Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.     

Amino acid sequence of protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25

The amino acid sequence of a protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25 (Haim II), which consists of 77 amino acid residues, including two disulfide bridges, was determined by conventional methods. One of the disulfide bridges was found to be located between Cys(6) and Cys(22), and the other between Cys(40) and Cys(67) from the results of structure analyses of the two cystine-containing peptides obtained from the thermolysin digest of the native inhibitor.     

Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae.

The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Nucleotide sequence of a carboxyphosphonoenolpyruvate phosphonomutase gene isolated from a bialaphos-producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans.

Summary The carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of bialaphos-producing Streptomyces hygroscopicus, which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.     

Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp.

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.     

Nucleotide sequence and regulated expression of a wound-inducible potato gene (wun1)

Summary Mechanical wounding of potato leaves, stems, roots and tubers leads to a rapid increase of wun1 mRNA. In potato leaves, the wound-induced accumulation of wun1 mRNA is inhibited by the addition of sucrose or other osmotically active agents. This inhibition is organ specific since sucrose does not prevent wun1 mRNA accumulation in wounded tubers. In contrast, expression of patatin was shown to be repressed in tubers by wounding and this repression was reversed by increasing osmotic pressure. Sequence data obtained from the analysis of a wun1 cDNA and a wun1 genomic clone show no homology to any gene known so far. Histochemical data demonstrate a striking analogy in cell specific expression of chimeric genes expressed under the control of the wun1 promoter and the cell specific production of callose in wounded tobacco leaves.     

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.     

Purification and properties of a proteinaceous metallo-proteinase inhibitor from Streptomyces nigrescens TK-23.

A novel metallo-proteinase inhibitor which is capable of inhibiting the activities of metallo-proteinases such as the thermolysin, was isolated from the culture filtrates of Streptomyces nigrescens TK-23. The inhibitor was purified batch-wise from the culture filtrate by Amberlite IRC-50 and column chromatographies on CM-Sephadex C-50 and Sephadex G-50. The purified inhibitor showed a single band on 15% polyacrylamide gel electrophoresis at pH 4.3, and at pH 7.5 on SDS-gels. The inhibitor retained 80% of its original activity after treatment of 100 degrees C for 5 min between pH and 7. The molecular weight was estimated to be 12 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, and calcuated as 11 950 from its amino acid composition. The isoelectric point was pH 10.3. The inhibitor showed a high content of hydrophobic amino acids, did not contain tryptophan, and had two disulfide bridges. It also showed specific inhibitory activity for metallo-proteinases but not for serine-, thio- and carboxyl-proteinases.