Investigating the biosynthetic enzymes of colibactin
Ontology highlight
ABSTRACT: Analysis of the biosynthetic enzyme, ClbH, that incorporates S-adenosylmethionine into the backbone of the growing colibactin natural product.
Project description:Diacylglycerol lipase-beta (DAGLB) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLb ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream pro-inflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to discover that disruption of DAGLb in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLb blockade, thereby directly supporting DAGLβ-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy sensing kinases to mediate cell biological and pain responses.
Project description:Diacylglycerol lipase-beta (DAGLβ) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLβ ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLβ in primary macrophages leads to LKB1–AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLβ blockade, thereby directly supporting DAGLβ–AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.
Project description:Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we hypothesized that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the “coenzyme” category have been examined, including a gene cluster encoding for the co-factor pyrroloquinoline quinone (PQQ). When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides a new engineering strategy for improving polyketide production and discovering new biosynthetic gene clusters.
2024-03-01 | GSE256209 | GEO
Project description:Evaluating Distribution of Bacterial Natural Product Biosynthetic Gene Clusters Across Lake Huron Sediment
Project description:bgc-gene-product model is a Named Entity Recognition (NER) model that identifies and annotates the protein products of Biosynthetic Gene Clusters (BGCs) in texts.
Project description:An improved understanding of the genome-wide regulation of natural compound biosynthesis in bacterial producers may accelerate the discovery of novel biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed the dynamic temporal variation of transcriptional activity, indicating that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and nonribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these surges in BGC transcriptional activity were associated with boosts in the production of known natural compounds, indicating that their biosynthesis was crucially regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provided unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under meager conditions for bacterial growth. The close association between BGC transcription and compound production suggested that the molecular manipulation of transcriptional activity may be a viable strategy to increase compound yields from myxobacterial producer strains, warranting increased efforts to develop genetic engineering tools for these organisms.
Project description:Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 – 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.