Shigezo Udaka

High-level secretion of heterologous proteins by Bacillus brevis.

Publisher Summary Among various host-vector systems for the production of foreign proteins in microorganisms, the use of Bacillus brevis ( B. brevis )as a host offers the advantage that proteins are secreted directly into the culture medium, where they are accumulated at high levels in a relatively pure state. B. brevis is isolated from soil as a protein-hyperproducing bacterium and is found to show little extracellular protease activity. Even when positive transformants of B. brevis are found, these cells sometimes cannot maintain the correct plasmid. Deletions of the foreign gene or cwp promoter are frequently found. Serial single-colony isolation of positive clones is helpful. The mutagenesis of B . brevis with N-methyl-N-nitro-N-nitrosoguanidine (NTG) prior to transformation is also helpful to obtain clones that can maintain the plasmid with the correct structure. When clones maintaining the plasmid can be obtained but the amount of foreign proteins produced is not large, mutants producing the protein with improved yields can be isolated by the mutagenesis of the clones with NTG.

Characterization of Bacillus brevis with descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov.

Thirty-five Bacillus brevis strains obtained from culture collections, including protein-producing isolates, were taxonomically studied by using numerical analysis, DNA base composition, and DNA-DNA hybridization. Six DNA relatedness groups were represented, and these groups correlated well with clusters based on the numerical analysis. The B. brevis strains were separated into B. brevis sensu stricto, four new species, and an unidentified species of the genus Bacillus. Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov. are proposed.

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

ABSTRACT We have developed a versatile Bacillus brevisexpression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

A Gene Encoding Phosphatidylethanolamine N-Methyltransferase from Acetobacter aceti and Some Properties of its Disruptant

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Efficient production of a functional mouse/human chimeric Fab′ against human urokinase-type plasminogen activator by Bacillus brevis

Abstract Expression/secretion vectors for the production of Fab′ and single-chain (sc) Fab′ by Bacillus brevis have been constructed. For the production of Fab′, the cDNAs encoding the L chain and Fd′ fragment (Fd with the hinge region) of a mouse-human chimeric Fab′ against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab′, the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab′ was efficiently produced by B. brevis, being accumulated at a level of 100u2009mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab′ remained at a level of a few milligrams per liter in the culture medium. The Fab′ produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd′ fragment, constituting the Fab′, had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

Proposal of Bacillus reuszeri sp. nov., Bacillus formosus sp. nov., nom. rev., and Bacillus borstelensis sp. nov., nom. rev.

Taxonomic studies of 26 putative Bacillus brevis strains (12 strains formerly assigned to groups 2, 4 through 7, and B and 14 ungrouped strains) were carried out. These organisms were placed in five distinct groups on the basis of phenotypic characteristics, the results of DNA base composition and reassociation analyses, and the results of cellular fatty acid and isoprenoid quinone composition analyses. Group 2, which contained three strains, was identified as Bacillus choshinensis, and group 4, which contained one strain, was identified as Bacillus parabrevis. The three remaining unidentified groups were clearly distinguishable phenotypically and genetically from the type strains of B. brevis and the recently described species Bacillus agri, Bacillus centrosporus, Bacillus migulanus, Bacillus choshinensis, and Bacillus parabrevis. Therefore, the names Bacillus reuszeri sp. nov., Bacillus formosus sp. nov., nom. rev., and Bacillus borstelensis sp. nov., nom. rev. are proposed for groups 5 (3 strains), 6 (3 strains), and 7 (16 strains), respectively. The S-layer proteins found in the three new species were immunologically and genetically similar to the S-layer proteins found previously in B. brevis, B. agri, B. centrosporus, B. choshinensis, and B. parabrevis. The G+C content of NRRL NRS-1206, the type strain of B. reuszeri sp. nov., was 46.5 mol%; the G+C content of NRRL NRS-863, the type strain of B. formosus sp. nov., nom. rev., was 47.2 mol%; and the G+C content of NRRL NRS-818, the type strain of B. borstelensis sp. nov., nom. rev., was 51.3 mol%.

Purification and characterization of intracellular esterases related to ethylacetate formation in Acetobacter pasteurianus

Acetobacter sp. produce ethylacetate during acetic acid fermentation. We found that high ethylacetate-producing strains contained more intracellular esterase than low ethylacetate-producing strains. Acetobacter pasteurianus N-23, a high ethylacetate-producing strain, formed two major intracellular esterases (esterase-1 and esterase-2) that were regulated by ethanol. These two esterases were purified to homogeneity by means of hydrophobic interaction, ion exchange, gel filtration, and hydroxyapatite chromatographies. The two esterases hydrolyzed various esters, but only esterase-1 showed ethylacetate synthesis activity at pH 3. The molecular weights of esterase-1 and esterase-2 were 72 kDa and 44 kDa, respectively, as determined by gel filtration, and 38 kDa and 42 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Esterase-1 was inhibited by cysteine enzyme inhibitors such as diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These esterases may play some role in the production of ethylacetate by Acetobacter sp. during vinegar fermentation.

The Efficient Production of Human Epidermal Growth Factor by Bacillus brevis

A system has been developed for the efficient production of heterologous proteins using Bacillus brevis as a host that secretes large amounts of cell wall protein into the medium. The promoter region and signal peptide-encoding region of the cell wall protein gene were used to construct an expression-secretion vector. Bacterial proteins such as amylases can be produced in large amounts by this system (1 g/l or more), but mammalian proteins such as human alpha-amylase are produced at a low level (one or two orders of magnitude less than for bacterial proteins). The highly efficient secretion of human epidermal growth factor (h-EGF, more than 1 g/l) was obtained with B. brevis HPD31 as the host and plasmid pHY481, derived from B. brevis 481, as the vector. Recombinant hEGF was purified easily from the culture supernatant by two steps. Purified hEGF had the identical NH2-terminal amino acid sequence and COOH-terminal amino acid sequence with those of the authentic hEGF, and it was fully active in biological assays. This recombinant hEGF has been shown to be successful for biological wool harvesting (CSIRO, Australia). These results, in combination with previous results, indicate that foreign proteins of diverse origins can be produced efficiently as functional proteins in B. brevis.

Bacillus aneurinolyticus sp. nov., nom. rev.

The taxonomic position of “Bacillus aneurinolyticus” was determined by numerical analyses based on phenotypic characteristics and whole-cell proteins profile, chemosystematic data, DNA base composition, and DNA relatedness. “B. aneurinolyticus” strains were separated into “B. aneurinolyticus” and Bacillus migulanus by DNA relatedness. This result correlated well with the clusters obtained from numerical analyses based on phenotypic characteristics and whole-cell proteins profile. “B. aneurinolyticus” was clearly distinct from other Bacillus species phenotypically and genetically. We propose the revival of the name Bacillus aneurinolyticus.