Identification and characterization of the vanillin dehydrogenase YfmT in Bacillus subtilis 3NA.

With vanillin as one of the most important flavoring agents, many efforts have been made to optimize its biotechnological production from natural abundant substrates. However, its toxicity against the hosts results in rather low yields and product concentrations. Bacillus subtilis as a soil-dwelling bacterium is a possible lignin-derived compound-degrading microorganism. Therefore, its vanillin and ferulic acid metabolism was investigated. With a rather high tolerance for vanillin up to 20 mM, it is a promising candidate to produce natural vanillin. In this study, the well-studied phenolic acid decarboxylases PadC and BsdBCD could be ascribed to function as the only enzymes in B. subtilis 3NA converting ferulic acid to 4-vinylguaiacol and vanillic acid to guaiacol, respectively. As vanillin also becomes converted to guaiacol, a previous conversion to vanillic acid was assumed. Usage of bioinformatic tools revealed YfmT, which could be shown to function as the only vanillin dehydrogenase in B. subtilis 3NA. Thus, YfmT was further characterized regarding its temperature and pH optima as well as its substrate range. Vanillin and ferulic acid metabolic routes in the tested B. subtilis strain were revealed, a direct conversion of ferulic acid to vanillin, however, could not be found.

Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate-dependent phosphotransferase system.

To optimize Bacillus subtilis as a production strain for proteins and low molecular substances by genome engineering, we developed a markerless gene deletion system. We took advantage of a general property of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular the mannose PTS. Mannose is phosphorylated during uptake by its specific transporter (ManP) to mannose 6-phosphate, which is further converted to fructose 6-phosphate by the mannose-6-phosphate isomerase (ManA). When ManA is missing, accumulation of the phosphorylated mannose inhibits cell growth. This system was constructed by deletion of manP and manA in B. subtilis Δ6, a 168 derivative strain with six large deletions of prophages and antibiotic biosynthesis genes. The manP gene was inserted into an Escherichia coli plasmid together with a spectinomycin resistance gene for selection in B. subtilis. To delete a specific region, its up- and downstream flanking sites (each of approximately 700 bp) were inserted into the vector. After transformation, integration of the plasmid into the chromosome of B. subtilis by single cross-over was selected by spectinomycin. In the second step, excision of the plasmid was selected by growth on mannose. Finally, excision and concomitant deletion of the target region were verified by colony PCR. In this way, all nine prophages, seven antibiotic biosynthesis gene clusters and two sigma factors for sporulation were deleted and the B. subtilis genome was reduced from 4215 to 3640 kb. Despite these extensive deletions, growth rate and cell morphology remained similar to the B. subtilis 168 parental strain.

The Bacillus subtilis mannose regulator, ManR, a DNA-binding protein regulated by HPr and its cognate PTS transporter ManP.

The transcriptional activator ManR of the Bacillus subtilis mannose utilization operon is composed of an N-terminal DNA-binding domain, two phosphotransferase system (PTS) regulation domains (PRDs), an EIIB(Bgl) - and an EIIA(Fru) -like domain. Site-specific mutagenesis of ManR revealed the role of conserved amino acids representing potential phosphorylation sites. This was investigated by ß-galactosidase activity tests and by mobility shift assays after incubation with the PTS components HPr and EI. In analogy to other PRD-containing regulators we propose stimulation of ManR activity by phosphorylation. Mutations in PRD1 lowered ManR activity, whereas mutations in PRD2 abolished ManR activity completely. The Cys415Ala (EIIB(Bgl)) and the His570Ala mutations (EIIA(Fru)) provoked constitutive activities to different degrees, whereas the latter had the greater influence. Addition of EIIBA(Man) reduced the binding capability significantly in a wild-type and a Cys415Ala background, but had no effect on a His570Ala mutant. The different expression levels originating from the two promoters PmanR and PmanP could be ascribed to different 5'-untranslated mRNA regions. Sequences of 44 bp were identified and confirmed as the ManR binding sites by DNase I footprinting. The binding properties of ManR, in particular the equilibrium dissociation constant KD and the dissociation rate kdiss, were determined for both promoter regions.

Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors.

BACKGROUND: Several vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis. RESULTS: Regulation of the promoters of mtlAFD operon (P(mtlA)) and mtlR (P(mtlR)) encoding the activator were investigated by fusion to lacZ. Identification of the P(mtlA) and P(mtlR) transcription start sites revealed the σ(A) like promoter structures. Also, the operator of P(mtlA) was determined by shortening, nucleotide exchange, and alignment of P(mtlA) and P(mtlR) operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in P(mtlA) constitutive expression demonstrating the inhibitory effect of EIICB(Mtl) and EIIA(Mtl) on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both P(mtlA) and P(mtlR) were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in P(mtlR) catabolite repression. Similarly, using P(groE) as a constitutive promoter, putative cre sites of P(mtlA) and P(mtlR) slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of P(mtlA) and P(mtlR) was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D) mutant. CONCLUSIONS: The mtl operon promoter (P(mtlA)) is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on low copy plasmids. It is tightly regulated by just one copy of the mtlR gene on the chromosome and subject to CCR. CCR can be switched off by mutations in MtlR and the glucose transporter. These properties and the low costs of the inducers, i.e. mannitol and glucitol, make the promoter ideal for designing regulated expression systems.

Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation.

A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ΔmanA (mannose metabolism) strain TQ281 and the ΔmanP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.