Metabolic engineering of Bacillus methanolicus and Corynebacterium glutamicum for the production of cadaverine from methanol

Pfeifenschneider J. Metabolic engineering of Bacillus methanolicus and Corynebacterium glutamicum for the production of cadaverine from methanol. Bielefeld: Universität Bielefeld; 2015

Transaldolase in Bacillus methanolicus: Biochemical Characterization and Biological Role in Ribulose Monophosphate Cycle

Pfeifenschneider J, Markert B, Stolzenberger J, Brautaset T, Wendisch VF. Transaldolase in Bacillus methanolicus: Biochemical Characterization and Biological Role in Ribulose Monophosphate Cycle. BMC Microbiology. 2020;20: 63.Background The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus. Results The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed KM of 0.74 mM and Vmax of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts. Conclusions While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP

Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l−1 during shake flask conditions. A volume-corrected concentration of 11.3 g l−1 of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol.publishedVersio

Erratum: Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains

Naerdal I, Pfeifenschneider J, Brautaset T, Wendisch VF. Erratum: Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains. Microbial Biotechnology. 2019;12(1):182-183

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate

Leßmeier L, Pfeifenschneider J, Carnicer M, Heux S, Portais J-C, Wendisch VF. Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate. Applied Microbiology and Biotechnology. 2015;99(23):10163-10176

The flexible feedstock concept in Industrial Biotechnology: metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources

Wendisch VF, Fernandes de Brito L, Gil Lopez M, et al. The flexible feedstock concept in Industrial Biotechnology: metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources. Journal of Biotechnology. 2016;234:139-157