Identification of Novel Unspecific Peroxygenase Chimeras and Unusual YfeX Axial Heme Ligand by a Versatile High‐Throughput GC‐MS Approach

Abstract

Catalyst discovery and development requires the screening of large reaction sets necessitating analytic methods with the potential for high-throughput screening. These techniques often suffer from substrate dependency or the requirement of expert knowledge. Chromatographic techniques (GC/LC) can overcome these limitations but are generally hampered by long analysis time or the need for special equipment. The herein developed multiple injections in a single experimental run (MISER) GC-MS technique allows a substrate independent 96-well microtiter plate analysis within 60 min. This method can be applied to any laboratory equipped with a standard GC-MS. With this concept novel, unspecific peroxygenase (UPO) chimeras, could be identified, consisting of subdomains from three different fungal UPO genes. The GC-technique was additionally applied to evaluate an YfeX library in an E. coli whole-cell system for the carbene-transfer reaction on indole, which revealed the thus far unknown axial heme ligand tryptophan.

Simultaneous screening of multiple substrates with an unspecific peroxygenase enabled modified alkane and alkene oxyfunctionalisations

“…We started our technology development using our previous m _ ultiple i _njections in a s _ingle e _xperimental r _un (MISER) GC-MS method. 26 As this method already provided the simultaneous assessment of three analytes, we further challenged the system by employing it to determine the cyclohexene epoxidation/allylic hydroxylation as well as the cyclohexane hydroxylation. Since the MISER GC-MS technique relies on analyte quantification via MS analysis, it is vital that all possible products exhibit different masses.…”

“…A tool to permit screenings of this kind would be our recently reported, substrateindependent high throughput analysis with an off-the-shelf GC-MS (gas chromatography-mass spectrometry). 26 This method enabled the quantification of an internal standard, the product and the byproduct simultaneously based on the MS separation within the mass analyser (quadrupole) with sample injection intervals of 33 s.…”

A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases

“…In fact, Mro UPO, Cgl UPO, Mth UPO and Tte UPO, which could be initially produced as non-engineered wild-type enzymes in yeast and characterised within the scope of this work, all belong to the class of short-type UPOs. Recent work in our laboratory suggests that gene shuffling of long-type UPOs can offer a viable option to obtain a library of active and structurally diverse long-type UPOs expanding the panel of available recombinant peroxygenases 55 .…”

Broadening the Biocatalytic Toolbox—Screening and Expression of New Unspecific Peroxygenases

“…Nonetheless, the results presented here increase the toolbox of UPOs available for genetic engineering and heterologous expression—a general requirement for the industrial application of this enzyme type. The described expression of new UPOs, the (evolutionary) optimization of the enzymes [ 6 , 9 , 24 , 29 , 30 , 31 ], as well as process-engineering approaches [ 32 , 33 , 34 , 35 , 36 , 37 ] will certainly make UPOs a key player in technical oxy-functionalization in the future.…”