Deep genome editing of Pseudomonas putida for rhamnolipid production using non-conventional substrates

Bator, Isabel; Blank, Lars M. (Thesis advisor); Jaeger, Karl-Erich (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2021)
Book, Dissertation / PhD Thesis

In: Applied microbiology 24
Page(s)/Article-Nr.: 1 Online-Ressource (XV, 184 Seiten) : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2020

Abstract

Biosurfactants, such as rhamnolipids, are valuable products with many potential applications. Their amphiphilic structure allows their use in different fields. The overall aim of this thesis was to produce rhamnolipids from renewable substrates using the nonpathogenic Pseudomonas putida KT2440, which would contribute to a sustainable bioeconomy. Various substrates were implemented, which allowed new insights into the respective metabolism. In order to reduce the costs for rhamnolipid production, lignocellulose-derived substrates, such as xylose and galacturonic acid, can be used. While P. putida can natively utilize galacturonic acid, the use of xylose does not lead to growth. Three bacterial xylose utilization pathways were introduced and the engineered strains were characterized regarding their growth and production. While the implementation of the oxidative xylose pathways resulted in higher biomass production and rhamnolipid titers, the product yield was higher for the Isomerase pathway caused by the different stoichiometries of the pathways. Rhamnolipid production from galacturonic acid was immediately efficient, however the production rate was improved by laboratory evolution. Ethanol is another sustainable carbon source as it can be produced out of lignocellulosic biomass by microbes. Laboratory evolution was applied to obtain a strain with a high growth rate on ethanol at elevated concentrations. Genome re-sequencing and gene expression studies revealed a rerouting of the metabolism for carbon-efficient ethanol utilization. The resulting biosurfactant producer was used in a fed-batch process, where the water-soluble ethanol served as carbon source and defoamer. This unique setup enabled a biosurfactant production of over 5 g/L in one day with a great space time yield of 0.23 g/L/h. In addition, P. putida KT2440 was modified to generate a genome-reduced chassis by targeted deletion of dispensable elements or processes. Several deletions led to a 66% improved rhamnolipid production. As precursor supply seemed to be a bottleneck, the overexpression of the phosphoglucomutase and rhamnose-operon was performed. The combination of a reduced genome and the overexpression further increased the rhamnolipid production up to 94%. In conclusion, this thesis clearly illustrates that rhamnolipid production from renewable substrates is feasible. Further, the application of P. putida KT2440 as microbial cell factory inindustrial processes is demonstrated. A combination of the improved chassis and the use of any renewable lignocellulose-derived substrate will truly contribute to establish a competitive production process.

Institutions

• Department of Biology [160000]
• Chair of Applied Microbiology [161710]