Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase.

BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Global transcriptional analysis of Bacillus licheniformis reveals an overlap between heat shock and iron limitation stimulon.

In this study, we characterized the heat shock stimulon of the important industrial microorganism Bacillus licheniformis using DNA microarrays. While sharing a high degree of homology with the closely related model organism Bacillus subtilis, the heat shock stimulon of B. licheniformis exhibited several novel and unexpected features. Most notably, heat shock in B. licheniformis resulted in decreased amounts of mRNA from the ytrABCEF operon, encoding a putative acetoin uptake system, and stimulated the transcription of purine biosynthesis and iron uptake genes. Unexpectedly, deletion of the ytrEF genes did not affect acetoin uptake, but increased heat sensitivity. To investigate the connection between heat stress and iron uptake further, we analyzed the iron limitation response of B. licheniformis by DNA microarrays and concluded that the response mostly involves the genes related to iron uptake and metabolism, while the only heat shock gene affected by iron limitation was clpE. We also attempted to delete the fur gene (encoding the ferric uptake repressor), but unexpectedly found it to be essential in B. licheniformis. Using the fluorescent protein-encoding reporter gene under control of the dhb promoter, which responded to both heat shock and iron-starvation, we confirmed the overlap between these responses.

Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to secretion of a heterologous protein by Bacillus subtilis.

Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

Structure of a Bacillus halmapalus family 13 alpha-amylase, BHA, in complex with an acarbose-derived nonasaccharide at 2.1 A resolution.

The enzymatic digestion of starch by alpha-amylases is one of the key biotechnological reactions of recent times. In the search for industrial biocatalysts, the family GH13 alpha-amylase BHA from Bacillus halmapalus has been cloned and expressed. The three-dimensional structure at 2.1 A resolution has been determined in complex with the (pseudo)tetrasaccharide inhibitor acarbose. Acarbose is found bound as a nonasaccharide transglycosylation product spanning the -6 to +3 subsites. Careful inspection of electron density suggests that the bound ligand could not have been formed through successive transglycosylations of acarbose and must also have featured maltose or maltooligosaccharides as an acceptor.

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species.

BACKGROUND: Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature. RESULTS: We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs. CONCLUSIONS: Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.