Link Lab

The metabolome of an E. coli CRISPRi library identifies benefits of minimal metabolite levels and targets for engineering Rapp et al. combine genome-scale CRISPRi and metabolomics in E. coli. CRISPRi of enzymes enforces metabolite accumulation, which leads to regulatory crosstalk and side reactions. This suggests that ma...

Technically this required metabolomics on ~3,000 samples from an arrayed CRISPRi library.

The bottleneck wasn’t MS or sampling.

It was sequencing the strains after arraying.

Paper: www.cell.com/cell-systems...

Example:
Knockdown of ispB increases farnesyl-PP.

Add a carotenoid pathway → more product. 🟠

Metabolite accumulation can reveal engineering targets.

This indicated that E. coli keeps metabolite pools low
Because high metabolite pools lead to problems:

They inhibit other enzymes

They cause regulatory crosstalk

They trigger promiscuous side reactions

These roadblocks reveal metabolites we normally never detect.
Many intermediates are at very low levels in wild-type cells.
Reduce an enzyme and substrates become measurable by MS. 🔬

What happens if you knock down every metabolic gene in E. coli?

We measured the metabolome of 1515 CRISPRi strains.

Result: metabolism behaves like traffic.
Block an enzyme and metabolites pile up upstream. 🚗🚗🚗
New paper 🧵

The Metabolic State of E. coli Influences Fosfomycin Efficacy and Promotes Resistance Evolution The phosphonic antibiotic fosfomycin is a bacterial cell wall synthesis inhibitor that targets MurA, the first enzyme in the peptidoglycan pathway. Transporter loss or enzymatic inactivation confers resistance to fosfomycin, but whether the metabolic state of a bacterium influences the efficacy of this antibiotic has not been characterized. Here, we used an Escherichia coli CRISPR interference library targeting 1,515 metabolic genes to identify metabolic activities that influence fosfomycin efficacy. We discovered that knockdowns of ATP synthase and pyruvate kinase genes lead to a regrowth phenotype, whereby cells resume growth after an initial phase of killing. By following up on this phenotype with population analysis profile tests and repeated treatment cycles, we found evidence that a heteroresistant population may promote the evolution of fosfomycin resistance. Whole-genome sequencing of the pykF CRISPRi strain after 24 h of fosfomycin exposure revealed that the acid stress protein-encoding gene ibaG, which is upstream of murA, carries a mutation that confers fosfomycin resistance. Metabolome analysis showed accumulation of the MurA substrate phosphoenolpyruvate in regrowing cells, which may compete with fosfomycin for binding to MurA. Transcriptome analysis provided further insight into the mechanism of cell regrowth, including upregulation of genes encoding cell envelope stress response regulators such as cpxP. These results suggest that the metabolic state can modulate the efficacy of fosfomycin and contribute to resistance evolution.

New paper from our group: Metabolism Influences Fosfomycin Efficacy pubs.acs.org/doi/10.1021/...

PhD and Postdoc positions opening in synthetic biology, CRISPR genome engineering of bacteria, and metabolomics.
Details and contact here: www.linkmetabolism.com/join-us

Community composition and strain identity drive metabolic competition and Staphylococcus aureus colonization resistance in Synthetic Nasal Communities The human nasal microbiome is a low-diversity ecosystem whose assembly principles and mechanisms of colonization resistance remain poorly understood. In particular, Staphylococcus aureus colonization ...

Preprint from our lab and @func-metabo-lab.bsky.social: In synthetic nasal communities a single Corynebacterium propinquum strain can exclude Staphylococcus aureus through nutrient competition. www.biorxiv.org/content/10.6...

Metabolic mutations influence E. coli's antibiotic susceptibility ➡️ www.embopress.org/doi/full/10....

Thanks Orestis Kanaris and Frank Schreiber for the very nice comment.

Hello Bluesky! We’re here to talk #microbes, #metabolism, #CRISPR, #metabolomics and more.