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Thioesterases for ethylmalonyl–CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1

Abstract

The ethylmalonyl–coenzyme A pathway (EMCP) is a recently discovered pathway present in diverse α-proteobacteria such as the well studied methylotroph Methylobacterium extorquens AM1. Its glyoxylate regeneration function is obligatory during growth on C1 carbon sources like methanol. The EMCP contains special CoA esters, of which dicarboxylic acid derivatives are of high interest as building blocks for chemical industry. The possible production of dicarboxylic acids out of the alternative, non-food competing C-source methanol could lead to sustainable and economic processes. In this work we present a testing of functional thioesterases being active towards the EMCP CoA esters including in vitro enzymatic assays and in vivo acid production. Five thioesterases including TesB from Escherichia coli and M. extorquens, YciA from E. coli, Bch from Bacillus subtilis and Acot4 from Mus musculus showed activity towards EMCP CoA esters in vitro at which YciA was most active. Expressing yciA in M. extorquens AM1 led to release of 70 mg/l mesaconic and 60 mg/l methylsuccinic acid into culture supernatant during exponential growth phase. Our data demonstrates the biotechnological applicability of the thioesterase YciA and the possibility of EMCP dicarboxylic acid production from methanol using M. extorquens AM1.

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Acknowledgments

This work was funded by the European Union in the context of PROMYSE research project (FP7-KBBE.2011.3.6-04). We thank Dr. Tobias Jürgen Erb (Swiss Federal Institute of Technology Zurich, Institute of Microbiology) for his very useful advices on the synthesis and analytics of CoA esters.

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Correspondence to Jens Schrader.

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Sonntag, F., Buchhaupt, M. & Schrader, J. Thioesterases for ethylmalonyl–CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1. Appl Microbiol Biotechnol 98, 4533–4544 (2014). https://doi.org/10.1007/s00253-013-5456-y

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Keywords

  • Methylobacterium extorquens
  • Thioesterases
  • Ethylmalonyl–CoA pathway
  • YciA
  • Dicarboxylic acid
  • Coenzyme A