Killing Two Birds With One Stone - Strain Engineering Facilitates the Development of a Unique Rhamnolipid Production Process

Abstract

High-titer biosurfactant production in aerated fermenters using hydrophilic substrates is often hampered by excessive foaming. Ethanol has been shown to efficiently destabilize foam of rhamnolipids, a popular group of biosurfactants. To exploit this feature, we used ethanol as carbon source and defoamer, without introducing novel challenges for rhamnolipid purification. In detail, we engineered the non-pathogenic Pseudomonas putida KT2440 for heterologous rhamnolipid production from ethanol. To obtain a strain with high growth rate on ethanol as sole carbon source at elevated ethanol concentrations, adaptive laboratory evolution (ALE) was performed. Genome re-sequencing allowed to allocate the phenotypic changes to emerged mutations. Several genes were affected and differentially expressed including alcohol and aldehyde dehydrogenases, potentially contributing to the increased growth rate on ethanol of 0.51 h^-1 after ALE. Further, mutations in genes were found, which possibly led to increased ethanol tolerance. The engineered rhamnolipid producer was used in a fed-batch fermentation with automated ethanol addition over 23 h, which resulted in a 3-(3-hydroxyalkanoyloxy)alkanoates and mono-rhamnolipids concentration of about 5 g L^-1. The ethanol concomitantly served as carbon source and defoamer with the advantage of increased rhamnolipid and biomass production. In summary, we present a unique combination of strain and process engineering that facilitated the development of a stable fed-batch fermentation for rhamnolipid production, circumventing mechanical or chemical foam disruption.

Keywords: Pseudomonas; adaptive laboratory evolution; biosurfactants; ethanol; fermentation; metabolic engineering; rhamnolipid; synthetic biology.