Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price limited formation acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA a key precursor number added-value products, in work we deployed an silico-guided rewiring program central carbon metabolism upgrading P. as host acetyl-CoA–dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets addition to manually-curated information enzyme mechanisms, identified targets that would lead increased levels. Based on these predictions, set plasmids based clustered regularly interspaced short palindromic repeats (CRISPR) dead CRISPR-associated protein 9 (dCas9) was constructed silence genes by CRISPR interference (CRISPRi). Dynamic reduction gene expression two (gltA, encoding citrate synthase, essential accA gene, subunit carboxylase complex) mediated 8-fold increase content rewired putida. Poly(3-hydroxybutyrate) (PHB) adopted proxy availability, synthetic pathways were engineered biopolymer accumulation. By including cell morphology extra target CRISPRi approach, fully strains programmed PHB accumulation had 5-fold titers bioreactor cultures using glucose. Thus, strategy described herein allowed rationally redirecting fluxes towards product biosynthesis—especially when deletion not option.