Enhanced rhamnolipid production in Pseudomonas aeruginosa 8D by systematic genetic engineering

Rhamnolipids are environmentally friendly biosurfactants primarily produced by Pseudomonas aeruginosa. The complex and tightly regulated network imposes limitations on rhamnolipid biosynthesis. Therefore, rational genetic engineering of P. aeruginosa to optimize metabolic flux holds significant promise for enhancing rhamnolipid production. A strain designated P. aeruginosa 8D was isolated and found to produce higher levels of rhamnolipids compared to the model strain PAO1. Promoter activity analysis revealed that the expression levels of regulatory genes rhlR and rhlI, biosynthetic genes rhlAB and rhlC, and the precursor synthesis gene algC were all significantly higher in 8D. To further enhance rhamnolipid production, we performed stepwise genetic modifications in 8D. First, deletion of two restriction modification (RM) systems, RM1 and RM2, led to a 4–6-fold increase in transformation efficiency. Next, the regulatory gene exsA of the type III secretion system (T3SS) was knocked out for reducing the strain’s pathogenicity. Subsequently, we disrupted the exopolysaccharide biosynthesis gene clusters pslA-O, algD-F, and pelA-G, which compete for metabolic precursors required for rhamnolipid synthesis. The engineered strain ΔRM1-RM2-exsA-pslA-O-algD-F-pelA-G produced 14.53 g/L of rhamnolipids, compared to 8.567 g/L for the wild-type 8D strain. To further boost production, we overexpressed rhlAB using two complementary approaches. The IPTG-inducible plasmid pMMB67EH-rhlAB increased rhamnolipid production 2.64-fold compared to 8D (pMMB67EH), reaching a final titer of 22.134 g/L, whereas chromosomal integration of CTX-rhlAB enabled stable expression and achieved 16.98 g/L of rhamnolipids, a 2.02-fold increase over 8D. This work presents a rational engineering strategy integrating reduced virulence and metabolic rewiring, offering a safe and efficient P. aeruginosa chassis for economical rhamnolipid production.