The bacterial genus Streptomyces is the producer of numerous bioactive natural products and a significant contributor of clinical antibiotics. However, the control of natural product biosynthesis is still unclear, and the characterization of the enzymes that synthesize the natural products is often complicated by the large number of candidates in the genome. In this thesis, shotgun proteomics was used to study the protein expression of three different Streptomyces species surrounding the onset of natural product biosynthesis. First, the proteome of Streptomyces coelicolor was monitored between replicates exhibiting different phenotypes. The expression of biosynthetic gene clusters (BGCs) was observed on a fine time resolution, revealing the activity of a lanthipeptide and ectoine BGCs w...[ Read more ]

The bacterial genus Streptomyces is the producer of numerous bioactive natural products and a significant contributor of clinical antibiotics. However, the control of natural product biosynthesis is still unclear, and the characterization of the enzymes that synthesize the natural products is often complicated by the large number of candidates in the genome. In this thesis, shotgun proteomics was used to study the protein expression of three different Streptomyces species surrounding the onset of natural product biosynthesis. First, the proteome of Streptomyces coelicolor was monitored between replicates exhibiting different phenotypes. The expression of biosynthetic gene clusters (BGCs) was observed on a fine time resolution, revealing the activity of a lanthipeptide and ectoine BGCs which has not been previously detected. More interestingly, the coelimycin BGC was observed to be expressed in a staggered manner where biosynthetic proteins would be expressed but the production was unlikely due to the lack of ScoT which releases coelimycin from the protein complex. Finally, the upregulation of several regulatory proteins, including the tellurium resistance protein TerD, were correlated with increased secondary metabolism. Then, a proteomining workflow exploiting the gene proximity of BGCs was used to detect the expression of active BGCs and link them to detected natural products. The proteomining workflow successfully detected active BGC expression in S. coelicolor, Streptomyces chrestomyceticus and Streptomyces tenebrarius during the stationary phase using the non-producing growth phase as background. Using the information gained from the proteomining, caerulomycin was detected and isolated for the first time from S. tenebrarius. Inactivation of the caerulomycin BGC resulted in the global repression of secondary metabolism. Finally, proteins involved in the biosynthesis of chresdihydrochalcone were proposed based on shotgun proteomics results. Chresdihydrochalcone was the first chalcone isolated from bacteria, and, unlike natural products produced by BGCs, was produced by a single protein but can be tailored by various stray enzymes. Candidate proteins were searched in the genome and the expression was correlated with chresdihydrochalcone. Several pathways were proposed based on reaction mechanism of other type III PKS. Overall, proteomics served as a valuable aid to genome mining approaches, narrowing down candidates of biosynthetic pathways well as being capable of providing valuable information for the optimization of natural product biosynthesis.