Cloning, characterization and expression of an α-amylase gene from Streptomyces griseus IMRU3570

Abstract

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.

Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene

“…The presence of a putative regulatory gene has been observed upstream of the ␣-amylase gene in S. limosus (27), S. venezuelae (45), and S. griseus (42). The homologous gene of S. lividans was isolated by using PCR to amplify chromosomal DNA of S. lividans (see Materials and Methods).…”

“…This led to the assumption that a regulatory protein(s) in S. lividans controlling the expression of aml might exist. A putative repressor, called open reading frame R (ORFR) in the present work, which is homologous to the regulatory proteins LacI and GalR of Escherichia coli has been located upstream of the ␣-amylase gene of S. venezuelae (45) and is also present upstream of the ␣-amylase genes of S. limosus (27) and S. griseus (42). Therefore, it was assumed that an ORF homologous to ORFR might be present in S. lividans.…”

Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases

“…Incubation was carried out for 3 h at 37˚C. (b) Coomassie-blue-R-stained SDS-PAGE showing the in vitro processing-degradation of different secreted proteins -p40 (Garda et al, 1997), Amy (Vigal et al, 1991) and Xyl30 (NCBI AAD32560) -by S. lividans SpB and SpC proteases. XC, S. lividans supernatant overexpressing the corresponding protein used in the in vitro experiment.…”

“…We therefore tested the effects of S. halstedii JM8 supernatant and pure SpB and SpC proteases on the following proteins: p40, a protein with no known catalytic activity and with a cellulose-binding domain at the carboxy terminus, isolated from S. halstedii JM8 (Garda et al, 1997), that is structurally similar to the xylanase Xys1L; Amy, an a-amylase from S. griseus IMRU3570 (Vigal et al, 1991), and Xyl30, a xylanase from Streptomyces avermitilis (NCBI AAD32560). In vitro experiments were carried out mixing S. lividans supernatants in which each protein of interest had been overexpressed with the S. halstedii JM8 supernatant or with pure SpB or SpC proteases.…”

Starch and Microbial α-Amylases: From Concepts to Biotechnological Applications

“…-Amylases are secreted by several species of Streptomyces, for example S. albus (Andrews & Ward, 1987), S. griseus IMRU3570 (Vigal et al, 1991), S. thermocyaneoviolaceus (Hang et al, 1996). Gene encoding extracellular -amylase has been cloned from many Streptomyces species (Bahri & Ward, 1990;Virolle et al, 1988).…”