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Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.

ABSTRACT: 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.

SUBMITTER: Pfeifenschneider J

PROVIDER: S-EPMC7092467 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.

Pfeifenschneider Johannes J Markert Benno B Stolzenberger Jessica J Brautaset Trygve T Wendisch Volker F VF

BMC microbiology 20200324 1

<h4>Background</h4>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 C<sub>1</sub> 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 ...[more]

PMID: 32204692

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Project description:Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D-mannitol, D-glucose and, as recently discovered, sugar alcohol D-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus. In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of B. methanolicus MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in B. methanolicus, two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to atlABC) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD). C. glutamicum is a natural D-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum ΔmtlD mutant with arabitol. Furthermore, D-arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate.

Project description:Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. Intriguingly, the only substrates known to support B. methanolicus growth as sole sources of carbon and energy are methanol, mannitol, and, to a lesser extent, glucose and arabitol. Because fluxomics provides the most direct readout of the cellular phenotype, we hypothesized that comparing methylotrophic and nonmethylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. In this study, we designed and performed a 13C metabolic flux analysis (13C-MFA) of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol, and arabitol to compare the associated metabolic states. On methanol, results showed a greater flux in the ribulose monophosphate (RuMP) pathway than in the tricarboxylic acid (TCA) cycle, thus validating previous findings on the methylotrophy of B. methanolicus New insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP variants were generated. Importantly, we demonstrated that the linear detoxification pathways and the malic enzyme shared with the pentose phosphate pathway have an important role in cofactor regeneration. Finally, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Overall, those data provide a better understanding of this strain under various environmental conditions.IMPORTANCE Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.

Dataset Information

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.

Publications

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.

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