Characterizing the native retrotransposons of Neocallimastix frontalis to enable genetic modification of anaerobic gut fungi

• Alex Hinton, Chemical Engineering, University of Delaware

• Kevin Solomon, Chemical and Biomolecular Engineering, University of Delaware

There are many organisms that produce useful enzymes and natural products that are unexploited due to lack of effective ways to genetically engineer them. One example is anaerobic gut fungi (AGF), which produce many enzymes that effectively degrade biomatter and are also hypothesized to produce useful natural products such as antibiotics. AGF as well as many other species of Basal fungi have been understudied so there are currently a lack of effective methods to engineer them. In order to develop a toolkit to genetically engineer these species, we have investigated long terminal repeat (LTR) retrotransposons native to the anaerobic gut fungus Neocallimastix frontalis. These elements have LTRs, repeated DNA sequences, flanking a pol gene. That gene encodes for the retrotransposase protein which is capable of binding to RNA encoding LTRs, reverse transcribing the RNA into DNA, and inserting that DNA back into the genome. The protein contains capsid, protease, reverse transcriptase, and integrase domains. From expressing an affinity tagged retrotransposase in E. coli, we have found that after the protein is produced it is cleaved into its separate domains, likely by its own protease domain. We have also expressed the capsid and reverse transcriptase domains independently in E. coli and have found that they are mostly insoluble; however, when expressed at lower temperatures and in media containing sorbitol, both of which slow protein folding, they are more soluble. To further investigate their solubility we will express them in Saccharomyces cerevisiae which is expected to improve folding and enzyme activity as Saccharomyces provides a folding environment closer to that of its native host. In the future we will also test the activity of the reverse transcriptase domain, both when folded in E. coli and in Saccharomyces as well as the capsid domain’s ability to bind to RNA. Characterizing these domains and finding optimal conditions for them to fold and function can lead to these elements being used for genetic modification of species such as AGF, advancing functional genomics and the development of enzyme overexpression systems enabling more efficient degradation of biomass.