Metabolic engineering to understand and control productionof l-glutamate from methanol by the methylotrophic thermotolerant bacterium Bacillus methanolicus
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The production of L-glutamate and L-lysine together excides 3.5 million tons annually and are the most important amino acids in production volume. Their production is based on fermentation processes with the bacterium Corynebacterium glutamicum utilizing sugar as carbon and energy source. The thermotolerant methylotroph bacterium Bacillus methanolicus is a candidate for amino acid production from methanol at 50 ºC, using an alternative carbon source. In this work, the genome sequences of the two wild type B. methanolicus strains, MGA3 (ATCC53907) and PB1 (NCIMB13113) are presented, representing the first genome sequences of gram positive methylotrophic bacteria. The gained information from the sequencing of the B. methanolicus sequences has led to the identification of genes encoding enzymes belonging to important biochemical pathways. Genes representing a dissimilatory ribulose monophosphate and a linear tetrahydrofolate pathway, D-mannitol and D-glucose uptake systems, genes representing a full TCA cycle and the anaplerotic node, genes encoding enzymes needed for the acetylase variant of the L-lysine biosynthetic pathway, putative L-lysine exporters and genes encoding enzymes and regulators in the L-glutamate biosynthetic pathway were identified, opening up for a wide range of opportunities for systems-level metabolic engineering of this organism. Three methanol dehydrogenases (MDH) encoding genes and one gene encoding an activator protein were identified in each of the two B. methanolicus genomes. All six MDHs displayed dehydrogenase activity on a range of primary alcohols and reductase activity on formaldehyde and acetaldehyde. They all showed enhanced enzymatic activity as a response to the addition of an activator protein. Sequence comparison of the MDH protein sequences clustered them into two subtypes which showed more than 90% sequence identity among the MDHs of each subtype, while the identity between the subgroups was about 60%. All MDHs were biochemically characterized and their kinetic constants for methanol and formaldehyde determined alone and in the presence of the activator protein. The MDH enzymes within each of the two subgroups were shown to have similar biochemical properties, which confirmed the presence of two subtypes of MDHs. B. methanolicus was shown to harbor one gene encoding a glutamate dehydrogenase and genes encoding two GOGAT enzymes, GltAB and GltA2, where the second GOGAT is a single subunit GOGAT enzyme. GDH and GOGAT from MGA3 were biochemical characterized, and their Km and Vmax for 2-oxoglutarate, ammonium, Lglutamine and L-glutamate determined, enlightening their role in L-glutamate synthesis and degradation. The individual enzymes impact on L-glutamate production rates were studied and all genes encoding enzymes catalyzing reactions leading the carbon flow from pyruvate to L-glutamate were cloned and overexpressed in the MGA3 wild type strain. Cultivations were performed in high cell density cultivations and in shake flasks and the growth media analyzed with respect to amino acid concentrations. Only the overexpression of glutamate synthase had a positive effect on the L-glutamate production level under the conditions tested, while the overexpression of 2-oxoglutarate dehydrogenase lead to significant reduced L-glutamate concentrations, pointing these to be key enzymes for L-glutamate production. Until now, no genetic knowledge was available regarding L-glutamate synthesis and degradation in B. methanolicus. This work provides insight into metabolic traits of B. methanolicus in general, which opens up for further metabolic engineering of diverse pathways of this bacterium, but especially for that of L-lysine and L-glutamate synthesis and degradation.