SEED FUND Projekt TPOT

A proof of concept for an E.coli hydroxylation platform with efficient terpene/terpenoid translocation through the outer membrane

Results

Microbial biotransformations represent a promising way to satisfy the growing demand for the scalable production of plant metabolite-based pharmaceuticals. Chemoselective hydroxylations are chemical dream reactions which are catalyzed by oxygenases and found in many secondary metabolic pathways of plants. Monooxygenases are for instance attractive catalysts to convert terpenes and terpenoids to highly valuable compounds/intermediates (e.g. taxol or verbenone). Industrial applications of monooxygenases (e.g. cytosolic expressed P450 BM3) have so far been restricted to more cost-effective whole-cell systems that enable efficient NAD(P)H recycling and have in general a higher process stability with lower downstream processing costs than cell-free systems. However, a general limitation for several compound classes is the limited substrate uptake and low efflux of products out of bacterial production systems such as E. coli. The latter limits often productivities and overall reaction rates. Therefore the main aim of this Seed fund project was to investigate the effect of a membrane barrel protein and its variant with deleted cork domain on the productivity of E. coli cells expressing target P450 monooxygenases during the oxidation of hydrophobic substrates. We demonstrated an innovative and general solution to improve substrate uptake and product efflux through an engineered passive diffusion channel, which is expressed in the outer E. coli membrane and harbors inside a ~1.3 nm pore which enables translocation of even single stranded DNA. Aromatic and terpenoid substrates of soluble P450 variants with low solubility in water (only several mg per liter) were chosen to prove the concept of this P450 and membrane barrel protein based whole cell catalyst. The groups of Prof. Schwaneberg and Prof. Urlacher characterized the coexpression of this membrane barrel protein acting as passive diffusion channel with different bacterial monooxygenases in relation to biochemical properties and protein localization. A significant improvement of the conversion of different aromatic and terpenoid substrates by the outer membrane barrel protein expressing cells could be shown. Microscopic and quantitative analyses proved the localisation of the barrel protein in the outer membrane where it acts as a pore to improve substrate and product translocation. In order to extend the system of a P450 monooxygenase and an outer membrane channel based whole cell catalyst to a broader substrate range and a sustainable production of e. g. plant secondary metabolites, the group of Prof. Usadel searched sequence and literature databases for appropriate P450 monooxygenases from plant sources. Those sequences were cloned into the whole cell catalyst construct and expressed in E. coli. The characterization of the plant monooxygenase based whole cell catalysts is ongoing in the groups of Prof. Schwaneberg and Prof. Urlacher.

Participating Core Groups

Coordinators
Prof. Dr. Ulrich Schwaneberg, Dr. Anna Joelle Ruff, Institute of Biotechnology, RWTH Aachen University
Worringerweg 3
522074 Aachen
Phone: +49 241 80 24176
Fax: +49 241 80 22387
email: u.schwaneberg@biotec.rwth-aachen.deaj.ruff@biotec.rwth-aachen.de

Partners
Prof. Vlada Urlacher, Institute of Biochemistry, Lehrstuhl II, Heinrich-Heine-Universität Düsseldorf
Prof. Dr. Björn Usadel, Lehrstuhl für Botanik und Institut für Biologie I, RTWH Aachen University

Funding
TPOT is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Innovation, Science and Research of the German State of North Rhine-Westphalia.