Universität Stuttgart
Permanent URI for this communityhttps://elib.uni-stuttgart.de/handle/11682/1
Browse
1 results
Search Results
Item Open Access Novel route to vanillin - an enzyme-catalyzed multi-step cascade synthesis(2016) Klaus, Tobias; Hauer, Bernhard (Prof. Dr.)The selective hydroxylation of aromatic compounds is one of the most challenging chemical reactions. As an alternative to traditional chemical catalysis, biocatalysis emerged during the past decades. Hence, in the present work, a number of biocatalysts was investigated with regard to the realization of a novel synthesis route to the valuable aromatic compound vanillin, starting from the simple low-cost aromatic substrate 3-methylanisole via the intermediate products 3-methoxybenzyl alcohol or 4-methylguaiacol and via vanillyl alcohol, as an example of consecutive enzyme-catalyzed oxidation reactions accomplished in a multi-enzymatic three-step cascade reaction. For this reason a preselected set of enzymes, namely the m-hydroxybenzoate hydroxylase MobA from Comamonas testosteroni GZ39 and the cytochrome P450 monooxygenases CYP116B3 from Rhodococcus ruber DSM 44319 and CYP102A1 from Bacillus megaterium ATCC 14581, was investigated towards the selective hydroxylation of the substrate 3-methylanisole. Beside the wild type enzymes, a variant of MobA, which was created by rational protein design, and an existing focused minimal mutant library of CYP102A1 were applied in initial biotransformation reactions, combined with an efficient cofactor recycling system. Though the wild type enzymes of CYP116B3 and CYP102A1 displayed only a basic level of activity towards 3-methylanisole, highly increased activity was detected for many of the CYP102A1 variants with a maximum of 59% total conversion for the double mutant F87V/A328L. With 3-methoxybenzyl alcohol and 4-methylguaiacol both intermediate compounds of the intended cascade synthesis were generated, though 4-methoxy-2-methylphenol was the main product in most of the reactions. However, none of the so far investigated variants accepted any of the intermediate compounds as substrate. As CYP116B3 was a good candidate for further protein engineering approaches, as a basic level of activity towards the substrate of interest was already present in the wild type enzyme, a focused mutant library of 20 single mutant variants of CYP116B3 was created based on literature, sequence and structure information in order to improve the enzymes activity and selectivity towards conversion of the substrate 3-methylanisole and in order to find variants for the conversion of 3-methoxybenzyl alcohol and/or 4-methylguaiacol. Therefore a homology model of the monooxygenase domain of CYP116B3 was generated. Though, compared to the wild type, variants with up to almost six time increased activity towards the model substrate 7-ethoxycoumarin were found, total activity towards 3-methylanisole was still much lower compared to the best CYP102A1 variants. In addition, none of the variants displayed appropriate conversion of the intermediate compounds 3-methoxybenzyl alcohol and 4-methylguaiacol. Moreover, additional mutation in the literature known amino acid position 437 of CYP102A1 variant F87V/A328L revealed no benefit towards conversion of any of the substrates, too. Molecular dynamics simulations of a CYP102A1 variant with 4-methylguaiacol as substrate revealed the bottleneck in the conversion of this compound. 4-Methylguaiacol was shown to be stabilized at the entrance of the substrate access channel by the polar amino acid residues R47 and Y51. Replacement of these residues by the hydrophobic residues leucine and phenylalanine, respectively, resulted in successful conversion of 4-methylguaiacol to vanillyl alcohol, the precursor of vanillin in the intended cascade synthesis. Though, as the yield of vanillyl alcohol synthesized from 4-methylguaiacol with CYP102A1 variants was rather low, a vanillyl alcohol oxidase from Penicillium simplicissimum and rationally designed variants thereof, described in literature, were investigated. As a result, not only vanillyl alcohol but also 4-methylguaiacol was converted in high yield to vanillin. Finally, a combination of the best 4-methylguaiacol producing variant, CYP102A1 variant A328L, with the best 4-methylguaiacol converting variant, VAO variant F454Y, in one reaction system both in vitro and in vivo yielded vanillin from 3-methylanisole with a maximal product formation of 2.0% and 1.1% vanillin, respectively. We demonstrated as a proof-of-principle the establishment of the proposed multi-enzymatic three-step cascade reaction pathway. Though further optimizations concerning increase of enzyme activity and improvement of enzyme selectivity are required, the above mentioned exemplary synthesis of vanillin illustrates the capability of biocatalysis.