caa a22 4500 605504199 CHVBK 20210128100614.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-015-6418-3 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6418-3 High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains [Elektronische Daten] [Frank Sonntag, Jonas Müller, Patrick Kiefer, Julia Vorholt, Jens Schrader, Markus Buchhaupt] 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 0.65g/l, representing a yield of 0.17g/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. Springer-Verlag Berlin Heidelberg, 2015 Dicarboxylic acids nationallicence Ethylmalonyl-CoA pathway nationallicence Methylobacterium extorquens nationallicence Methanol nationallicence PHB nationallicence Sonntag Frank DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany aut Müller Jonas Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland aut Kiefer Patrick Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland aut Vorholt Julia Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland aut Schrader Jens DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany aut Buchhaupt Markus DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3407-3419 0175-7598 99:8<3407 2015 99 253 https://doi.org/10.1007/s00253-015-6418-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6418-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sonntag Frank DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany aut NATIONALLICENCE 700 1- Müller Jonas Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland aut NATIONALLICENCE 700 1- Kiefer Patrick Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland aut NATIONALLICENCE 700 1- Vorholt Julia Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland aut NATIONALLICENCE 700 1- Schrader Jens DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany aut NATIONALLICENCE 700 1- Buchhaupt Markus DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3407-3419 0175-7598 99:8<3407 2015 99 253