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

Jia Mi,Patrice Lubuta,Hendrik Schewe,Markus Buchhaupt,Kerstin Tusch,Jens Schrader,Daniela Becher,Sarah Dany

doi:10.1186/s12934-014-0170-8

Jia Mi, Patrice Lubuta + Show 6 more

Open Access

https://doi.org/10.1186/s12934-014-0170-8

Copy DOI

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.

Highlights

Production of monoterpenoids as valuable chemicals using recombinant microbes is a growing field of interest
Suitability of P. putida as a host for geranic acid production To verify the expected advantage of P. putida for monoterpenoid production compared with conventional host strains, i.e. an assumed superior product tolerance, growth assays with P. putida DSM 12264, E. coli DH5α and S. cerevisiae CEN.PK2-1C in the presence of geranic acid were performed (Figure 1)
According to Brennan and colleagues, shake flasks with screw caps were used for growth experiments to avoid monoterpenoid evaporation amounts of geraniol were added to growing cells in E2 medium and resulting geranic acid concentrations were determined at different time points within 64 h (Figure 2)

Summary

Introduction

Production of monoterpenoids as valuable chemicals using recombinant microbes is a growing field of interest. A couple of industrial processes with P. putida have already been established [20] and more wildtype and recombinant P. putida strains have been recently described for potential industrial de novo production of toxic aromatic compounds such as phenol [21] and p-hydroxybenzoate [22] or for biotransformation of toxic precursors such as toluene to o-cresol [23] or the monoterpenoid limonene to perillic acid [24] This illustrates the potential of this species as an alternative host for biotechnological applications, especially if toxic substrates or products are dealt with. De novo monoterpenoid production with P. putida has not been reported until the present work

Objectives

Methods

Results

Discussion

Conclusion