<p>A robust three-tier cocktail of random mutagenesis involving atmospheric and room temperature plasma, gamma-rays, and <i>N</i>-methyl-<i>N’</i>-nitro-<i>N</i>-nitrosoguanidine exposures was applied to induce glycolipopeptide overproduction in <i>Pseudomonas aeruginosa</i> strain IKW1 (GenBank Accession No.: PV664482.1). Auxanographic investigations suggested unrepaired metabolic blocking of the threonine, and hence, isoleucine branches of the aspartate pathway in the overproducing mutant, Thr⁻Ile⁻PGN4. Glycolipopeptide overproduction significantly correlated with increases in urease activity, Ni²⁺ uptake, cytosolic Ni²⁺ localization and upregulation of histidine biosynthesis. Scalability by batch fermentation in modified optimized medium in 5-L bioreactor led to 1.24-fold higher volumetric oxygen transfer coefficient, <i>k</i>L<i>a</i>, with an oxygen uptake rate (OUR) of 18.6 mmol O₂ gDCW^− 1 h^− 1, which resulted in 3.86-fold higher glycolipopeptide productivity. Periodic biochemical and thin layer chromatographic analysis revealed trophophasic synthesis of a functional surface-active rhamnolipid base with surface (SFT) and interfacial (IFT) tension reductions to 29.74 dynes cm^− 1 and 2.22 dynes cm^− 1, respectively. A late idiophasic peptidation of the rhamnolipid base further reduced SFT and IFT to 24.41 and 0.87 dynes cm^− 1, respectively. Superior catalysis by catechol 1,2-dioxygenase favored glycolipopeptide-mediated crude oil degradation by <i>Pseudomonas putida</i> strain ATCC 49,182 compared to commercial biosurfactants giving rate constants of 0.115 d^− 1 (glycolipopeptide), 0.087 d^− 1 (di-rhamnolipid), 0.096 d^− 1 (surfactin) and 0.065 d^− 1 (without surfactant). The technology is recommended for sustainable glycolipopeptide production toward efficient crude oil-impacted ecosystems’ bioremediation.</p>