Actinobacteria Metabolic Engineering
This group is located at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS)
The growing resistance towards established antibiotics presents a serious problem especially with infectious diseases. The development of new drugs is mainly based on known molecules and mechanisms, which allows bacteria to assimilate rapidly. Hence, scientists are looking for novel drugs. At the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), a branch of the Helmholtz Centre for Infection Research (HZI) at Braunschweig, the researchers develop new pathways, by which they force actinomycetes to produce hitherto unknown compounds.
Regulatory gene networks responsible for the heterologous expression in S. albus and S. lividans
The expression of secondary metabolic gene clusters is controlled by many different families of regulatory proteins, some of which are found only in actinomycetes, and is elicited by both extracellular and intracellular signalling molecules. Using systematic in vivo transposon mutagenesis combined with gusA based screening, we identify gene networks responsible for the up and down regulation of natural product biosynthesis in streptomycetes.
The in vivo transposon-based strategy is a valuable tool to identify regulatory genes of secondary metabolism, including gene loci which cannot be detected using current in silico approaches. Even though a derivative of the transposon Tn5 was successfully developed and applied in our laboratory to perform random mutagenesis in actinomycetes, using different transposons minimizes untargeted gaps due to insertion-site specificities. We have developed an additional transposon, a derivative of Himar1, a mariner family element isolated from the horn fly Haematobia irritans. The Himar1 encoding synthetic gene has been synthesized and cloned under the inducible tipA and tcp promoters, respectively.
The DNA fragments containing an apramycin resistance gene and spectinomycin resistance gene linked to the R6Kγ ori have been used as minitransposons. In addition, we have introduced ermE and tcp outwards promoters at the end of minitransposon to overexpress downstream and upstream genes in the chromosome. Thus our minitransposon systems will not only disrupt genes via integration, but also will activate (overexpress) open reading frames within actinomycete chromosome. The frequency of insertion for hyperactive Tn5 derivative and Himar1 was more that 98 % of transformed S. lividans and S. coelicolor cells.
The used transposon also contains an apramycin resistance marker, which is very efficient in most streptomycetes, and R6Kg replication origin, which allows quick and easy cloning of the insertion site directly from the chromosome.
Bachelor & Master
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