In situ monitoring of the Shikimate pathway: A combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging.

Our research is focused on evaluating the potentials of Raman reverse stable isotope probing for sensing and visualizing the Shikimate pathway activity in prokaryotic and eukaryotic model systems in a cost-effective and non-destructive manner at community and single cell level in situ. In this work, we have successfully demonstrated that by using unlabeled 12C-glucose as the sole carbon source in the 13C labelled microbial culture medium the Shikimate pathway can be monitored qualitatively and quasi-quantitatively. The Shikimate Pathway links central carbon metabolism to biosynthesis of aromatic ring containing metabolites such as aromatic amino acids, folates, salicylic acid, and others. The incorporation 12C in the 13C labelled metabolome pool causes a blue shift in the Raman spectra. Phenylalanine was selected as representative band shikimate pathway activity marker. Temporal changes in the intensity of representative bands over a period of 0 to 24 hours have been analyzed to demonstrate the feasibility of this approach. We have also visualized the Shikimate pathway activity and phenylalanine turnover using Raman hyperspectral imaging aided by multivariate curve resolution. The novelty of our study lies in the fact that for the first time Shikimate pathway activity has been monitored in situ in a cost-effective, extraction free, and non-destructive manner. The overall findings are very encouraging and support the prospective utility of this approach for monitoring pathways related to aromatic rings containing metabolites. This can have a wide range of applications in sensing commercially and medically relevant microbial metabolites in situ.