Cytochrome P450 monooxygenases : a study of the synthesis of industrial relevant aliphatic ω-hydroxy products (2013)

The ω-regioselective hydroxylation of aliphatic compounds like alkanes or fatty acids with different chain lengths is a longstanding problem in chemistry. Chemo-catalyzed reactions suffer from subterminal hydroxylation and overoxidation which can only be solved by harsh reaction conditions. Biocatalysis offers interesting tools for these complex questions. In the present thesis several biocatalysts were investigated with the focus on cytochrome P450 monooxygenases (CYP or P450). These widely spread enzymes accept a variety of substrates and perform C–H hydroxylations with high regio- and stereospecificity. Particularly members of the bacterial CYP153A subfamily show interesting abilities in substrate specificity (alkanes) and terminal hydroxylation selectivity. Several CYP153A candidates were characterized in vitro towards alkanes, primary alcohols, mono (un)- and saturated fatty acids to determine the substrate specificity of the biocatalysts. Suitable enzymes have been selected to oxidize n butane as well as dodecanoic acid. Further enzyme improvements were achieved by employing optimization techniques like rational protein design, directed evolution experiments and establishment of self-sufficient fusions. CYP153AP. sp. from Polaromonas sp. and CYP153AM. aq. from Marinobacter aquaeolei were selected for a detailed in vitro analysis. CYP153AP. sp. was identified as predominant alkane ω hydroxylase which hydroxylates C5-C12 alkanes combined with ω-regioselectivity of up to 91 %. In contrast CYP153AM. aq. showed predominantly fatty acid ω-hydroxylase activity with a broad substrate spectrum (C8:0-C20:0 and 9(Z)/9(E) C14:1-C18:1). For the purpose of applying CYP153AM. aq. and CYP153AP. sp. in a bacterial whole cell system, a rational design approach was used to identify positions which are important for substrate selectivity and activity. CYP153AM. aq.(G307A) and CYP153AP. sp.(G254A) showed up to 10-fold higher activity against smaller substrates with further increased ω regioselectivity (more than 95 %). To optimize the coupling efficiency as well as protein expression, self-sufficient fusion constructs were established. The heme domain of the monooxygenase was fused to the reductase domain (CPR) of P450 BM3 from Bacillus megaterium. The measured coupling efficiency (more than 70 %) with the test substrate dodecanoic acid (C12-FA) was higher for CYP153AM. aq.(G307A)-CPRBM3 in comparison to the use of single redox proteins (ca. 20 %). Different accessible biocatalysts for the hydroxylation of gaseous n-butane to liquid 1 butanol were compared. These experiments resulted in final concentrations of 0.74-0.88 g per liter butanol after 24 h (more than 90 % ω regioselectivity) for CYP153AP. sp. and CYP153A6-BMO1 (CYP153A6-butane monooxygenase 1) applied in a E. coli resting cell experiment with their natural redox partners. After the improvements of enzymes via fusion establishment and optimization of the reaction conditions by a high-pressure tank, product concentrations of up to 4 g per liter after 24 hours were achieved with a cell mass of 18.7 gcdw. For further enzyme optimization, directed evolution with a SeSaM-library was applied. This strategy was combined with a viability in vivo screening based on Pseudomonas putida KT2440. This strain offers the opportunity to select mutants with the ability to hydroxylate alkanes at the terminal position because they are able to grow on primary alcohols as carbon source. The screening with butane enabled the identification of two new hotspots (Ala184 and Thr300) in the used enzyme CYP153AP. sp.. With a solid method in hand for the oxidation of gaseous n butane in whole cells we turned our attention towards dodecanoic acid as substrate. After initial shaking flask experiments, the performance of a non-engineered non solvent adapted E. coli resting cell system was increased in a small scale bioreactor (1 L). After first experiments with C12 FA yielding 1.2 g per liter ω-hydroxylated product in 30 h using a cell mass of 15.1 gcdw, dodecanoic acid methyl ester was used as substrate for bioconversion experiments. An additional outer membrane transporter in form of AlkL from P. putida was coexpressed to overcome the transfer limitation of the substrates. Parallel feeding with a glucose/glycerol mix and fine regulation of fermentation parameters (e.g. pO2) lead to maximum production concentrations of 4 g per liter in 28 h using 18.2 gcdw cell mass. The present studies successfully demonstrated new possibilities to optimize different CYP153A enzymes via rational design, directed evolution and fusion experiments. With CYP153A CPRBM3 a new promising way for the production of 1-butanol based on butane was shown. The flexibility of the biocatalyst was proven with the highest ever reported end concentrations for the synthesis of ω-dodecanoic acids with bacterial production hosts.