Abstract
BACKGROUND: Green fluorescent protein (GFP) is a valuable macromolecule widely utilized in biomedical diagnostics and diverse biotechnological applications due to its low toxicity and intrinsic fluorescence, which does not require exogenous substrates or cofactors. Nevertheless, the conventional Aequorea victoria–derived GFP exhibits limitations, including relatively low brightness and restricted applicability under low-oxygen conditions. Consequently, there is increasing interest in identifying alternative and less explored microbial sources of GFP, particularly from marine environment. RESULTS: In the current study, eleven fluorescent bacteria were isolated from different anatomical parts of squid collected from Miami Beach (Alexandria, Egypt) and from seawater sample from Atubia island (Safaga, Egypt). Among the three isolation media evaluated, Sea Water Complete agar medium (SWC) supported the highest recovery of fluorescent bacteria. Fluorescence-based primary and secondary screening identified isolate E2 as the most efficient GFP producer. It was identified phenotypically using the VITEK(®) 2 automated system and genotypically using 16 S rRNA studies as Pseudomonas gessardii with 99.34% similarity. Sequences of the 16 S rRNA gene were deposited in the GenBank as OQ285875. Optimization of environmental condition using a one-factor-at-a-time approach revealed the maximum GFP production was achieved after 30 h of incubation at 25 °C and pH 7.5 under static conditions (0 rpm). These optimized parameter enhanced the fluorescence intensity of GFP by 1.51-fold compared to the basal medium. Subsequent purification using an aqueous two-phase system and fast protein liquid chromatography successfully recovered GFP with a high purity of 96.3%. CONCLUSION: Overall, the current study identifies Pseudomonas gessardii E2 strain as a novel marine-derived source of high-quality pure GFP and establishes an effective strategy for its production and purification. The findings provide a promising foundation for the potential application of this GFP in bioimaging, biosensing, and fluorescence-based reporter systems, including applications in oxygen-limited environments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-026-02960-9.