title: High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains.
abstract: 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.
authors: DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany.
authors: Frank
authors: F
authors: Sonntag
authors: Jonas E N
authors: JE
authors: Müller
authors: Patrick
authors: P
authors: Kiefer
authors: Julia A
authors: JA
authors: Vorholt
authors: Jens
authors: J
authors: Schrader
authors: Markus
authors: M
authors: Buchhaupt
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D003035:Cobalt / Q000172:deficiency* / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D003998:Dicarboxylic Acids / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006885:Hydroxybutyrates / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D020601:Methylobacterium extorquens / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D000214:Acyl Coenzyme A; D003035:Cobalt; D003470:Culture Media; D003998:Dicarboxylic Acids; D006885:Hydroxybutyrates; D011091:Polyesters; C102829:ethylmalonyl-coenzyme A; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates