Secretory overproduction of Streptomyces cholesterol oxidase by Streptomyces lividans with a multi-copy shuttle vector

Molnár, István; Choi, Kwang Pil; Hayashi, Naoaki; Murooka, Yoshikatsu

doi:10.1016/0922-338x(91)90089-y

Cited by 31 publications

(8 citation statements)

References 21 publications

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“…The improved yield times vs. wild-strain ATCC25486 recombinant pIJ702 plasmids frequently showed deletions of cloned inserts [28]. Moreover, the expression of multiple copies of cloned genes leads to unexpected variations in secondary metabolite production [16].…”

Section: Highly Resistant Exconjugantsmentioning

confidence: 99%

Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis

Jin

2009

Appl Biochem Biotechnol

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Improving pristinamycin production from Streptomyces pristinaespiralis was performed by introducing the resistance gene ptr followed by selection for enhanced tolerance to pristinamycin and fermentation test. To transfer ptr into S. pristinaespiralis, an effective method was established for the first time by using the intergeneric conjugation of DNA from Escherichia coli to S. pristinaespiralis. The procedure was optimized with heat treatment, spore concentration, optimum medium used in conjugation, concentration of MgCl(2), etc. With the optimized conditions, the conjugation frequency was up to 1.36 x 10(-3) exconjugants per recipient. The procedure was used to transfer the ptr gene into S. pristinaespiralis, resulting in 146 exconjugants. These exconjugants were screened on the pristinamycin-resistant plates, and then the fermentation test subsequently. Finally, two strains (SPR1 and SPR2) were obtained with a high yield of 0.11 and 0.15 g/l, respectively, which is about six to eight times more than that of wild-strain ATCC25486. The subculture experiments indicated that the hereditary character of the high-producing S. pristinaespiralis SPR1 and SPR2 was stable. Our work suggests that introducing resistance gene ptr into S. pristinaespiralis could be the way to improve the production of pristinamycin through the enhancement of antibiotic tolerance.

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Section: Highly Resistant Exconjugantsmentioning

confidence: 99%

Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis

Jin

2009

Appl Biochem Biotechnol

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“…Moreover, high-copy-number vectors such as pIJ702 were shown to be structurally unstable, and recombinant pIJ702 plasmids frequently showed deletions of cloned inserts (14,20). One of the best ways to stably clone a single copy of a gene is to integrate the gene of interest in the chromosome itself.…”

Section: Discussionmentioning

confidence: 99%

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp . Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Senthamaraikannan

Dharmalingam

2003

Appl Environ Microbiol

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Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.Streptomyces spp. are gram-positive spore-forming soil bacteria, and they synthesize a wide array of antibiotics and other secondary metabolites. Genetic approaches to improve secondary-metabolite production in antibiotic producer strains were hampered by restriction barriers, the absence of efficient gene transfer systems in industrial strains, and a lack of suitable cloning vectors (4). To circumvent these problems, there has been considerable interest in the use of intergeneric conjugation as a means of plasmid transfer, using Escherichia coli as the donor. This technique allows one to construct and manipulate recombinant plasmids in E. coli and subsequently transfer them to Streptomyces. This mode of gene transfer has been efficient even in recipient strains containing restriction systems (5, 33).Intergeneric conjugation between E. coli and Streptomyces was first reported in 1989 (18). Since then, the process has been demonstrated to work in several Streptomyces species, such as Streptomyces fradiae, Streptomyces ambofaciens (5), Streptomyces coelicolor, Streptomyces lividans (8), and Streptomyces nanchangensis (28); in Saccharopolyspora spinosa (17); and in various strains of actinomycetales (32). In all these cases, the donor species was E. coli. The conjugative functions of plasmid RP4, (5,10,16,18,21,26,32), the RP1 derivative pMB307 (8), and the RK2 derivative pUZ8002 (23) were used to mobilize the resident plasmids. Several cloning vectors which could be transferred from E. coli to Str...

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“…The Streptomyces ChOx gene was also overexpressed in S. lividans by subcloning the choP ‐ choA operon into a multicopy shuttle vector, which produced a 70‐fold greater amount of ChOx than the original organism (i.e. up to 4400 U·L −1 in the fermentation broth) [16]. Interestingly, more than 90% of the protein produced is extracellular.…”

Section: Sources Of Choxmentioning

confidence: 99%

Cholesterol oxidase: biotechnological applications

2009

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Cholesterol oxidase is a bacterial FAD‐containing flavooxidase that catalyzes the first reaction in cholesterol catabolism. Indeed, this enzyme catalyzes two reactions: the oxidation of the C3‐OH group of cholesterol (and other sterols) to give cholest‐5‐en‐3‐one; and its isomerization to cholest‐4‐en‐3‐one. In the past several years, the structural and functional characterization of cholesterol oxidase has been developed together with its application as a biological tool. Cholesterol oxidase has been used in biocatalysis for the production of a number of steroids, as an insecticidal protein against boll weevil larvae and, in particular, as a diagnostic enzyme for determining serum levels of cholesterol. These applications prompted various laboratories worldwide to isolate this flavooxidase from different sources and to improve its properties by protein engineering, further increasing our knowledge on its structure–function relationships. These studies also discovered new physiological roles for cholesterol oxidase (e.g. in virulence and as an antifungal sensor). We assume that the investigations of cholesterol oxidase and its applications will continue to grow quickly in the near future, in particular to uncover unexpected, new areas of application.

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Secretory overproduction of Streptomyces cholesterol oxidase by Streptomyces lividans with a multi-copy shuttle vector

Cited by 31 publications

References 21 publications

Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis

Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp . Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Cholesterol oxidase: biotechnological applications

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