The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric 22 acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires 23 suitable (R)-3-hydroxybutyryl-CoA specific coenzyme B12-dependent mutases (RCM). Here, 24 we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic 25 RCM, closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae 26 DSM 2912 (M.-T. Weichler, N. Kurteva-Yaneva, D. Przybylski, J. Schuster, R. H. Müller, H. 27 Harms, and T. Rohwerder, Appl Environ Microbiol 81:4564-4572, 2015, 28 http://dx.doi.org/10.1128/AEM.00716-15). Using both RCM variants, 2-HIBA production 29 from methanol was studied in fed-batch bioreactor experiments with recombinant 30 Methylobacterium extorquens AM1. After complete nitrogen consumption, concomitant 31 formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were 32 successfully expressed. However, although identical vector systems and incubation conditions 33 were chosen, metabolic activity of the variant bearing the RCM genes from strain DSM 2912 34 was severely inhibited, likely due to negative effects caused by the heterologous expression. 35 In contrast, biomass yield of the variant expressing the JC6 genes was close to wild-type performance and 2-HIBA titers of 2.1 g L-1 36 could be demonstrated. In this case, up to 24% of 37 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 38 were achieved. 39 Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as 40 methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly down-regulated 41 after exponential growth which currently prevents a prolonged overflow phase and, thus, 42 higher product yields with strain AM1.