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

Microbial Cell Factories

Research

Markus Buchhaupt¹  Jens Schrader¹  Jia Mi¹  Daniela Becher¹  Patrice Lubuta¹  Kerstin Tusch¹  Hendrik Schewe¹  Sarah Dany¹ 

[1] DECHEMA Research Institute, Frankfurt am Main, Germany;

关键词: Pseudomonas putida;    de novo;    DSM12264;    Microbial cell factory;    Monoterpene;    Monoterpenoid;    Mevalonate pathway;    Metabolic engineering;    Geranic acid;    Geraniol;   

DOI  :  10.1186/s12934-014-0170-8

 received in 2014-07-25, accepted in 2014-11-19,  发布年份 2014

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Mi et al.; licensee BioMed Central Ltd. 2014. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.