Optimizing rhamnolipid biosynthesis: evaluating predictive methods using Pseudomonas aeruginosa mutants.

Area of Science:

• Microbiology
• Biotechnology
• Metabolic Engineering

Background:

• Rhamnolipids (RLs) are biosurfactants with industrial applications, but high production costs hinder widespread use.
• Genetic engineering strategies aim to improve RL yields, yet rely on often inaccurate qualitative assays.
• Previous studies frequently used unreliable phenotypic methods for screening RL-producing mutants.

Purpose of the Study:

• To systematically evaluate the reliability of traditional phenotypic assays for assessing RL production in *P. aeruginosa* mutants.
• To compare traditional assays with quantitative liquid chromatography-mass spectrometry (LC/MS) for accurate strain screening.
• To identify reliable genetic targets for metabolic engineering to enhance RL production.

Main Methods:

• Generated 29 *P. aeruginosa* PA14 mutants with targeted gene knockouts.
• Assessed RL production using traditional methods: Siegmund-Wagner blue plates and swarming motility assays.
• Quantified RL titers and analyzed mutant phenotypes using liquid chromatography-mass spectrometry (LC/MS).

Main Results:

• Traditional assays exhibited a high misprediction rate (~35-38%) due to confounding factors like motility and growth rate.
• LC/MS revealed that *rpoN* and *pvdQ* knockouts significantly increased total RL titers.
• *crc*, *dksA*, and *dspI* knockouts led to decreased RL production, with increased titers linked to biomass, not per-cell production.

Conclusions:

• Qualitative phenotypic assays are insufficient for reliable screening of RL production mutants.
• Quantitative LC/MS is essential for accurate assessment and identification of genetic targets in metabolic engineering.
• This study clarifies the impact of specific genes (*rpoN*, *pvdQ*, *crc*, *dksA*, *dspI*) on RL production and provides a validated framework for future research.