De Novo Production of Monoterpenoic Acids with Pseudomonas Putida

Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more difficult with conventional host strains due to product toxicity. Hence, we set out to investigate the potential of a solvent tolerant Pseudomonas putida strain for de novo monoterpene production. Our target molecules are geranic acid and perillic acid, plant monoterpenoic acids which show broad antimicrobial activity and are of commercial interest, e.g. as natural preservatives for cosmetics or antifungal agrochemicals [1,2].

Since P. putida DSM12264 wild type is able to oxidize geraniol to geranic acid and limonene to perillic acid, the functional expression of plant geraniol or limonene synthases already led to the production of small amounts of the desired acids in shake flask cultures indicating exploitation of terpene precursors derived from the endogenous MEP pathway. Precursor supply was improved by expression of the heterologous mevalonate (MVA) pathway, an elegant concept of engineering the terpene biosynthesis in microbes first published by Martin et al. 2003 [3]. As donor strain of the MVA genes, we chose Myxococcus xanthus for two reasons: the ease of the cloning procedure (5 of 6 genes are organized in one operon) and the fact that it is a prokaryot which may facilitate functional expression in the prokaryotic host. P. putida strains coexpressing Mentha spicata limonene synthase and MVA pathway showed a 17-fold increase in formation of perillic acid (0.68 mg/L). Strains coexpressing Ocimum basilicum geraniol synthase and MVA pathway showed a 13-fold increase in formation of geranic acid (16.8 mg/L); after doubling the concentration of the C source glycerol a product concentration of 36.1 mg/L was achieved in shake flasks. Further improvements of productivity and final product concentration can therefore be expected with an optimized fed-batch process on bioreactor scale. This is, to our best knowledge, the first example of de novo monoterpenoic acid production with an engineered microbe. We intend to further improve the product titers in the medium by pathway and bioprocess engineering to eventually take advantage of the host’s pronounced monoterpene tolerance. Comparing growth of E. coli, S. cerevisiae and P. putida showed, that P. putida has a several times higher tolerance towards limonene, perillic acid and geranic acid and an equal tolerance towards geraniol. We are also investigating the cellular mechanisms to identify those responsible for the monoterpene robustness of this strain. We assume the involvement of energy-dependent efflux pumps and a cellular response by adapting the membrane composition [4].

[1] Mirata MA et al. (2009) Integrated bioprocess for the oxidation of limonene to perillic acid with Pseudomonas putida DSM 12264, Process Biochem  44: 764–771

[2] Yang T et al. (2011) Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns, Metabolic Engineering 13(4):414-25

[3] Martin VJ et al. (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nat Biotechnol 21(7):796-802.

[4] Segura A et al. (2012) Solvent tolerance in Gram-negative bacteria, Curr Opin Biotechnol 23(3):415-21