Biopharma Insights Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.
Total time
Several weeks to months
Model organism
Streptomyces diastatochromogenes Tü6028
Steps
1
Identify antibiotic natural product from culture
Screen Streptomyces fermentation extracts for antibiotic activity using a susceptible indicator strain to detect the bioactive compound.
▶ 00:54
2
Propose biosynthetic model of compound
Based on the purified compound's chemical structure, predict plausible enzymatic steps and precursor building blocks for its biosynthesis.
▶ 03:54
3
Identify candidate biosynthetic gene cluster
Sequence the producing strain's genome and mine the data for gene clusters encoding enzymes consistent with the proposed biosynthesis.
▶ 05:02
4
Verify biosynthetic gene cluster by inactivation
Inactivate a candidate cluster gene and compare metabolite profiles to confirm its role in compound production.
▶ 06:34
5
Analyze HPLC of inactivated mutant
Run HPLC analysis on the pokPI-inactivated strain to demonstrate loss of the target compound, confirming cluster function.
▶ 07:40
6
Conclude gene cluster verification
Summarize how the integrated workflow links a natural product to its biosynthetic gene cluster.
▶ 08:07