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

• Published in: Applied microbiology and biotechnology Vol. 99; no. 8; pp. 3407 - 3419
• Main Authors: Sonntag, Frank, Müller, Jonas E. N, Kiefer, Patrick, Vorholt, Julia A, Schrader, Jens, Buchhaupt, Markus
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.04.2015; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: acetyl coenzyme A; Acids; Acyl Coenzyme A - metabolism; Biomedical and Life Sciences; Biosynthesis; Biotechnological Products and Process Engineering; Biotechnology; Biotechnology - methods; Carbon; Carbon sources; Carboxylic acids; Cobalt; Cobalt - deficiency; Cobalt - metabolism; culture media; Culture Media - chemistry; Dehydrogenases; dicarboxylic acids; Dicarboxylic Acids - metabolism; E coli; Enzymes; Gene Knockout Techniques; Hydroxybutyrates - metabolism; Influence; Life Sciences; Metabolic Engineering - methods; Metabolism; Methanol; Methylobacterium extorquens; Methylobacterium extorquens - metabolism; Methylotrophs; Microbial Genetics and Genomics; Microbiology; Physiological aspects; Plasmids; Polyesters - metabolism; Polyhydroxyalkanoates; Polyhydroxybutyrate; Process engineering; Productivity; sodium; Studies; Sustainable development; Sustainable production; Trace elements; Europe; PHB; Dicarboxylic acids; Ethylmalonyl-CoA pathway; Methanol
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6418-3

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.