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Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest M.J.W., A.K., and N.H. thank the Bundesministerium für Bildung und Forschung (“Biotechnologie 2020+ Strukturvorhaben: Leibniz Research Cluster”, 031A360B) for generous funding. P.P. thanks the Landesgraduiertenförderung Sachsen- Anhalt for a Ph.D. scholarship. M.G.-B. thanks the Generalitat de Catalunya AGAUR for a Beatriu de Pinós H2020 MSCACofund 2018-BP-00204 project and the Spanish MICINN (Ministerio de Ciencia e Innovación) for PID2019-111300GAI00 project, and J.S. thanks the Spanish MIU (Ministerio de Universidades) for a predoctoral FPU fellowship FPU18/ 02380. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged (RES-QSB-2019-3-0009 and RES-QSB-2020-2-0016)

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations., Püllmann et al developed a modular Golden Gate-based secretion system, which enabled production and one-step purification of active fungal unspecific peroxygenases (UPOs) in yeast. Their system was applied to an enantioselective conversion on a preparative scale and may be used in the future for other genes of interest that are suitable for production in yeast.

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N-H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si-H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si-H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

A high throughput GC-MS approach was developed, permitting the simultaneous analysis of up to three substrates and six products quantitatively from one reaction mixture. This screening approach was applied to site-saturation libraries of the novel unspecific peroxygenaseMthUPO. Using this setup enabled substantial insights from a small mutant library. Enzyme variants were identified exhibiting selective alkene epoxidation and substantially shifted regioselectivities to 2- and 1-octanol formations. Computational modelling rationalised the observed selectivity changes M. J. W, A. K., and N. H. thank the Bundesministerium für Bildung und Forschung (“Biotechnologie 2020+ Strukturvorhaben: Leibniz Research Cluster”, 031A360B) for generous funding. P. P. thanks the Landesgraduiertenförderung Sachsen-Anhalt for a PhD scholarship. The authors thank Eugen Schell for fruitful discussions. M. G. B. thanks the Generalitat de Catalunya AGAUR for a Beatriu de Pinós H2020 MSCA-Cofund 2018-BP-00204 project, the Spanish MICINN (Ministerio de Ciencia e Innovación) for PID2019-111300GA-I00 project, and J. S. thanks the Spanish MIU (Ministerio de Universidades) for a predoctoral FPU fellowship FPU18/02380. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged (RES-QSB-2019-3-262 0009 and RES-QSB-2020-2-0016)

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

Thirty-one different 3-O-acetyl-OA derived amides have been prepared and screened for their cytotoxic activity. In the SRB assays nearly all the carboxamides displayed good cytotoxicity in the low μM range for several human tumor cell lines. Low EC50 values were obtained especially for the picolinylamides 14–16, for a N-[2-(dimethylamino)-ethyl] derivative 27 and a N-[2-(pyrrolinyl)-ethyl] carboxamide 28. These compounds were submitted to an extensive biological testing and proved compound 15 to act mainly by an arrest of the tumor cells in the S phase of the cell cycle. Cell death occurred by autophagy while compounds 27 and 28 triggered apoptosis.

The screening effort of large protein variant libraries renders the probability of coincidental discovering a new enzyme with non-natural activity to almost zero - the so-called numbers problem. Insights into the origin of life, evolution and enzymatic promiscuity, combined with the inspiration of methods from organic chemistry, offer solutions for this problem. With the newly discovered enzymes synthetic micro production units shall be established in a Leibniz Research Cluster where engineering and biotechnology are combined.

The functionalization of C-H bonds with non-precious metal catalysts is an important research area for the development of efficient and sustainable processes. Herein, we describe the development of iron porphyrin catalyzed reactions of diazoacetonitrile with N-heterocycles yielding important precursors of tryptamines, along with experimental mechanistic studies and proof-of-concept studies of an enzymatic process with YfeX enzyme. By using readily available FeTPPCl, we achieved the highly efficient C-H functionalization of indole and indazole heterocycles. These transformations feature mild reaction conditions, excellent yields with broad functional group tolerance, can be conducted on gram scale, and thus provide a unique streamlined access to tryptamines.

C—H insertion reactions with organometallic and enzymatic catalysts based on earth-abundant iron complexes remain one of the major challenges in organic synthesis. In this report, we describe the development and application of these iron-based catalysts in the reaction of two different carbene precursors with N-heterocycles for the first time. While FeTPPCl showed excellent reactivity in the Fe(III) state with diazoacetonitrile, the highest activities of the YfeX enzyme could be achieved upon heme-iron reduction to Fe(II) with both diazoacetonitrile and ethyl diazoacetate. This highlights unexpected and subtle differences in reactivity of both iron catalysts. Deuterium labeling studies indicated a C—H insertion pathway and a marked kinetic isotope effect. This transformation features mild reaction conditions, excellent yields or turnover numbers with broad functional group tolerance, including gram-scale applications giving a unique access to functionalized N-heterocycles.