Takeshi Uozumi

Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus.

A-factor is a potent pleiotropic effector produced by Streptomyces griseus and is essential for streptomycin production and spore formation in this organism. Its production is widely distributed among various actinomycetes including Streptomyces coelicolor A3(2). Genetic analysis of A-factor production was carried out with S. coelicolor A3(2), and two closely linked loci for A-factor mutations (afsA and B) were identified between cysD and leuB on the chromosomal linkage map. In contrast, genetic crosses of A-factor-negative mutants of S. griseus, using a protoplast fusion technique, failed to give a fixed locus for A-factor gene(s) and suggested involvement of an extrachromosomal or transposable genetic element in A-factor synthesis in this organism.

Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties

We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3-4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

Restriction and modification in Bacillus species

SummaryHost specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage Φ105C. These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction. M-type strains (B. subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting Φ105C from other groups of Bacillus by ratios of 10-1 to 10-3. Strains of groups H,C,N,E,F,G, and P restricted Φ105C from other groups by ratios of 10-2 to 10-8. It was confirmed with some of the strains that type-specific modification was endowed only by the last host. Furthermore, we isolated one restriction deficient mutant of B. subtilis marburg 168-YS11, which had also lost its modification phenotype.

Replacement of an amino acid residue of cyclodextrin glucanotransferase of Bacillus ohbensis doubles the production of γ-cyclodextrin

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin (CD) from starch through an intramolecular transglucosylation reaction. To obtain a better understanding of the amylolytic and cyclization mechanisms of CGTase, and furthermore to improve the production of gamma-CD, mutant CGTases were constructed by site-directed mutagenesis of the CGTase gene of Bacillus ohbensis replacing Tyr at position 188 by 19 other amino acids. All mutant enzymes retained both starch-degrading and CD synthesizing activities to various extents. Among them, a mutant enzyme having Trp instead of Tyr-188 produced 15% of gamma-CD from soluble starch, which is about twice as much as the amount produced by the wild-type enzyme.

Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli

SummaryA cyclcodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at −108 to −67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80 000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55°C; and stability below 45°C. The yields of the products from starch as the substrate were 25% for β-and 5% for γ-cyclodextrin.

Cloning and sequencing of an α-glucosidase gene from Aspergillus niger and its expression in A. nidulans

We have cloned an extracellular alpha-glucosidase gene from Aspergillus niger with oligonucleotide probes synthesized on the basis of the determined peptide sequences. The nucleotide sequence revealed an open reading frame of 985 amino acids split with three introns, and the deduced amino acid sequence was nearly identical to that of the alpha-glucosidase previously determined. The cloned gene was introduced into Aspergillus nidulans, and its expression in the transformants was shown to be regulated by the carbon sources in the medium, suggesting that a common regulatory expression system is shared by these two species as is the case of other starch-degrading enzymes of Aspergillus species.

Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter

To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300 000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lacUV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.

Renaturation and activation of calf prochymosin produced in an insoluble form in Escherichia coli

Abstract Calf prochymosin produced in Escherichia coli cells harboring the expression plasmids was insoluble and formed large inclusion bodies, which were solubilized by 8 M urea. The conditions allowing correct refolding of denatured prochymosin were investigated. Dialysis at pH 10 in the presence of 500 mM NaCl was found to give the maximum renaturation, and subsequent acidic treatment for autocatalytic processing of refolded prochymosin allowed almost 100% recovery of chymosin.

Correlation between cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea cellobiohydrolase 1

The cellulose‐binding domains (CBDs) of fungal cellulases interact with crystalline cellulose through their hydrophobic flat surface formed by three conserved aromatic amino acid residues. To analyze the functional importance of these residues, we constructed CBD mutants of cellobiohydrolase 1 (CBH1) of the thermophilic fungus Humicola grisea, and examined their cellulose‐binding ability and enzymatic activities. High activity on crystalline cellulose correlated with high cellulose‐binding ability and was dependent on the combination and configuration of the three aromatic residues. Tyrosine works best in the middle of the flat surface, while tryptophan is the best residue in the two outer positions.

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells.

SummaryThe aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH2-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH2-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.