* Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3
In this study, we used label-free quantitative proteomics to generate reference proteome data for B. methanolicus MGA3 and compared the proteome on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50C and 37C). From a total of approximately 1200 different detected proteins, approximately 1000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested,the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the synthesis of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional tricarboxylic acid cycle, the proteins of which are differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. This work was recently published Müller J. E.N et al., 2014, Proteomics 14:725-737.
* Biochemical characterization of key enzymes of B. methanolicus RuMP pathway:
This work has involved careful selection of genes expected to encode key enzymes of the central RuMP pathway for formaldehyde assimilation and the work has generated new and surprising insight into the biochemistry of methylotrophy in model organism B. methanolicus highly useful for the engineering of methylotrophy into new bacterial hosts. The work was recently accepted for publication (Stolzenberger et al. 2013).
* Publication of a novel class of methanol dehydrogenases:
From the genome sequences we discovered that B. methanolicus strains surprisingly possess three different mdh genes; two chromosomal and one located on a plasmid. The organization of the mdh genes in PB1 and MGA3 were different and also biochemical characterizations of all six MDHs revealed that they are different with respect to kinetic properties, substrate preferences and responses to the common activator protein ACT. This discovery has unravelled new insight into the complexity of methanol oxidation as well as provided several new promising candidate genes useful for engineering methylotrophy into any bacterial host species. This work was recently published (Krog et al. 2013. PLOS ONE vol 8 issue 3:e59188).
* Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum:
In this study, gene-directed deletion mutagenesis was employed to decipher the functions of the genes present in the main carotenogenic gene cluster of C. glutamicum and in a second cluster encoding putative phytoene synthase and phytoene desaturase paralogs. Moreover, the potential of C. glutamicum to produce carotenoids was estimated by metabolic engineering of the conversion of GGPP to lycopene. This work was published by Heider et al., 2012 in journal BMC Microbiol.
* Publication of two Bacillus methanolicus genome sequences as basis for PROMYSE progress:
Comparative methanol fermentations unravelled major differences in the methylotrophic properties of two B. methanolicus (methanol uptake, CO2 evolution, glutamate secretion) wild-type strains MGA3 and PB1. We previously genome sequenced MGA3 and we chose to also genome sequenced PB1 and both genomes were published as a full research paper (Heggeset et al. 2012. Appl Environ Microbiol 78:5170 – 81). These data have represented a crucial basis and source in PROMYSE for understanding physiological and genetic/regulatory aspects of bacterial methylotrophy.
* Publication of new knowledge related to methylotrophic pathways and regulation of important PROMYSE model methylotroph Methylobacterium extorquens.
As B. methanolicus has been our main model organism for the gram-positive methylotrophs, M. extorquens has been the major model organism for gram-negative methylotrophs and during the project period crucial new information on methylotrophic pathways and their regulation has been generated and published also for M. extorquens (Peyraud et al. 2012 PLoS ONE 7:e48271; Schneider et al. 2012 J. Biol. Chem. 287:757-766).