Biochemical and genetic basis of natural product biosynthesis in social amoebae
Zusammenfassung der Projektergebnisse
Most of the 40 functional PKS genes present in the D. discoideum genome contain DH (dehydratase), ER (enol reductase), and KR (keto reductase) domains, suggesting that D. discoideum mostly produces highly reduced polyketides that may be hard to detect by the conventional HPLC-UV approach. In this project we focused on the identification of the polyketide product generated by PKS26, because pks26 knockout cells were reported by Shaulsky and colleages (Santorelli et al., Nature 2008) to possess a „loser“ phenotype and produce less spores than their fair share when mixed whith an equal amount of wild-type cells. This phenotype may indicate a role of the putative PKS26 product in terminal cell differentiation of D. discoideum multicellular development. We found during the analysis of pks26 mutants that a metabolomic UPLC-HRMS-based approach analyzing the whole spectrum of metabolites in appropriate extracts produces high-quality data that were reproducible between different biological replicates of the same strain and even between different strains. Unfortunately, however, we have not been able to detect the product of PKS26 by comparing the wild-type cells to either a knockout or an overexpressor of pks26. This result may imply that the PKS26 product is produced at too low level to be detectable even with highly sensitive methods and in a supposed overexpressor strain, although we cannot exclude that we missed a particular extraction method required to isolate this compound or that the compound is very unstable. We have set up a biological assay that had the potential to reproduce the „loser“ phenotype of pks26 knockout cells reported by Shaulsky and colleages. In our hands, the phenotype was not observed, neither in the original Shaulsky strain nor in our pks26 knockout. Thus, the possibility exists that pks26 function is not related to the regulation of stalk-spore-ratios during terminal differentiation in D. discoideum. Instead, we observed that pks26 is in fact a cAMP-regulated gene that is maximally expressed during aggregation. Thus, the question remains as to whether the regulation of pks26 is coincidental in a way that genome evolution has „accidentally“ placed the pks26 gene under control of a cAMP-induced promoter without a dedicated biological function, or whether the putative PKS26 product controls cellular functions during early development that are not related to aggregation.
