THESIS
2022
1 online resource (xiii, 170 pages) : illustrations (some color)
Abstract
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...[
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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.
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