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

Abstract

BackgroundSeveral 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.ResultsRegulation of the promoters of mtlAFD operon (PmtlA) and mtlR (PmtlR) encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the σA like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D) mutant.ConclusionsThe mtl operon promoter (PmtlA) 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.

Metabolic engineering of Bacillus subtilis forl‐valine overproduction

“…To determine if l ‐valine overproduction may be limited by 2‐acetolactate levels in AW007‐5, we inserted P grac .UPmod in place of P alsSD in strain AW008‐5. However, a severe growth defect was observed for AW008‐5, prompting us to insert a weaker inducible promoter in place of P alsSD , that is, the mannitol‐inducible promoter P mtlA (Heravi, Wenzel, & Altenbuchner, ), in strain AW009‐5 (Figure ). The peak cell density and final l ‐valine titer decreased to OD 600 3.6 after 8 hr (Figure a) and 1.27 g/L (Figure b), respectively, in cultures of AW009‐5.…”

“…To determine if L-valine overproduction may be limited by 2-acetolactate levels in AW007-5, we inserted P grac.UPmod in place of P alsSD in strain AW008-5. However, a severe growth defect was observed for AW008-5, prompting us to insert a weaker inducible promoter in place of P alsSD , that is, the mannitol-inducible promoter P mtlA (Heravi, Wenzel, & Altenbuchner, 2011), in strain AW009-5…”

An Alternative Bacterial Expression System Using Bacillus pumilus SG2 Chitinase Promoter

“…This could be due to the shortened untranslated region of the lacZ mRNA on pChi2 and pChi3. Similarly, shortening of the untranslated region of lacZ mRNA expressed by P mtlA increased the β-galactosidase activity in the B. subtilis host strain (26). Replacing the LB medium with M9 medium led to weak growth of the strains (data not shown).…”

Current development in genetic engineering strategies of Bacillus species

“…These plasmids were successfully applied to integrate the lipase gene of Bacillus thermocatenulatus into a B. subtilis glucose-regulated system. Morabbi et al also used this system to perform markerless integration of P mtlR -mtlR-H342D into the chromosome of B. subtilis [36]. …”