Der methylotrophe Bacillus methanolicus als eine synthetische Zellfabrik für die L-Prolin-Produktion

The thermotolerant methylotrophic Bacillus methanolicus MGA3 was originally isolated from freshwater marsh soil and it grows optimally at 50 °C. The organism can use the C1-compound methanol as the sole carbon- and energy sources and is therefore explored as a cell factory for the production of amino acids, fine chemicals, and proteins. During high cell density fed-batch fermentation processes, the excretion of metabolites leads to an increase of the external osmolarity, which can negatively affect the productivity of the cell factory. Hence, this paper presents the analysis of the core physiological adjustment process of B. methanolicus at high osmolarity surroundings. In consensus with the ecophysiological conditions of its natural habitat (freshwater marsh soil), the organism possesses only a restricted ability to cope with osmotic stress. None of the externally provided compatible solutes or proline containing peptides rescue the growth of B. methanolicus at high salinity. At hyperosmotic conditions, B. methanolicus produces exclusively the moderately effective compatible solute glutamate. Strikingly, most of the newly synthesized glutamate was secreted by B. methanolicus. The expression of the gene of glutamate synthase (gltAB, gltA2) and the regulatory gene (gltC) was upregulated at hyperosmotic conditions. Such a transcription pattern of the glutamate synthesis genes has not been observed before. Due to the various benefits of B. methanolicus in terms of fermentation processes, the present work demonstrates the development of a methanol-based proline production strain. Among various Bacillus species, the proteinogenic amino acid is produced for anabolic purpose and as an osmostress protectant to cope with high salinities. At first, the osmoadaptive proline synthesis enzyme of B. licheniformis (ProJ-ProA-ProH) was heterologously expressed in B. methanolicus and strikingly, the overexpression led to an accumulation of citrulline. This result suggests that the ProH ∆1-pyrroline-5-carboxylate reductase from the mesophile organism is the underlying cause for the inability of the recombinant B. methanolicus to produce proline. Alternatively, the anabolic proline biosynthetic genes of B. methanolicus was used to develop a proline production strain. However, this route is strictly regulated on the transcriptional and post-transcriptional levels. The transcription of the proBA operon and proI gene is controlled in response to intracellular proline levels via a T-box regulatory element. The Glutamate-5 Kinase ProB is feedback regulated by the product proline. By bioinformatic analysis a key amino acid (E142) for the feedback regulated mechanism was observed. This amino acid is located within a flexible loop, which modulates the enzyme-inhibitor interaction in the active centre. By replacement of the negatively charged glutamate by a positively charged arginine (E142/R) the allosteric regulation is significantly reduced. The overexpression of a synthetic anabolic proline biosynthetic route (proB*AI) led to successful proline production in B. methanolicus. Overall, in high cell density fed batch fermentation processes a proline production of 263 mg L-1 was reached. This study represents the first microbial conversion of methanol to proline. For a side project of this work, the restriction modification system of B. methanolicus MGA3 was analysed in more detail. The system is located on the native plasmid pBM69. In vivo studies have shown that the absence of the native plasmid led to a 10-fold increase of the transformation efficiency.