SEED FUND 2.0 perspective project DesignR

Tailoring Biosurfactants – Production of Designer Rhamnolipids

Results

Surfactants are molecules that lower liquids' surface tension, leading to increased miscibility of different liquid phases (e.g., water and oil). This unique feature is exploited in various applications, including household detergents, pharmaceuticals or cosmetics. Biosurfactants, including glycolipids like rhamnolipids (RL), can be produced via fermentation using renewable resources, avoiding the use of crude oil.

RLs, as an industrially established example of glycolipids, have been investigated within the FocusLab Bio². They are composed of up to two rhamnose moieties (hydrophilic part) and two β-hydroxyalkanoic acids (HAAs) with varying chain lengths between C₈-C₂₄ (hydrophobic region). Different congeners of RLs are expected to have different features; however, the relationship between the structural diversity of RLs and their physicochemical properties remains unclear.

The DesignR project provides a modular production platform for different rhamnolipid (RL) congeners from varying carbon sources. Combining RL genes rhlA, rhlB, and rhlC of different origin in different copy numbers, integration mode, and expression strength resulted in new P. putida production strains, which produce tailored RL mixtures differing in the number of rhamnose units and the length of fatty acid chains. The respective expression modules, complemented with eYFP to enable online expression monitoring, were introduced into engineered P. putida strains to allow RL production from alternative carbon sources present in renewable resources (xylose, ethanol, or 1,4‑butanediol). The RL production with the different carbon sources was comparable to the production with glucose. An innovative approach applying Co-fed of CO₂ helped to increase the titers by another 10%. The eYFP monitoring appeared here as a valuable tool for high-throughputRL production screening.

Since a complete conversion toward particular RL species is so far not achievable with the biological systems, normal-phase chromatography was established for efficient compound purification. Adsorption isotherms showed a stronger interaction for dRL with the Silica matrix than mRL and HAA enabling successful separation of HAA, mRL and dRL from fermentation broth extracts in a semi-preparative chromatography scale. Finally, novel glycosyltransferase genes from unknown producer strains were identified to further expand the DesignR glycolipid platform in the future.

The production and screening platform based on P. putida developed in this project constitutes a further step towards sustainable production of tailored RL compositions.

SEED FUND 2.0 Coordinator

Carl Brehl
Chair of Biochemical Engineering (AVT.BioVT)
RWTH Aachen University

Partners

Prof. Jaeger, Dr. Stephan Thies and Sonja Kubicki, Institute for molecular enzyme technology (IMET), Heinrich Heine University Düsseldorf
Prof. Andreas Jupke and Andreas Biselli, Fluid Process Engineering (AVT.FVT), RWTH Aachen University
Prof. Jochen Büchs and Dr. Nina Ihling, Biochemical Engineering (AVT.BioVT), RWTH Aachen University

Funding period

01.08.2020 - 31.10.2021

Funding

DesignR is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia.

Publications

Gauthier, C, Lavoie, S, Kubicki, S, Piochon, M, Cloutier, M, Dagenais-Roy, M, Groleau, M-C, Pichette, A, Thies, S and Déziel, E (2024). Structural characterization of a nonionic rhamnolipid from Burkholderia lata. Carbohydrate Research 535: 108991.  

Filbig, M, Kubicki, S, Bator, I, Hausmann, R, Blank, LM, Henkel, M, Thies, S and Tiso, T (2023). Chapter 8 - Metabolic and process engineering on the edge—Rhamnolipids are a true challenge: A review. Biosurfactants. Soberón-Chávez, G., Academic Press: 157-181.