In the spliceotide, work, we uncovered new biosynthetic gene clusters encoding for spliceotides from Eubacterium rectale and a number of Bacteroidetes strains. A number of experimental designs were tested to obtain enzymatic activity for splicease enzymes: heterologous expression in E. coli and Microvirgula aerodenitrificans, and homologous and heterologouns expression in Bacteroides thetaiotaomicron. Most recently, we attempted to express the protein precursor and splicease individually to optimize expression and purification conditions. We hoped that by exploring the in vitro activity of the splicease, we could determine what are the critical parameters for catalysis. However, attempts to get in vitro activity failed.
To tackle the function of spliceotide products, we turned to characterizing better behaved systems. We tested the activities of spliceotide products from Cystobacter fuscus, Pseudoalteromonas phenolica, Rheinheimera aquimaris, and Pseudoalteromonas piscicida against a number of bacterial strains and proteases. While no antibacterial activity was observed, all spliceotides had some protease inhibitory activity:
• C. fuscus spliceotide: IC50 of 2.6 mM against cathepsin B and of 2.6 nM against human neutrophil elastase (HNE)
• P. piscicida spliceotide: IC50 of 0.4 mM against chymotrypsin
• R. aquimaris spliceotide: IC50 of 0.5 mM against chymotrypsin, 1.3 against Cathepsin B and 36 nM against HNE
• P. phenolica spliceotide: IC50 of 20 mM against HNE
These results are detailed in the manuscript: “Widespread microbial utilization of ribosomal β-amino acid-containing peptides and proteins“, currently under review
In the origamin work, we discovered origamin BGCs from gut microbes such as E. coli, Bifidobacterium longum, and Collinsella aerofaciens. We had previously demonstrated the activities for two enzymes in origamin biosynthesis in E. coli, but were not able to reproduce those results in origamins from gut bacteria. In the E. coli system, we were also unable to characterize all biosynthetic enzymes and therefore were not able to obtain the final origamin product.
Nonetheless, we expanded our search of origamin clusters with the goal of isolating a final origamin product by obtaining activity of all key biosynthetic enzymes. In our bioinformatic search, we discovered a new type of origamin cluster which contains 3 key biosynthetic enzymes, in addition to the precursor peptide: an epimerase rSAM, a SAM-dependent N-methyltransferase, and a B12-dependent C-methyltransferease. We identified this cluster in organisms such as Polymorphum gilvum and Actinosynnema sp. We then expressed the precursor peptide and tailoring enzymes from P. gilvum. Since E. coli does not have the metabolic capability to produce the B12 cofactor, we turned to an alternative heterologous expression host, Microvirgula aerodenitrificans. M. aerodenitrificans is a Gram-negative denitrifier and was isolated from activated sludge. Our research group engineered M. aerodenitrificans for heterologous expression, through the development of a suitable expression plasmid and transformation procedures. We thus heterologously expressed the partial or whole P. gilvum cluster, and, upon co-expression of all tailoring enzymes, we observed 18 epimerizations, 13 N-methylations, and additional 13 C-methylations. Our group is currently working on the full structural elucidation of this final product and on measuring its biological activity.
A manuscript detailing these findings is in preparation and will be submitted to publication later this year.
