Horizontal pathway transfer to Escherichia coli from Rhodopseudomonas palustris: Transcription, translation, pathway extension

Metabolic engineering attempts to alter cellular pathways by the overexpression or deletion of genes associated with enhancing the yield of desired cellular products. The field focuses on improving the yield of metabolites within a cell, which sometimes includes the expression of heterologous genes to extend native pathways towards new metabolic targets. In this work we seek to expand the concept of heterologous gene expression to include pathway transfer from one organism to another as opposed to the practice of cloning single genes, one by one. We approach this goal of pathway transfer using two strategies, both of which aim to move a large piece of DNA from one organism to a new host such as Escherichia coil and have the DNA readily integrated with the underlying metabolic network.;The first approach modifies the E. coli host's transcription and translation apparatus to recognize native, unaltered DNA from the donor organism, Rhodopseudomonas palustris, a high GC photosynthetic alphaproteobacterium. The two E. coli host targets which we improve include the transcription apparatus, the sigma factor of RNA polymerase, and the translation apparatus, ribosomal protein S1, both of which enhance expression of foreign DNA.;Recognizing that the first approach to this problem may encounter some limitations, the second approach to this same problem utilizes DNA synthesis techniques to recreate a pathway from R. palustris. The design encompasses features amenable to expression in E. coli, including reduced GC content in both the coding genes and in the 5'-untranslated region important for expression of high GC DNA in a host such as E coli. The cyclohexane carboxylate degradation pathway from R. palustris was chosen as a model pathway enabling the degradation of this compound to pimeloyl-CoA, which feeds into the biotin production pathway of E. coli.;Finally, we present transcription profiling data from mutants in the R. palustris photosynthesis regulators PpsR1 and PpsR2. The data allow us to understand the components of this important metabolic function and to elucidate two photosynthetic regulons, whose characterization will assist in defining pathways which are available for future mobilization to E. coli or other organisms.