Chassis Optimization for Recombinant Rhamnolipid Synthesis

Previously recombinant rhamnolipid production with P. putida KT2440 was established [1], delivering an alternative for the synthesis of this secondary metabolite with the pathogenic P. aeruginosa. While production rate and yield already compete with the conventional process based on P. aeruginosa, titers are far from being industrially interesting. Metabolic engineering of P. putida KT2440 to become a rhamnolipid synthesis chassis is presented here.

First, futile pathways were deleted. A strain deficient in the synthesis of the storage polymer polyhydroxyalkanoate (ΔphaC), and a knockout strain (ΔfadB) featuring a decelerated β-oxidation were  tested. The novel chassis shows enhanced rhamnolipid production.

Second, precursor pathways were enhanced. For rhamnose synthesis phosphoglucomutase and the whole rml operon encoding the dTDP-rhamnose pathway were overexpressed. To facilitate the increased synthesis of the lipid precursor, one subunit of the pyruvate dehydrogenase complex was overexpressed, leading to triplication in surfactant titers compared to the control strain. Third, a mutant devoid of the energy intensive flagellar machinery [2] was tested, with the aim to reduce metabolic energy for maintenance.

Last, the export of rhamnolipids was improved using a putative transporter from P. aeruginosa.

The additive effects on rhamnolipid synthesis of the engineering targets identified here are investigated.

References

1.         Wittgens A, Tiso T, Arndt TT, Wenk P, Hemmerich J, Müller C, Wichmann R, Küpper B, Zwick M, Wilhelm S, et al: Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440. Microb Cell Fact 2011, 10.

2.         Martinez-Garcia E, Nikel PI, Aparicio T, de Lorenzo V: Pseudomonas 2.0: Genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb Cell Fact 2014, 13.