Applied Microbiology and Biotechnology

, Volume 99, Issue 8, pp 3407–3419

High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains

  • Frank Sonntag
  • Jonas E. N. Müller
  • Patrick Kiefer
  • Julia A. Vorholt
  • Jens Schrader
  • Markus Buchhaupt
Biotechnological products and process engineering


Bio-based production of dicarboxylic acids is an emerging research field with remarkable progress during the last decades. The recently established synthesis of the ethylmalonyl-CoA pathway (EMCP)-derived dicarboxylic acids, mesaconic acid and (2S)-methylsuccinic acid, from the alternative carbon source methanol (Sonntag et al., Appl Microbiol Biotechnol 98:4533–4544, 2014) gave a proof of concept for the sustainable production of hitherto biotechnologically inaccessible monomers. In this study, substantial optimizations of the process by different approaches are presented. Abolishment of mesaconic and (2S)-methylsuccinic acid reuptake from culture supernatant and a productivity increase were achieved by 30-fold decreased sodium ion availability in culture medium. Undesired flux from EMCP into polyhydroxybutyrate (PHB) cycle was hindered by the knockout of polyhydroxyalkanoate synthase phaC which was concomitant with 5-fold increased product concentrations. However, frequently occurring suppressors of strain ΔphaC lost their beneficial properties probably due to redirected channeling of acetyl-CoA. Pool sizes of the product precursors were increased by exploiting the presence of two cobalt-dependent mutases in the EMCP: Fine-tuned growth-limiting cobalt concentrations led to 16-fold accumulation of mesaconyl- and (2S)-methylsuccinyl-CoA which in turn resulted in 6-fold increased concentrations of mesaconic and (2S)-methylsuccinic acids, with a combined titer of 0.65 g/l, representing a yield of 0.17 g/g methanol. This work represents an important step toward an industrially relevant production of ethylmalonyl-CoA pathway-derived dicarboxylic acids and the generation of a stable PHB synthesis negative Methylobacterium extorquens strain.


Dicarboxylic acids Ethylmalonyl-CoA pathway Methylobacterium extorquens Methanol PHB 


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Frank Sonntag
    • 1
  • Jonas E. N. Müller
    • 2
  • Patrick Kiefer
    • 2
  • Julia A. Vorholt
    • 2
  • Jens Schrader
    • 1
  • Markus Buchhaupt
    • 1
  1. 1.DECHEMA Research InstituteFrankfurt am MainGermany
  2. 2.Institute of MicrobiologyETH ZurichZurichSwitzerland

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