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Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid

Paper ID Volume ID Publish Year Pages File Format Full-Text
31819 44841 2006 16 PDF Available
Title
Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid
Abstract

In this study strains of Ralstonia eutropha H16 and Pseudomonas putida KT2440 were engineered which are suitable for biotechnological production of 2-methylcitric acid (2MC). Analysis of a previous mutant of R. eutropha able to accumulate 2MC recommended this strain as a candidate for fermentative production of 2MC. This knowledge was used for construction of strains of R. eutropha H16 and P. putida KT2440 capable of enhanced production of 2MC. In both bacteria the chromosomal genes encoding the 2-methyl-cis-aconitate hydratase (acnM) were disrupted by directed insertion of a copy of an additional 2-methylcitrate synthase gene (prpC) yielding strains R. eutropha ΔacnMReΩKmprpCPp and P. putida ΔacnMPpΩKmprpCRe. In both strains 2-methylcitrate synthase was expressed under control of the constitutive kanamycin-resistance gene (ΩKm) resulting in up to 20-fold higher specific 2-methylcitrate synthase activities in comparison to the wild type. The disruption of the acnM gene by insertion of prpC led to a propionate- and levulinate-negative phenotype of the engineered strains, and analysis of supernatant of these strains revealed overproduction and accumulation of 2MC in the medium. A two stage cultivation regime comprising an exponential growth phase and a 2MC production phase was developed and applied to both engineered strains for optimum production of 2MC. Whereas gluconate, fructose or succinate were provided as carbon source for the exponential growth phase, a combination of propionate or levulinate as precursor substrate for provision of propionyl-CoA and succinate or fumarate as precursor substrate for provision of oxaloacetate were used in the production phase to make sure that the 2-methylcitrate synthase was provided with their substrates. Employing the optimised feeding regime P. putida ΔacnMPpΩKmprpCRe and R. eutropha ΔacnMReΩKmprpCPp produced 2MC up to maximal concentrations of 7.2 g/L or 26.5 mM and 19.2 g/L or 70.5 mM, respectively, during 144 h of cultivation.

Keywords
Levulinate; Metabolic engineering; 2-Methyl-cis-aconitate hydratase; 2-Methylcitrate; 2-Methylcitric acid cycle; Propionate; PrpC cluster; Pseudomonas putida; Ralstonia eutropha
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Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 8, Issue 6, November 2006, Pages 587–602
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us