A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.

A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.

An in vitro assay for (1 --> 6)-beta-D-glucan synthesis in Saccharomyces cerevisiae.

(1 --> 6)-beta-D-glucan is a key cell wall component of Saccharomyces cerevisiae and Candida albicans. Many genes are known to affect the levels or structure of this glucan, but their roles and a molecular description of the synthesis of (1 --> 6)-beta-D-glucan remain to be established and a method to measure (1 --> 6)-beta-D-glucan synthase activity in vitro would provide an enabling tool. Here, conditions for the detection of in vitro synthesis of this polymer are described. Crude membrane preparations from S. cerevisiae were isolated, and incubated in the presence of UDP-glucose and GTP. With anti-(1 --> 6)-beta-D-glucan-specific antibodies, a time-dependent increase in the amount of this glucan was demonstrated in a dot-blot assay, or through an inhibition enzyme immunoassay. Antibody specificity was validated by competition experiments using pustulan, a (1 --> 6)-beta-D-glucan, laminarin, a (1 --> 3)-beta-D-glucan, yeast mannan and glycogen. The identity of the reaction product was also demonstrated by its sensitivity to a recombinant (1 --> 6)-beta-D-glucanase. Extracts from mutants in 10 genes with a wide range of altered cell wall (1 --> 6)-beta-D-glucan levels were assayed for in vitro synthesis of the polymer. A strong correlation of in vitro synthase activity with in vivo glucan levels was found, providing genetic support for the specificity of the assay. The basis for the GTP-dependence of the synthase reaction was studied. Extracts from rho2, rho3, rho4 and rho5 null mutants had wild-type in vitro activity. In contrast, Rho1p overproduction led to increased in vitro synthesis, implicating Rho1p in the regulation of (1 --> 6)-beta-D-glucan synthesis.

Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae.

The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1-Rho1-controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall-related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes. Transcript profiles were analysed using two recently developed algorithms, viz. REDUCE, which correlates upstream regulatory motifs with expression, and Quontology, which compares expression of genes from functional groups with overall gene expression. Both methods indicated upregulation of Rlm1p-controlled cell wall genes and STRE-controlled genes, and downregulation of ribosomal genes and rRNA genes. Comparison of these expression profiles with the published profiles of two constitutively active upstream activators of the Slt2p-MAP kinase pathway, viz. Pkc1-R398A and Rho1-Q68A, revealed significant similarity. In addition, a new putative regulatory motif, CCC(N)(10)GGC, was found. In Zymolyase-treated cells a regulatory site was identified, ATGACGT, which resembles the AFT/CRE binding site. Interestingly, Sko1p, a downstream regulator of the high osmolarity pathway is known to bind to the AFT/CRE binding site, suggesting a possible role for the Hog1 pathway in the response to cell wall stress. Finally, using REDUCE, an improved version of the Rlm1 binding motif, viz. TA(W)(4)TAGM, was discovered. We propose that this version can be used in combination with REDUCE as a sensitive indicator of cell wall stress. Taken together, our data indicate that cell wall stress results in activation of various signalling pathways including the cell wall integrity pathway.

The protein kinase Kic1 affects 1,6-beta-glucan levels in the cell wall of Saccharomyces cerevisiae.

KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-beta-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-beta-glucan-deficient strain fks1delta, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-beta-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-beta-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-beta-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-beta-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-beta-glucan levels in the wall.

Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance.

The protein kinase C (PKC1) pathway is essential for maintaining cell integrity in yeast. Here it is shown that various forms of cell wall damage result in activation of the downstream MAP kinase Slt2/Mpk1. Several cell wall mutants displayed enhanced FKS2-lacZ expression, a known output of Slt2 activation. A similar response was obtained with wild-type cells grown in the presence of the cell wall perturbants Calcofluor white and Zymolyase. Upregulation of FKS2-lacZ in response to sublethal concentrations of these agents fully depended on the presence of Slt2. The same cell wall stress conditions resulted in dual threonine and tyrosine phosphorylation of Slt2. Both Slt2 phosphorylation and FKS2-lacZ induction could be largely prevented by providing osmotic support to the plasma membrane. Interestingly, Slt2 phosphorylation in response to cell wall damage required the putative plasma-membrane-located sensor Mid2 but not Hcs77/Wsc1. Finally, cell wall perturbation gave rise to cells with increased resistance to glucanase digestion and heat shock. These responses depended on the presence of Slt2. These results indicate that weakening of the cell wall activates the Slt2/Mpk1 MAP kinase pathway and results in compensatory changes in the cell wall.