λ Red-Mediated Genetic Manipulation of Antibiotic-Producing Streptomyces (original) (raw)
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Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences
The Journal of Antibiotics, 1988
Wehave developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. Weconstructed the plasmid pMSB212which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoKI site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of knowngenes and to identify new genes encoded on a cloned fragment. Streptomycetes are very important microorganisms whichproduce manyantibiotics and enzymes of commercial value. Recent developments in Streptomyces gene cloning have resulted in cloning of the resistance, biosynthetic, and regulatory genes of antibiotics, and elucidation of Streptomyces gene organization and regulation1~7). Mutant analysis gives us muchinformation about gene function and regulation. Mutants usually have been obtained by using chemical or physical mutagens, but there have been reports recently about another mutagenic approach, gene replacement techniques in Escherichia coli8), Bacillus subtilis9~12:> or Saccharomyces cerevisiae13 > 14\
Proceedings of the National Academy of Sciences, 2000
The filamentous soil bacterium Streptomyces coelicolor undergoes a complex cycle of morphological differentiation involving the formation of an aerial mycelium and the production of pigmented antibiotics. We have developed a procedure for generating insertional mutants of S. coelicolor based on in vitro transposition of a plasmid library of cloned S. coelicolor DNAs. The insertionally mutated library was introduced into S. coelicolor, and transposon insertions were recovered at widely scattered locations around the chromosome. Many of the insertions revealed previously uncharacterized genes, and several caused novel mutant phenotypes, such as altered pigment production, enhanced antibiotic sensitivity, delayed or impaired formation of aerial hyphae, and a block in spore formation. The sporulation mutant harbored an insertion in one of three adjacent genes that are apparently unique to Streptomyces but are each represented by at least 20 paralogs at dispersed locations in the chromosome. Individual members of the three families often are found grouped together in a characteristic arrangement, suggesting that they have a common function.
Excisable Cassettes: New Tools for Functional Analysis of Streptomyces Genomes
Applied and Environmental Microbiology, 2006
The functional analysis of microbial genomes often requires gene inactivation. We constructed a set of cassettes consisting of single antibiotic resistance genes flanked by the attL and attR sites resulting from site-specific integration of the Streptomyces pSAM2 element. These cassettes can easily be used to inactivate genes by in-frame deletion in Streptomyces by a three-step strategy. In the first step, in Escherichia coli , the cassette is inserted into a cloned copy of the gene to be inactivated. In the second step, the gene is replaced by homologous recombination in Streptomyces , allowing substitution of the wild-type target gene with its inactivated counterpart. In the third step, the cassette can be removed by expression of the pSAM2 genes xis and int . The resulting strains are marker-free and contain an “ attB -like” sequence of 33, 34, or 35 bp with no stop codon if the cassette is correctly chosen. Thus, a gene can be disrupted by creating an in-frame deletion, avoiding...
Isolation of restriction-reduced mutants from Streptomyces
Agricultural and biological chemistry, 1990
Restriction-reduced mutants were isolated from Streptomyces rosa subsp. notoensis KA301 and S. tanashiensis strain Kala which produce the benzoisochromanequinone antibiotics nanaomycin and kalafungin, respectively. The mutants of S. rosa, which can be transformed with a multi-copy plasmid and in which the actinophage Pa16 can propagate, were selected. They were transformed with a single-copy plasmid propagated in S. lividans TK24, and with its modified plasmid propagated in the mutant at higher efficiency. The mutants of S. tanashiensis were selected by their capability to be transformed with a multi-copy plasmid. The efficiency of transformation with a single-copy plasmid propagated in S. lividans TK24 was low, but was much increased by heating the protoplasts at 42 degrees C for 15 min prior to the transformation. These mutants derived from both strains probably lack at least one of their restriction systems.
An improved plasmid for the isolation and analysis of Streptomyces promoters
Gene, 1988
A Streptomyces promoter-probe plasmid vector was developed to isolate and characterize nucleotide sequences involved in transcriptional regulation. This pCLL34 vector was derived from plasmid pARC1 carrying the SLP1.2 origin of replication [Horinoucbi and Beppu, J. Bacterial. 162 (1985) 406-4121. Important featm-es of the new vector include moderate host range, low copy number, simple id~nt~cation of promoter activity by brown pigment production, an upstream ~~sc~ption~ terminator, and a polylinker sequence containing a unique BarnHI site for flexible cloning and fragment reisolation. The vector also provides the capability to directly determine the sequence of promoter-active inserts by dideoxy chain-termination sequencing methods using double-stranded plasmid templates and the appropriate oligodeoxynucleotide primers.
Cloning Streptomyces genes for antibiotic production
Trends in Biotechnology, 1983
The cloning and recombination of the genes of Streptomyces bacteria offer a method of increasing antibiotic yields and generating new antibiotics. Novel vectors, both plasmids and phages, have been developed for use with Streptomyces. This article describes some of these vectors and relevant cloning and screening techniques.
Scientific Reports, 2021
Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including the production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by the full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5.19% genome reduction. DEL2 can be characterized by faster growth and inability to produce three main native metabolites...