Rational Metabolic Engineering strategies design for the co-production of pyocyanin and rhamnolipids in Pseudomonas aeruginosa IGPR1 for the bioremediation of oil-polluted water bodies

Abstract

Fossil fuels are a central part of life, most human activities are powered by them. However, their increasing demand and manipulation suppose a constant threat of accidental release to the environment. The consequences of oil spills impact the environmental, social, and economic spheres of life, generating biodiversity, health, and monetary losses. Among oil remediation techniques, bioremediation is the most promising strategy to mitigate oil spills negative effects. Nonetheless, the implementation of bioremediation strategies is slow and uncertain as successful bioremediation efforts require deep comprehension of the strains used. The study and application of environmental strain Pseudomonas aeruginosa IGPR1 to bioremediation brings the opportunity to design rational metabolic engineering strategies to improve oil emulsification. IGPR1 is a metabolically flexible strain that produces pyocyanin and rhamnolipids, two metabolites involved in the emulsification and degradation of oil. Here we describe the characterization of IGPR1 strain through the metabolic reconstruction of its genome and the implementation of classic and novel metabolic engineering strategies such as homologous recombination and the use of CRISPR/Cas9-APOBEC1 coupled with ribozymes-mediated maturation of sgRNAs to induce premature stop codons and generate doble knockouts. This is the first study describing the CRISPR/Cas9-APOBEC1 system with ribozymes, furthermore, iCDC1103 is the first metabolic reconstruction of a P. aeruginosa environmental strain with bioremediation applications. This work sets the foundations for future research with this complex yet industrially interesting strain.