Abstract
The thermotolerant methylotroph Bacillus methanolicus MGA3 was originally isolated from freshwater marsh soil. Due to its ability to use methanol as sole carbon and energy source, B. methanolicus is increasingly explored as a cell factory for the production of amino acids, fine chemicals, and proteins of biotechnological interest. During high cell density fermentation in industrial settings with the membrane-permeable methanol as the feed, the excretion of low molecular weight products synthesized from it will increase the osmotic pressure of the medium. This in turn will impair cell growth and productivity of the overall biotechnological production process. With this in mind, we have analyzed the core of the physiological adjustment process of B. methanolicus MGA3 to sustained high osmolarity surroundings. Through growth assays, we found that B. methanolicus MGA3 possesses only a restricted ability to cope with sustained osmotic stress. This finding is consistent with the ecophysiological conditions in the habitat from which it was originally isolated. None of the externally provided compatible solutes and proline-containing peptides affording osmostress protection for Bacillus subtilis were able to stimulate growth of B. methanolicus MGA3 at high salinity. B. methanolicus MGA3 synthesized the moderately effective compatible solute L-glutamate in a pattern such that the cellular pool increased concomitantly with increases in the external osmolarity. Counterintuitively, a large portion of the newly synthesized L-glutamate was excreted. The expression of the genes (gltAB and gltA2) for two L-glutamate synthases were upregulated in response to high salinity along with that of the gltC regulatory gene. Such a regulatory pattern of the system(s) for L-glutamate synthesis in Bacilli is new. Our findings might thus be generally relevant to understand the production of the osmostress protectant L-glutamate by those Bacilli that exclusively rely on this compatible solute for their physiological adjustment to high osmolarity surroundings.
Highlights
Several members of the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, and Bacillus megaterium) are used as industrial cell factories for the manufacturing of bulk and fine chemicals and proteins of biotechnologically interest (Schallmey et al, 2004; Korneli et al, 2013; Van Dijl and Hecker, 2013; Su et al, 2020)
Given that enhanced L-glutamate production in B. methanolicus MGA3 is triggered by high osmolarity (Figure 3A), we considered the possibility that the transcription of the genes encoding the two GOGAT enzymes, their putative regulatory gene gltC (Figure 4B) and the gene for the glutamate dehydrogenase (GDH) enzyme were upregulated in response to sustained osmotic stress
The thermophilic Gram-positive methylotroph B. methanolicus MGA3 is increasingly recognized as a potential industrial workhorse (Brautaset et al, 2007; Müller et al, 2015a) for the commodity production of various amino acids, fine chemicals, and recombinant proteins using the readily replenishable and non-food C1 feed-stock methanol (Brautaset et al, 2003, 2010; Naerdal et al, 2015, 2017; Irla et al, 2016, 2020; Hakvag et al, 2020)
Summary
Several members of the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, and Bacillus megaterium) are used as industrial cell factories for the manufacturing of bulk and fine chemicals and proteins of biotechnologically interest (Schallmey et al, 2004; Korneli et al, 2013; Van Dijl and Hecker, 2013; Su et al, 2020). Bacillus methanolicus (Schendel et al, 1990; Arfman et al, 1992; Heggeset et al, 2012) is a thermotolerant natural methylotroph and assimilates methanol via the ribulose monophosphate (RuMP) pathway (Müller et al, 2015b; Carnicer et al, 2016). It can grow at high temperature (optimally at 50◦C) in minimal and rich media, and can use methanol as sole carbon and energy source. The natural ability of the B. methanolicus strain MGA3 (Naerdal et al, 2017) to synthesize and excrete large amounts (up to 59 g L−1 under fed-batch conditions) of the biotechnological important amino acid L-glutamate, makes this bacterium an interesting candidate to serve as an industrial cell factory for the conversion of the commodity chemical methanol into value-added products (Brautaset et al, 2007; Müller et al, 2015a)
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