The overall goal of this workflow is to identify a natural product with antibiotic activity, purify the compound, and determine its biosynthetic gene cluster through genome mining. New antibiotics are urgently needed because of the growing number of myto resistant pathogens. Streptomycin strains are known antibiotic producers and we should utilize that potential.
With the knowledge of an antibiotic biocine heterogene cluster we can propose a biosynthetic pathway and further modify the compound. These procedure help you identify the corresponding gene cluster. The radio is demonstrated with streptomycin strain.
But it can be applied to other micro organism. To begin, throw the streptomycin strain under different conditions like different media or incubation time. Harvest the cells by centrifugation for 10 minutes at 3, 000 times G.And transfer the supernatants into a new flask.
For compound extraction from the culture broth, adjust the supernatant to PH4 by the addition of one molar hydrochloric acid. And transfer it into a separating funnel. Then add the same volume of ethal acetate and shake the mixture for 30 minutes at 180 rpm.
Next, collect the ethal acetate phase and transfer it into a round-bottom flask through a filter paper. Evaporate the ethal acetate by rotary evaporation at 40 degree Celsius and 240 bar. For compound extraction from the mycelia resuspend the cells in a twofold volume of acetone.
Shake the cells in a tube at 180 rpm for 30 minutes. Filter the liquid through filter paper. Then evaporate the acetone using a rotary evaporator at 40 degree Celsius and 550 bar.
Dissolve the dried extract in 20 milliliters of water ethal acetate and continue extraction of compounds as before. Then dissolve the dried extracts in methanol. Filter them through a 0.45 micrometer pore-size filter.
And transfer the extracts into HPLC vials. Inject the samples into the HPLC machine and analyze the crude extract as described in the text protocol. To identify the antibiotic activity using the disc diffusion assay, spread various test strain precultures onto respective agar plates.
Then pipette 20 to 50 microliters of the dissolved extract onto sterile paper discs. Also prepare a negative control with solvent and a positive control with an appropriate antibiotic. After drying the paper discs for 30 minutes, transfer them onto the plates with the test cultures.
Incubate the test strain plates under adequate conditions. Then determine the apparent inhibition zones for each test strain. If an inhibition zone exists, purify the responsible compound from large scale cultivation and elucidate the structure as described in the text protocol.
During purification a compound may change due to oxidation, radiation or temperature. The more purification tabs used, the higher the chance of degradation. Up to five milligrams of compound, which is the minimum needed for analysis common genogene.
Analyze the chemical structure of the isolated compound and predict enzymes that may be involved in it's biosynthesis. Here poly cidomycin is used as a example to demonstrate the the procedure. First, subdivide the structure into obvious single moieties.
Poly cidomycin is composed of a tetracyclic moiety, two monosaccharides, and dimethylsalicylic acid. Next predict putative genes in the cluster where the moieties may be derived from. Here the tetracyclic moiety could be derived from a poly key type synphase type II.Whereas the dimethylsalicylic moiety may be derived from a iterative poly keytype synphase type I.Likewise the two sugar moieties, which are 6 deoxy sugars might be synthesized from glucose involving a dNDP-glucose-4, 6-dehydratase during biosynthesis and attached by two glycosyltransferases.
Sequence the genomic DNA by next generation sequencing technologies. For the identification of the secondary metabolite gene cluster run programs such as antiSMASH, Natural Product Domain seeker, or NRPS predicter. Submit the genome sequence as fasta format.
After several hours, the results are sent via an email link. In the draft genome of streptimyoces diastatochromogenes, antiSMASH identified 22 putative gene clusters. Analyze the putative gene clusters for their enzymatic pathways.
Search for clusters containing genes which might be involved in the synthesis of your compound. In astatochromogenes, cluster II contains poly keytype synphase type II genes, three genes for an iterative PKS type I, and dTDP-glucose-4, 6-dehydratase gene, and two glycosyltransferases encoding genes. To compare the cluster with other clusters, antiSMASH provides a link to the MIBiG database which includes annotations and further information about similar biosynthetic gene clusters and their products.
Compare the structure of the compound with other known compounds to check for similarities. The identification of the responsible biosynthetic gene cluster may be straight forward. But in a few cases it can also be very difficult.
For verification that the proposed gene cluster is responsible for the biosynthesis of the compound search for a gene within the cluster and coding for an enzyme with a central function for biosynthesis. To confirm the poly cidomycin gene cluster, the gene pokPI one coding for ketosynthase alpha from the PKS type II was selected and inactivated by an out of frame deletion. After cloning and conjugation of the single crossover construct as described in the text protocol.
Check the single crossover mutants. To do so, inoculate 100 milliliters of production media with single clones containing the verified gene interruption. Incubate the cells under the same conditions as used previously.
Proceed to extract the crude extract as shown before. Finally, check the production by HPLC MS Analysis as described in the text protocol. The corresponding peak in the HPLC chromatogram and the mass for the compound should not be detectable.
The poly cidomycin biosynthetic gene cluster was verified by a gene deletion experiment. Shown here is the HPLC chromatogram of the crude extract of the wildtype S.Diastatochromogenes and the mutant strain with interrupte pokPI one gene. The mutant strain does not produce poly cidomycin anymore.
Once mastered, this procedure can be done within several months. Many years ago, the identification of a biosynthetic gene cluster took much time. Today genome sequencing and genome mining strongly accelerate this process.
After watching this video, you should have a good understanding how to get from a novel compound to its biosynthetic gene cluster. We showed the whole procedure in single steps so you can assign them now to any other natural product producer. The knowledge about the gene cluster and it's synthesized compounds gives us insight into biosynthetic pathways.
This gives us further opportunities to modify already existing molecules in order to improve them.
