Results

Natural secondary metabolites encompass a multitude of bioactive compounds. These can have numerous applications, e.g. in the agricultural field for pest control or in the pharmaceutical industry. Since the total chemical synthesis is often tedious, recombinant production in a robust microbial host can facilitate access to such compounds and derivatives thereof. The VIOLA project established the production of indole alkaloids, namely (deoxy)violacein, in the bacterium Pseudomonas putida KT2440. The violacein-type bisindoles are of interest due to various relevant bioactivities including inhibition of devastating fungal plant pathogens like Phytophthora, Fusarium or Botrytis. Therefore, the respective biosynthesis was implemented in P. putida by genomic integration of genes from Chromobacterium violaceum into the rDNA. P. putida intrinsic metabolism provided L-tryptophan as precursor, which was successfully converted in several catalytic steps to the product. Using robotic workflows for cultivation, sampling and at-line analytics, different cultivation conditions for the production host were evaluated. Thereby,  the process procedure for an effective conversion of L-tryptophan into deoxyviolacein was optimized. In addition, isolation protocols were established. Moreover, the mutasynthesis for the conversion of L-tryptophan analogs to new violacein derivatives was explored. The deletion of intrinsic L-tryptophan biosynthesis, and a controlled fed-batch strategy allowed the production of biomass as catalyst for mutasynthesis without initial deoxyviolacein formation. This facilitated the feeding of synthesized L-tryptophan analogs, such as 1-methyl-tryptophan and 7-chloro-tryptophan. In summary, a concept for the effective recombinant production of deoxyviolacein and derivatives thereof was developed.