in standard mesophilic hosts (e.g. E. coli). This group of enzymes are commercially and economically relevant due to their unique properties. However, the lack of specialised expression systems continue to hamper development of new psychrophilic enzymes to the market. Typically, low expression, misfolded/non-functional protein and inclusion bodies hinder successful protein production.
We have recently identified bacterial isolates that are promising hosts for expressing psychrophilic and challenging proteins. We have performed basic characterization such as growth at different temperatures, up take of genetic material and expression of fluorescent (reporter) proteins from plasmids. Interestingly, one isolate was highly suitable as an expression host. We are currently working with difficult enzyme targets that has shown to fail when expressed in E.coli.