De novo production of the monoterpenoid geranic acid by metabolically engineered Pseudomonas putida

Abstract

BackgroundProduction of monoterpenoids as valuable chemicals using recombinant microbes is a growing field of interest. Unfortunately, antimicrobial activity of most monoterpenoids hampers a wide application of microorganisms for their production. Strains of Pseudomonas putida, a fast growing and metabolically versatile bacterium, often show an outstanding high tolerance towards organic solvents and other toxic compounds. Therefore, Pseudomonas putida constitutes an attractive alternative host in comparison to conventionally used microorganisms. Here, metabolic engineering of solvent tolerant Pseudomonas putida as a novel microbial cell factory for de novo production of monoterpenoids is reported for the first time, exemplified by geranic acid production from glycerol as carbon source. The monoterpenoic acid is an attractive compound for application in the flavor, fragrance, cosmetics and agro industries.ResultsA comparison between Escherichia coli, Saccharomyces cerevisiae and Pseudomonas putida concerning the ability to grow in the presence of geranic acid revealed that the pseudomonad bears a superior resilience compared to the conventionally used microbes. Moreover, Pseudomonas putida DSM 12264 wildtype strain efficiently oxidized externally added geraniol to geranic acid with no further degradation. Omitting external dosage of geraniol but functionally expressing geraniol synthase (GES) from Ocimum basilicum, a first proof-of-concept for de novo biosynthesis of 1.35 mg/L geranic acid in P. putida DSM 12264 was achieved. Doubling the amount of glycerol resulted in twice the amount of product. Co-expression of the six genes of the mevalonate pathway from Myxococcus xanthus to establish flux from acetyl-CoA to the universal terpenoid precursor isopentenylpyrophosphate yielded 36 mg/L geranic acid in shake flask experiments. In the bioreactor, the recombinant strain produced 193 mg/L of geranic acid under fed-batch conditions within 48 h.ConclusionMetabolic engineering turned Pseudomonas putida DSM 12264, a versatile monoterpenoid oxidation biocatalyst, into an efficient microbial cell factory for de novo geranic acid production. Improvements by metabolic and process engineering are expected to further increase the product concentration. To the best of the authors’ knowledge, this is the first example of a de novo production of a monoterpenoid with Pseudomonas putida and of a microbial monoterpenoic acid synthesis in general.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0170-8) contains supplementary material, which is available to authorized users.

Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli

“…The present work showed that geraniol production was increased to 48.5 ± 0.9 mg L −1 by engineering tCaGES and the foreign genes mentioned above. The geraniol yield of this study is higher than those in transgenic tobacco and yeast; also, parallel to or higher than the yields of other terpenes production in metabolically engineered microorganisms [23,32,39,40,42]. Recently it has been demonstrated that deletion of YjgB, an endogenous dehydrogenase responsible for geraniol loss in E. coli MG1655, will increase geraniol production to 182.5 mg L −1 [38].…”

“…In addition, the fed-batch fermentation was proved to increase the yields of other terpenes [23,39,40,42]. Thus, this study will be an alternative to selectively produce geraniol in engineered E. coli, together with other metabolic engineering methods such as balancing the whole biosynthetic pathway and using fed-batch fermentation.…”

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

“…Concerning tolerance to hydrophobic and toxic molecules such as the monoterpene limonene, which, for example, may serve as a precursor for jet biofuels in the future [10,66], P. putida should have a clear advantage over monoterpene-sensitive S. cerevisiae [16,17,54]. However, the first de novo production of a monoterpene with P. putida has been described only very recently [53]. By introducing a heterologous bacterial mevalonate pathway from Myxococcus xanthus together with a plant geraniol synthase, P. putida GS1 produced about 200 mg/L geranic acid in a bioreactor.…”

Microbial production of scent and flavor compounds

“…Geranic acid has a sweet, woody or leafy flavor with hints of citrus. Incorporation of the mevalonate pathway as well as geraniol synthase enables flux towards geraniol in P. putida, which is natively oxidized to geranic acid [46] (Figure 4). The optimization of the mevalonate pathway and transposition of this system to a fed-batch bioreactor yielded 193 mg/L geranic acid from 4.6 g/L glycerol over 2 days (Table 1) [46].…”

“…Pseudomonas putida is a host organism that displays high solvent tolerance [45], and has been utilized as a host for geranic acid production from glycerol ( Figure 4) [46]. Geranic acid has a sweet, woody or leafy flavor with hints of citrus.…”