Konstruktion neuer Produktionsstämme für die heterologe Rhamnolipidsynthese in dem nicht-pathogenen Wirt Pseudomonas putida KT2440

Abstract

Rhamnolipids are the best characterized class of biosurfactants and feature a great potential for a wide variety of industrial applications. The major disadvantage is the origin of their production in the opportunistic human-pathogen Pseudomonas aeruginosa and the complex regulation of the biosynthesis by the Quorum sensing system.

In this work the synthesis of mono- and di-rhamnolipids was established by heterologous expression of relevant rhamnolipid synthase genes from P. aeruginosa PAO1 in the non-pathogenic Pseudomonas putida KT2440. Also species from the genus Burkholderia are able to produce rhamnolipids, which are characterized by their long chain fatty acids. For the first time the heterologous synthesis of these rhamnolipid species could be established in recombinant P. putida by expression of corresponding genes from B. glumae PG1. It could be verified, that the acyltransferase RhlA define a specifity for the length of fatty acids linked to the HAA used for the synthesis of mono-rhamnolipids. It could be shown that RhlB is the rhamnosyltransferase I responsible for the synthesis of mono-rhamnolipids by using only HAA, which were synthezed by RhlA. Therefore, RhlB must not necessarily form an enzyme complex together with RhlA for its activity. The experiments have revealed, that extracellular HAA as well as mono-rhamnolipids could be taken up by the cells and transported to the cytoplasm. The established rhamnolipid synthesis was optimized by the construction of a plasmid-based library of synthetic promoters with various expression strengths. The rhamnolipid synthesis was further increased by metabolic engineering. The relevant genes for the synthesis of educts for rhamnolipid production were coexpressed and deletion mutants were constructed to abolish metabolic pathways in competition with the rhamnolipid synthesis. The new created P. putida strain reached a higher rhamnolipid concentration than the P. aeruginosa PAO1 wild type under similar conditions.