Date of Award
2026
Degree Name
Biomedical Research
College
Joan C. Edwards School of Medicine
Type of Degree
Ph.D.
Document Type
Dissertation
First Advisor
Dr. Hongwei Yu
Second Advisor
Dr. Timothy Long
Third Advisor
Dr. Vincent Sollars
Fourth Advisor
Dr. Lydia Bogomolnaya
Fifth Advisor
Dr. Richard Egleton
Abstract
The increasing demand for novel antimicrobial agents and biologically derived therapeutic materials has intensified interest in microbial biosurfactants such as rhamnolipids. However, translational application of rhamnolipids for biomedical use is severely restricted by the virulence of the natural producer, Pseudomonas aeruginosa. This dissertation explores the mechanisms that determine rhamnolipid biosynthesis in P. aeruginosa strain PAO1 and examines them in genetically attenuated strains of P. aeruginosa. A review of the characteristics of P. aeruginosa that make it the most proficient source of rhamnolipids is presented and the use of genetically attenuated PGN strains as a chassis for biosynthesis is advocated. This research validates a simplified screening assay using PIA-CTAB methylene blue agar.
The engineered PGN strains were characterized for suitability for rhamnolipid production. The rhamnolipid/cell mass yields (grams per gram biomass) 1,2 from PAO1 were 5.1 ± 3.5 grams per gram biomass, PGN4 was 3.7 ± 1.5 grams per gram biomass and PGN5 was 4.3 ± 3.6 grams per gram biomass when M9 mineral salt media with 2% glycerol was used for fermentation. Statistical analysis showed that there was no significant difference in the yield or titer of rhamnolipids produced by PAO1, PGN4 and PGN5 strains. The rhlABRI gene cluster was deleted in PAO1, PGN4 and PGN5 to create rhamnolipid deficient mutants PAO1ΔrhlABRI, PGN4ΔrhlABRI and PGN5ΔrhlABRI. Rescue strains were created by insertion of pHERD20TrhlAB in these deletion mutants to create PAO1ΔrhlABRI-pHERD20TrhlAB, PGN4ΔrhlABRI-pHERD20TrhlAB and PGN5ΔrhlABRI-pHERD20TrhlAB. The rhamnolipid yield and titer was then determined in these rescue mutants.
PAO1ΔrhlABRI-pHERD20TrhlAB, PGN4ΔrhlABRI-pHERD20TrhlAB and PGN5ΔrhlABRI-pHERD20TrhlAByielded rhamnolipids at 0.22 ± 0.11grams per gram biomass,
0.28 ± 0.20grams per gram biomass, 0.28 ± 0.02 grams per gram biomass. The rhamnolipid yields by these rescue strains were significantly lower than that of PAO1 at 1.18 ± 0.24 gram per gram biomass when PIB with glycerol was used as the media. Disconnecting the intrinsic Rhl- QS apparently failed to increase the rhamnolipid yield.
The interplay of metabolic pathways has been highlighted by examining the effects deletion of algD gene. The rhamnolipid yield from PAO1 and PAO1ΔalgD were 2.75 ± 1.45 grams per gram biomass and 1.9 ± 0.97 grams per gram biomass. Statistical analysis showed that the difference in the yield was not significant. There was no increase in rhamnolipid yield by blocking alginate synthesis.
Novel genetic regulators of rhamnolipid production have been uncovered by transposon mutant screen, which include PA4657(NAD/FAD dependent oxidoreductase) and mmsB genes (3-hydroxyisobutyrate dehydrogenase). The screen also revealed the role of algC, estA and fabI in rhamnolipid production.
Future work in this research will address the methodology limitations and explore the novel genes detected. The research explores the feasibility of using genetically attenuated strains as a chassis for rhamnolipid production. This would overcome the bottleneck created by the pathogenicity of the wild type P. aeruginosa strains. This dissertation is a first step towards establishing a safe microbial cell factory that bridges clinical safety with industrial biotechnology.
Subject(s)
Medicine -- Research.
Biology -- Research.
Bacteria.
Gene editing.
Biosurfactants.
Bacterial genetics.
Pathogenic bacteria.
Genetic engineering.
Microbial biotechnology.
Pseudomonas aeruginosa -- Genetics.
Recommended Citation
Vijayamohana Das, Parvathy, "Genetic engineering of attenuated strains of Pseudomonas aeruginosa for rhamnolipid production" (2026). Theses, Dissertations and Capstones. 2159.
https://mds.marshall.edu/etd/2159
Included in
Cell Biology Commons, Genetic Processes Commons, Medical Biotechnology Commons, Medical Cell Biology Commons, Medical Genetics Commons, Medical Microbiology Commons, Medical Molecular Biology Commons, Microbiology Commons, Other Medical Specialties Commons
