Dataset Information

Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.

ABSTRACT: The major Thermomonospora fusca YX extracellular protease gene (tfpA) was cloned into Escherichia coli and Streptomyces lividans and was sequenced. The open reading frame encoded 375 residues, including a 31-residue potential signal sequence, an N-terminal prosequence containing 150 residues, and the 194-residue mature protease that belongs to the chymotrypsin family. The protease was secreted by S. lividans, but evidence suggested that it was bound to an extracellular protease inhibitor. An inhibitor-deficient mutant was selected to produce protease for purification.

SUBMITTER: Lao G

PROVIDER: S-EPMC168250 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.

Lao G G Wilson D B DB

Applied and environmental microbiology 19961101 11

The major Thermomonospora fusca YX extracellular protease gene (tfpA) was cloned into Escherichia coli and Streptomyces lividans and was sequenced. The open reading frame encoded 375 residues, including a 31-residue potential signal sequence, an N-terminal prosequence containing 150 residues, and the 194-residue mature protease that belongs to the chymotrypsin family. The protease was secreted by S. lividans, but evidence suggested that it was bound to an extracellular protease inhibitor. An inh ...[more]

PMID: 8900021

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Project description:BACKGROUND:Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS:In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION:The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Dataset Information

Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.

Publications

Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.

Similar Datasets

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