Overproduction of glycolipopeptide bio-amphiphile by Pseudomonas aeruginosa mutant generated through three-tier cocktail random mutagenesis

A robust three-tier cocktail of random mutagenesis involving atmospheric and room temperature plasma, gamma-rays, and N-methyl-N'-nitro-N-nitrosoguanidine exposures was applied to induce glycolipopeptide overproduction in Pseudomonas aeruginosa 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, kLa, 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 Pseudomonas putida 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.