Journal: bioRxiv

Article Title: Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

doi: 10.1101/2025.03.18.643695

Figure Lengend Snippet: A Growth-Coupled Isoprenol Biosensor for High-Throughput Screening and Targeted Strain Engineering (A) Biosensor repurposing pipeline sketch to enhance heterologous isoprenol production. A pool of ∼16,500 gRNA targets were generated to use a dCas12a/CRISPRi system targeting nearly all genes in P. putida . The isoprenol biosensor was modified to replace the pedF-inducible mCherry gene by pyrF to enable the growth on M9 glucose of a ΔpyrF strain in the presence of isoprenol. The PpedF-RBS-pyrF Δ pyrF strain containing the gRNA library was grown on M9 minimal medium with 2% of glucose and assayed for isoprenol production. Enriched gRNAs at the end of the experiment were amplified, sequenced, and verified by generating the corresponding deletion mutant to role of the deleted gene on isoprenol titer. (B) RBS mutagenesis was performed to optimize the pedF-inducible expression of pyrF to constrain growth to higher concentrations of isoprenol. The response of the construct was evaluated measuring the growth of the strain under different isoprenol concentrations on M9 minimal medium with glucose as the carbon source. All datapoints are shown and the error bars indicate standard deviation from the mean. (C) The enriched gRNA sequencing results were compared between all growth assays performed using multiple optimized RBS and two strains with different isoprenol production productivities. The coverage of the gRNA library as detected with the rapid ONT sequencing method at the start of the experiment was also included. Each line indicates a different enriched gRNA detected from a representative sample and is plotted against its location in the P. putida KT2440 genome.

Article Snippet: Representative Plasmidsaurus ONT gRNA amplicon sequencing reads.

Techniques: High Throughput Screening Assay, Generated, Modification, Amplification, Mutagenesis, Expressing, Construct, Standard Deviation, Sequencing

Journal: bioRxiv

Article Title: Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

doi: 10.1101/2025.03.18.643695

Figure Lengend Snippet: (A) Strain lineage sketch. This graphic describes the combinatorial modifications evaluated to increase the heterologous production of isoprenol. Over 165 deletions and overexpression constructs were tested and a representative strain lineage is shown. Deletion candidate targets were identified in the dCpf1/gRNA PpedF-RBS-pyrF growth assay and isoprenol productivity was evaluated after generating each modification. Candidate modifications were classified based on known gene annotations and are represented with different icons (refer to legend in the bottom left corner). Genes were also color-coded to indicate the isoprenol productivity of such modification (refer to heat-map legend in the top right corner). i: Isoprenol production timecourse of selected strains. Production strains were grown in M9 2% glucose and prepared for isoprenol titer analysis and sampled at the indicated timepoints. All datapoints are shown and the error bar indicates standard deviation from the mean. (B) Growth curve of two of the best isoprenol producing strains, TEAM-3174 and TEAM-3185, compared to the control strain TEAM-2595 in M9 2% glucose and 1µM crystal violet as inducer. n=4 biological replicates and the shaded area indicates standard deviation from the mean. (C and D) Volcano plots representing the ∼2,500 proteins detected by LC-MS/MS in TEAM-3185 (C) and TEAM-3174 (D) compared to the control strain, TEAM-2595. The proteome was analyzed in both growth and production phase. (E) Scatter plot indicating the percentage abundance of the selected proteins overlaid on all detected proteins in strains TEAM-2595, TEAM-3174, and TEAM-3185. Selected proteins were grouped according to the indicated functions and are colored according to the legend. (F) Metabolic map representing the differential log2 fold abundance change of the indicated metabolites in TEAM-3174 and TEAM-3185 compared to the control strain, TEAM-2595. The squares represent the log2 fold abundance of the metabolites in TEAM-3174 and TEAM-3185 in growth and production phase. Blue arrows indicate the predicted flux to isoprenol based on the variations of the log2 fold metabolite concentrations in production phase samples (24 hour timepoint). Mean values from 3 biological replicates for each sample are reported. A key production phase metabolite, phenylalanine, is highlighted in yellow. Abbreviations: GP: growth phase samples. PP: production phase samples. Pyruvate (PYR), leucine (LEU), tryptophan (TRP), phenylalanine (PHE), tyrosine (TYR), fumarate (FUM), malate (MAL), oxalacetate (OAA), citrate (CIT), aconitate (ACON), isocitrate (ICIT), alpha-ketoglutarate (AKG), glutamate (GLT), succinate (SUC), glyoxylate (GLX), acetyl-coenzyme A (Ac-CoA), acetoacetyl-coenzyme A (Acc-CoA), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), mevalonate (MVA), mevalonate monophosphate (MVAP), isopentenyl monophosphate (IP), Entner-Doudoroff-Embden-Meyerhof-Parnas cycle (EDEMP).

Article Snippet: Representative Plasmidsaurus ONT gRNA amplicon sequencing reads.

Techniques: Over Expression, Construct, Growth Assay, Modification, Standard Deviation, Control, Liquid Chromatography with Mass Spectroscopy