A gene, amy, encoding an alpha-amylase, was cloned on a 4.8 kb Sau3A fragment from the DNA of Streptomyces griseus IMRU3570. The gene was localized to a 2.27 kb fragment by subcloning and deletion mapping experiments. The gene contained an open reading frame (ORF) of 1698 nucleotides that encoded a protein of 566 amino acids with a deduced Mr of 59713 Da. Dot-blot analysis revealed that the copy number of the transcript in S. lividans transformed with the amy gene was 2.8-fold higher than in the donor S. griseus strain in good agreement with the proportionally higher secretion of amylase in S. lividans. A transcription initiation site was found approximately 64 bp upstream from the ATG translation start codon. The promoter of the amy gene was subcloned on a 290 bp HindIII--EcoRI fragment. Expression of a neomycin resistance gene from the amy promoter was negatively regulated by glucose. A 219 nucleotide fragment extending from the single BstEII site to the end of the amy gene was dispensable since active alpha-amylase was secreted after deletion of this region and coupling of a TGA translation stop codon.

Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. gniseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.A large number of extracellular enzymes are produced by Streptomyces species that are used by these saprophytic microorganisms to degrade polymeric nutrients in soil originating from decaying plant material. Several genes encoding extracellular enzymes have been cloned from Streptomyces species. These include endoglycosidase H (25), tyrosinase (16), agarase (4, 17), P-lactamase (8), a-amylase (2,14,20,21,32,33), and xylanase (15,22). We have cloned in Streptomyces lividans the gene encoding a-amylase of Streptomyces griseus IMRU 3570 (31), a strain which is known to overproduce several extracellular enzymes (7). It contains an open reading frame of 1,698 nucleotides which encodes a protein with a deduced molecular mass of 59,713 Da.Very little is known, however, about the mechanisms of posttranslational modification and secretion of extracellular enzymes of Streptomyces species. Secretion of extracellular enzymes in Bacillus species and other bacteria proceeds by membrane anchoring of the amino-terminal end of the newly synthesized intracellular protein (24). The interaction of the amino-terminal hydrophobic region of the protein (the leader peptide) produces a modification of the protein phospholipid arrangement of the lipid bilayer that results in the opening of channels through which the protein is secreted. Passage of the extracellular protein through the secretion channels results in the cleavage of the leader peptide by the signal peptidase (23,24).In Streptomyces species, the leader peptides of extracellular proteins are poorly known, and no detailed studies on the protein-processing mechanisms prior to or during the secretion have bee...

Five different mutations were introduced into the leader peptide region of the alpha-amylase gene of Streptomyces griseus IMRU 3570. A mutation which increased the positive charge of the N-terminal region of the leader peptide enhanced the secretion of alpha-amylase by two- to threefold. Replacement of the native promoter of the amylase gene by the promoter of the Tn5 neo gene or by the promoter of the saf gene resulted in a 16-fold increase in alpha-amylase secretion. The enhanced secretion of alpha-amylase obtained by using the most efficient promoters was due to a correlated increase in the amount of transcript formed. The translation and secretion processes in S. lividans are not a bottleneck for enzyme secretion even at very high transcription rates, since stimulation of transcription of the alpha-amylase gene results in a proportionate increase in secretion of the enzyme.

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