High-level overproduction of Thermus enzymes in Streptomyces lividans

Díaz, Margarita; Ferreras, Eloy; Moreno, Renata; Yepes, Ana; Berenguer, José; Santamaría, Ramón I.

doi:10.1007/s00253-008-1495-1

Cited by 20 publications

(15 citation statements)

References 25 publications

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“…For example, recent studies have demonstrated that the use of a variety of host expression strains can assist in the expression of the desired metabolite [118–121]. Specifically, these strains are often chosen based on possible similarities with the producer bacteria (if known), such as, for example, the similarities in codon usage, as well as presence of specific machinery necessary for the production of particular metabolites [119,120]. …”

Section: The Challenges Of Microbial Bioactive Natural Product Devmentioning

confidence: 99%

Development of Novel Drugs from Marine Surface Associated Microorganisms

2010

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While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future.

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Section: The Challenges Of Microbial Bioactive Natural Product Devmentioning

confidence: 99%

Development of Novel Drugs from Marine Surface Associated Microorganisms

2010

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“…This bacterium is GC rich and represents one of the best alternative hosts for expression of thermophilic enzymes, encoded by genes that also carry high GC content [20,27]. Therefore, a few publications have demonstrated successful use of this bacterium for expression of heterologous proteins [9,28,50].…”

Section: Introductionmentioning

confidence: 98%

Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in Streptomyces lividans

Côté

Shareck

2010

J Ind Microbiol Biotechnol

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Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment in a fed-batch bioreactor submitted to high temperature (50-70 degrees C) and alkaline pH (7-8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10-164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The optimum pH (8.5) and temperature (60 degrees C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore, the enzyme was thermostable for as long as 30 min at 70 degrees C. The maximum activity of the purified lipase was 4,287 IU/mg towards p-nitrophenyl (p-NP) butyrate (60 degrees C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was slightly inhibited by 10 mM CoCl2 (14%), HgCl2 (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are quite encouraging for further use of this enzyme in different industrial processes.

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“…As a host, Streptomyces has the following advantages over other systems: 1) the formation of inclusion bodies has not been described in the literature; 2) it is a well-suited host for the expression of very GC-rich genes without codon adaptation [12]; 3) it has high protein secretion efficiency, which makes it feasible to obtain the protein of interest in the culture supernatant, facilitating the protein folding and downstream procedures of extraction and purification [11]; 4) the species used to express proteins (see below) display a relatively low level of endogenous extracellular proteolytic activity in comparison with other hosts such as Bacillus , where deletion of up to seven extracellular proteases had to be done to improve protein production [13,14]. …”

Section: Introductionmentioning

confidence: 99%

“…The system has proved useful for the expression of genes from different Streptomyces species, from Aspergillus nidulans and from Thermus thermophilus ([12,16]). This system uses multi-copy mono-functional ( Streptomyces ) or bi-functional ( E. coli and Streptomyces ) plasmids that are selected with antibiotic resistance genes (mainly thiostrepton or neomycin).…”

Section: Introductionmentioning

confidence: 99%

Stable expression plasmids for Streptomyces based on a toxin-antitoxin system

2013

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BackgroundBacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. One of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. To date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. However, the use of antibiotics to produce proteins at industrial level is currently becoming more restricted owing to the possibility of contamination of the final product. In this report, we describe the use of the S. lividans yefM/yoeBsl toxin-antitoxin system to develop a stable plasmid expression system.ResultsIn order to use the yefM/yoeBsl system to stabilize expression plasmids in Streptomyces, a S. lividans mutant strain that contained only the toxin gene (yoeBsl) in its genome and the antitoxin gene (yefMsl) located in a temperature-sensitive plasmid was constructed and used as host. This strain was transformed with an expression plasmid harbouring both the antitoxin gene and the gene encoding the protein of interest. Thus, after elimination of the temperature-sensitive plasmid, only cells with the expression plasmid were able to survive. On using this system, two proteins - an α-amylase from S. griseus and a xylanase from S. halstedii - were overproduced without the addition of antibiotic to the culture medium. The production of both proteins was high, even after long incubations (8 days), and after serial subcultures, confirming the stability of the plasmids without antibiotic selection.ConclusionsThis is the first report that describes the use of a toxin-antitoxin system to maintain high -copy plasmids in Streptomyces. This finding could be a valuable tool for using Streptomyces as a host to produce proteins at the industrial and pharmaceutical levels without the use of antibiotics in the production step.

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High-level overproduction of Thermus enzymes in Streptomyces lividans

Cited by 20 publications

References 25 publications

Development of Novel Drugs from Marine Surface Associated Microorganisms

Development of Novel Drugs from Marine Surface Associated Microorganisms

Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in Streptomyces lividans

Stable expression plasmids for Streptomyces based on a toxin-antitoxin system

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