51 research outputs found

    Design, development and application of whole cell based antibiotic specific biosensor

    No full text
    Synthetic biology techniques hold great promise for optimising the production of natural products by microorganisms. However, evaluating the phenotype of a modified bacterium represents a major bottleneck to the engineering cycle particularly for antibiotic producing actinobacteria strains, which grow slowly and are challenging to genetically manipulate. Here, we report the generation and application of antibiotic specific whole cell biosensor derived from TetR transcriptional repressor for use in identifying and optimising antibiotic producers. The constructed biosensor was successfully used to improve production of polyketide antibiotic pamamycin. However, an initial biosensor based on native genetic elements had inadequate dynamic and operating ranges. To overcome these limitations, we fine tuned biosensor performance through alterations of the promoter and operator of output module and the ligand affinity of transcription factor module, which enabled us to deduce recommendations for building and application of actinobacterial biosensor

    Evaluation of heterologous promoters for genetic analysis of Actinoplanes teichomyceticus--Producer of teicoplanin, drug of last defense

    No full text
    Actinoplanes teichomyceticus is the only known producer of the valuable glycopeptide antibiotic teicoplanin. Random mutagenesis and selection were extensively applied to teicoplanin producers, while the gene engineering methods were not used, because of the paucity of genetic tools for A. teichomyceticus. Particularly, availability of promoters of different strength that are functional in Actinoplanes would be very useful for overexpression of beneficial genes. Here we report the use of a glucuronidase reporter system (gusA) for studying transcriptional activity in A. teichomyceticus and describe the behavior of a set of heterologous promoters in this strain. We reveal several elements that exceed in their strength the well-established Streptomyces promoter ermEp, underscoring the utility of the gusA reporter for Actinoplanes sp. Remarkable overproduction of teicoplanin was achieved by constructing strains carrying additional copies of the regulatory gene tcp28 under the control of one of the two most active promoters, moeE5p and actp, discovered in this study

    The pathway-specific regulatory genes, tei15* and tei16*, are the master switches of teicoplanin production in Actinoplanes teichomyceticus

    No full text
    Pathogenic antibiotic-resistant bacteria are an unprecedented threat to health care worldwide. The range of antibiotics active against these bacteria is narrow; it includes teicoplanin, a \u201clast resort\u201d drug, which is produced by the filamentous actinomycete Actinoplanes teichomyceticus. In this report, we determine the functions of tei15* and tei16*, pathway-specific regulatory genes that code for StrR- and LuxR-type transcriptional factors, respectively. The products of these genes are master switches of teicoplanin biosynthesis, since their inactivation completely abolished antibiotic production. We show that Tei15* positively regulates the transcription of at least 17 genes in the cluster, whereas the targets of Tei16* still remain unknown. Integration of tei15* or tei16* under the control of the aminoglycoside resistance gene aac(3)IV promoter into attB\u3d5C31 site of the A. teichomyceticus chromosome increased teicoplanin productivity to nearly 1 g/L in TM1 industrial medium. The expression of these genes from the moderate copy number episomal vector pKC1139 led to 3\u20134 g/L teicoplanin, while under the same conditions, wild type produced approximately 100 mg/L. This shows that a significant increase in teicoplanin production can be achieved by a single step of genetic manipulation of the wild-type strain by increasing the expression of the tei regulatory genes. This confirms that natural product yields can be increased using rational engineering once suitable genetic tools have been developed. We propose that this new technology for teicoplanin overproduction might now be transferred to industrial mutants of A. teichomyceticus
    corecore