Broadening the Biocatalytic Toolbox-Screening and Expression of New Unspecific Peroxygenases.

Unspecific peroxygenases (UPOs) catalyze the selective transfer of single oxygen atoms from peroxides to a broad range of substrates such as un-activated hydrocarbons. Since specific oxyfunctionalizations are among the most-desired reactions in synthetic chemistry, UPOs are of high industrial interest. To broaden the number of available enzymes, computational and experimental methods were combined in this study. After a comparative alignment and homology modelling, the enzymes were expressed directly in P. pastoris. Out of ten initially selected sequences, three enzymes (one from Aspergillus niger and two from Candolleomyces aberdarensis) were actively expressed. Cultivation of respective expression clones in a bioreactor led to production titers of up to 300 mg L-1. Enzymes were purified to near homogeneity and characterized regarding their specific activities and pH-optima for typical UPO substrates. This work demonstrated that directed evolution is not necessarily required to produce UPOs in P. pastoris at respective titers. The heterologous producibility of these three UPOs will expand the toolbox of available enzymes and help to advance their synthetic application.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α-promoter. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

Modeling and simulation-based design of electroenzymatic batch processes catalyzed by unspecific peroxygenase from A. aegerita.

Unspecific peroxygenases have attracted interest due to their ability to catalyze the oxygenation of various types of C-H bonds using only hydrogen peroxide as a cosubstrate. Due to the instability of these enzymes at even low hydrogen peroxide concentrations, careful fed-batch addition of the cosubstrate or ideally in situ production is required. While various approaches for hydrogen peroxide addition have been qualitatively assessed, only limited kinetic data concerning enzyme inactivation and peroxide accumulation has been reported so far. To obtain quantitative insights into the kinetics of such a process, a detailed data set for a peroxygenase-catalyzed benzylic hydroxylation coupled with electrochemical hydrogen peroxide production is presented. Based on this data set, we set out to model such an electroenzymatic process. For this, initial velocity data for the benzylic hydroxylation is collected and an extended Ping-Pong-Bi-Bi type rate equation is established, which sufficiently describes the enzyme kinetic. Moreover, we propose an empirical inactivation term based on the collected data set. Finally, we show that the full model does not only describe the process with sufficient accuracy, but can also be used predictively to control hydrogen peroxide feeding rates To limit the concentration of this critical cosubstrate in the system.

H2 O2 Production at Low Overpotentials for Electroenzymatic Halogenation Reactions.

Various enzymes utilize hydrogen peroxide as an oxidant. Such "peroxizymes" are potentially very attractive catalysts for a broad range of oxidation reactions. Most peroxizymes, however, are inactivated by an excess of H2 O2 . The electrochemical reduction of oxygen can be used as an in situ generation method for hydrogen peroxide to drive the peroxizymes at high operational stabilities. Using conventional electrode materials, however, also necessitates significant overpotentials, thereby reducing the energy efficiency of these systems. This study concerns a method to coat a gas-diffusion electrode with oxidized carbon nanotubes (oCNTs), thereby greatly reducing the overpotential needed to perform an electroenzymatic halogenation reaction. In comparison to the unmodified electrode, with the oCNTs-modified electrode the overpotential can be reduced by approximately 100 mV at comparable product formation rates.

Co-production of acetone and ethanol with molar ratio control enables production of improved gasoline or jet fuel blends.

The fermentation of simple sugars to ethanol has been the most successful biofuel process to displace fossil fuel consumption worldwide thus far. However, the physical properties of ethanol and automotive components limit its application in most cases to 10-15 vol% blends with conventional gasoline. Fermentative co-production of ethanol and acetone coupled with a catalytic alkylation reaction could enable the production of gasoline blendstocks enriched in higher-chain oxygenates. Here we demonstrate a synthetic pathway for the production of acetone through the mevalonate precursor hydroxymethylglutaryl-CoA. Expression of this pathway in various strains of Escherichia coli resulted in the co-production of acetone and ethanol. Metabolic engineering and control of the environmental conditions for microbial growth resulted in controllable acetone and ethanol production with ethanol:acetone molar ratios ranging from 0.7:1 to 10.0:1. Specifically, use of gluconic acid as a substrate increased production of acetone and balanced the redox state of the system, predictively reducing the molar ethanol:acetone ratio. Increases in ethanol production and the molar ethanol:acetone ratio were achieved by co-expression of the aldehyde/alcohol dehydrogenase (AdhE) from E. coli MG1655 and by co-expression of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from Z. mobilis. Controlling the fermentation aeration rate and pH in a bioreactor raised the acetone titer to 5.1 g L(-1) , similar to that obtained with wild-type Clostridium acetobutylicum. Optimizing the metabolic pathway, the selection of host strain, and the physiological conditions employed for host growth together improved acetone titers over 35-fold (0.14-5.1 g/L). Finally, chemical catalysis was used to upgrade the co-produced ethanol and acetone at both low and high molar ratios to higher-chain oxygenates for gasoline and jet fuel applications. Biotechnol. Bioeng. 2016;113: 2079-2087. © 2016 Wiley Periodicals, Inc.

Caldariomyces fumago DSM1256 Contains Two Chloroperoxidase Genes, Both Encoding Secreted and Active Enzymes.

Inspection of transcriptome data from the chloroperoxidase (CPO)-producing fungus Caldariomyces fumago DSM1256 led to the discovery of two distinct CPO mRNA sequences. This strain could be shown to contain the newly identified isogene as well as produce and secrete both isoenzymes. The CPO2 enzyme bears high sequence similarity to the well-characterized CPO (87% identity for the mature proteins). It shows two insertions in the signal peptide and in the C-terminal propeptide, and one deletion in the mature polypeptide close to the C-terminus. Furthermore, it lacks one of the serine residues known to be O-glycosylated in the CPO sequence. The demonstration of a CPO isogene which is expressed as a secreted and active CPO clarifies the nature of this isoenzyme already identified in earlier reports. A structure model comparison shows a high conservation of the active site and the substrate channel, suggesting very similar catalytic properties.

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.

Integrated bioprocess for the stereospecific production of linalool oxides from linalool with Corynespora cassiicola DSM 62475.

Linalool oxides are of interest to the flavour industry because of their lavender notes. Corynespora cassiicola DSM 62475 has been identified recently as a production organism because of high stereoselectivity and promising productivities [Mirata et al. (2008) J Agric Food Chem 56(9):3287-3296]. In this work, the stereochemistry of this biotransformation was further investigated. Predominantly (2R)-configured linalool oxide enantiomers were produced from (R)-(-)-linalool. Comparative investigations with racemic linalool suggest that predominantly (2S)-configured derivatives can be expected by using (S)-(+)-configured substrate. Substrate and product inhibited growth even at low concentrations (200 mg l⁻¹). To avoid toxic effects and supply sufficient substrates, a substrate feeding product removal (SFPR) system based on hydrophobic adsorbers was established. Applying SFPR, productivity on the shake flask scale was increased from 80 to 490 mg l⁻¹ day⁻¹. Process optimisation increased productivity to 920 mg l⁻¹ day⁻¹ in a bioreactor with an overall product concentration of 4.600 mg l⁻¹ linalool oxides.