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Titel (primär) Production of 2-hydroxyisobutyric acid from methanol by Methylobacterium extorquens AM1 expressing (R)-3-hydroxybutyryl coenzyme A-isomerizing enzymes
Autor Rohde, M.-T.; Tischer, S.; Harms, H.; Rohwerder, T.;
Journal / Serie Applied and Environmental Microbiology
Erscheinungsjahr 2017
Department UMB;
Band/Volume 83
Heft 3
Sprache englisch;
POF III (gesamt) R31; T15;
UFZ Querschnittsthemen RU4;
Abstract The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B12-dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564–4572, 2015, https://doi.org/10.1128/AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter−1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g−1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1.
ID 18533
dauerhafte UFZ-Verlinkung http://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=18533
Rohde, M.-T., Tischer, S., Harms, H., Rohwerder, T. (2017):
Production of 2-hydroxyisobutyric acid from methanol by Methylobacterium extorquens AM1 expressing (R)-3-hydroxybutyryl coenzyme A-isomerizing enzymes
Appl. Environ. Microb. 83 (3), e02622-16