An enzymatic route to α-tocopherol synthons: Aromatic hydroxylation of pseudocumene and mesitylene with P450 BM3Bio VI
Dennig*, A., Weingartner*, A. M., Kardashliev, T., Müller, C. A., Tassano, E., Schürmann, M., Ruff, A. J., Schwaneberg, U. (2017). Chemistry - European Journal , first published online: October 9 2017, DOI: 10.1002/chem.201703647
P450 BM3 variants for the production of key phenolic building blocks for α-tocopherol synthesis
Alternative enzymatic routes for vitamin E synthesis are important to match increasing demands. Here, we show the first direct aromatic hydroxylation of pseudocumene and mesitylene in water, with O2 and under mild reaction conditions to access five key phenolic α -tocopherol synthons including a direct route to trimethylhydroquinone, TMHQ. The P450 BM3 wild-type catalyzed a 94% selective aromatic hydroxylation of mesitylene, whereas pseudocumene was hydroxylated to a large extent ranging from 46-64% on benzylic positions. Site-saturation mutagenesis generated a new P450 BM3 mutant, named “variant M3”, with a 3 to 8fold increased coupling efficiency and 75 to 230-fold increased activity for pseudocumene and mesitylene conversion. Additional π - π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also a 61 to 75% selectivity toward aromatic hydroxylation of pseudocumene. Detailed product pattern analysis, substrate docking and mechanistic considerations support the hypothesis that pseudocumene binds in an inverted orientation in the active site of P450 BM3 WT as compared to P450 BM3 variant M3 to allow this change in chemo-selectivity.
Under continuous NADPH-recycling the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor TMHQ at 35% selectivity at concentrations up to 0.18 mg ml-1 directly from pseudocumene, which is a significant step toward more sustainable synthesis of vitamin E. The reaction can be performed in one-pot without the need for intermediate purification or additional catalysts. In case of mesitylene over-oxidation leads to dearomatization and formation of a valuable p-quinol synthon that can directly serve as educt for synthesis of TMHQ. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2.
Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-HydroxyalkanoatesBio VI
Yunus Ensari, Gaurao V. Dhoke, Mehdi D. Davari, Marco Bocola, Anna Joëlle Ruff, Ulrich Schwaneberg, Chemistry – A European Journal 2017, 23, 51, Issue, 12636-12645.
Enlargment of substrate binding pocket of cpADH5 led the conversion of medium chain methyl 3-hydroxyalkanoates and inverted enantiopreference for short chain methyl 3-hydroxyalkanoates.
Selective oxidation of primary or secondary alcohols to carbonyl compounds, i.e. aldehydes and ketones, is a key reaction in organic synthesis and industry. Oxidation of alcohol to ketones can be performed efficiently by a great number of chemical oxidation methods. Selectivity, undesired by-product formation, substrate scope and conversion rates are still major challenges in oxidation of alcohols. Alcohol dehydrogenases are promising alternatives to metal catalysts and perform reversible oxidations of primary or secondary alcohols to their corresponding aldehydes or ketones.
Ketone-functionalized fatty acid derivatives are versatile compounds in organic chemistry, are used as intermediates and building blocks in the production of pharmaceutically active compounds, detergents, and so on.
This study reports the first reengineered Candida parapsilosis alcohol dehydrogenase - cpADH5 - for the efficient oxidation of methyl 3-hydroxyhexanoate and methyl 3-hydroxyoctanoate, which were not converted by the cpADH5 WT, to their corresponding ketones. In this study, we mutated and redesigned the substrate binding pocket of cpADH5 in order to extend its substrate scope toward medium-chain 3-hydroxy fatty acid methyl esters – FAME- which contain 6--12 carbon atoms in their alkyl chains. Furthermore, we describe the inverted enantiopreference for the oxidation of methyl 3-hydroxybutyrate which was observed for our engineered cpADH5 variant.
Research highlight: Micro delivery service for fertilizersWiley-VCH
Plants can absorb nutrients through their leaves as well as their roots. However, foliar fertilization over an extended period is difficult. In the journal Angewandte Chemie, German researchers have now introduced an efficient delivery system for micronutrients based on biohybrid microgels. Special peptides anchor the "microcontainers" onto the leaf surface while binding sites inside ensure gradual release of the "cargo".
Foliar fertilization is already commonly used in areas such as viniculture, when the leaves on the vines turn yellow due to a mineral deficiency. Yet, despite the use of detergents, adhesives, and humectants, controlled delivery of nutrients through foliar fertilization over several weeks is nearly impossible to achieve. Up to 80% of the nutrients are washed away, winding up in the soil and being converted into forms that the plant cannot use. In addition, they can be washed into bodies of water and cause environmental problems. An additional problem is that strong sunlight evaporates the water out of the applied fertilizer solution. This results in a high salt concentration that draws water out of the leaf and causes burn damage.
A team from DWI-Leibniz Institute for Interactive Materials in Aachen, RWTH Aachen University, and the University of Bonn has now developed a foliar fertilization system based on biocompatible microgels that adhere selectively to leaves for a long period and slowly deliver nutrients in a controlled fashion. Microgels are tiny particles of cross-linked macromolecules that can bind water and other molecules, such as fertilizers very efficiently.
Led by Ulrich Schwaneberg, Andrij Pich and Felix Jakob, the researchers equipped the interiors of gel particles with binding sites modeled on the iron-binding proteins of bacteria. These ensure that the iron ions are released slowly. The microgels are loaded with an iron-containing solution at a pH of 3. When the pH rises to 7, the microgels shrink, releasing water and binding the iron.
The surface of the gel particles is equipped with anchor peptides from lactic acid bacteria. These bind securely to leaf surfaces to hinder rinsing away of the microgels. The water in the gel provides an aqueous microenvironment that allows the iron to diffuse into the leaves. Yellow leaves of iron-deficient cucumber plants rapidly turned green in spots where the new foliar fertilizer was applied.
By incorporating different binding sites, the microgel "containers" can be loaded with a multitude of other metal ions or agents. A controlled delivery of agents as required would minimize the applied quantities as well as the release of fertilizers and pesticides into the environment. Low production costs, high levels of loading, easy application, and adjustable adhesive properties should make broad industrial applications possible. The goal is to make self-regulating delivery systems for sustainable agriculture.
Richard A. Meurer et al, Biofunctional Microgel-Based Fertilizers for Controlled Foliar Delivery of Nutrients to Plants, Angewandte Chemie International Edition (2017). DOI: 10.1002/anie.201701620
Generation of building blocks for synthesis of filter membranesBio VI
The iron transporter FhuA - Ferric hydroxamate uptake protein component A is a channel protein produced by the bacterium Escherichia coli which was tailored for the generation of synthetic membranes using FhuA proteins and polymers. The FhuA protein forms a permeable channel with a uniform pore size from 2.5 to 3.0 nm and a barrel-like structure. Lysine residues were specifically located in a rim on the outer surface of the channel above the transmembrane region for the attachment of polymer chains. This design enabled the grafting of PNIPAAm polymer chains from the outer FhuA channel surface. In the future, the synthesized building blocks of the FhuA channel proteins and polymer chains will be used for generation of hybrid membranes for nanofiltration processes. These generated membranes can be applied in the downstream processing as molecular sieves for the separation of different components which is essential for synthesizing sweeteners, pesticides and pharmaceuticals.
In vitro flow cytometry-based screening platform for cellulase engineeringBio VI
Screening technologies are of pivotal importance for tailoring biocatalysts in directed evolution, as millions of mutant enzyme variants could be generated in every trial. Hence, ultrahigh throughput screening techniques have been developed in order to still complete such trials in a reasonable amount of time. These techniques are well capable of analyzing up to 107 events per hour and thus can analyze the complete coverage of a generated protein sequence with high efficiency.
This technology becomes even more powerful if it is coupled with a cell-free enzyme expression technique. This expression method enables the experimentator to reduce diversity loss when transforming mutant libraries into expression hosts, to design enzymes of animal or human origin or even perform directed evolution of toxic enzymes.
The first ever combination of such a cell-free compartmentalization platform with a flow cytometry-based screening has been achieved in the InVitroFlow technology and successfully applied to directed evolution of cellulose enzymes.
Screening through the PLICable promoter toolbox enhances protein production in Escherichia coliBio VI
Escherichia coli is a common host for recombinant protein production in which product titers are highly dependent on the employed expression system. Thereby, promoters are a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed. It enables the identification of the most suitable promoter out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) for high level protein production in a single cloning step and after a screening experiment.