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Publikation

Mahor, D.; Cong, Z.; Weissenborn, M. J.; Hollmann, F.; Zhang, W.; Valorization of small alkanes by biocatalytic oxyfunctionalization ChemSusChem 15, e202101116, (2022) DOI: 10.1002/cssc.202101116

The oxidation of alkanes into valuable chemical products is avital reaction in organic synthesis. This reaction, however, ischallenging, owing to the inertness of C−H bonds. Transitionmetal catalysts for C−H functionalization are frequently ex-plored. Despite chemical alternatives, nature has also evolvedpowerful oxidative enzymes (e.g., methane monooxygenases,cytochrome P450 oxygenases, peroxygenases) that are capableof transforming C−H bonds under very mild conditions, withonly the use of molecular oxygen or hydrogen peroxide aselectron acceptors. Although progress in alkane oxidation hasbeen reviewed extensively, little attention has been paid tosmall alkane oxidation. The latter holds great potential for themanufacture of chemicals. This Minireview provides a conciseoverview of the most relevant enzyme classes capable of smallalkanes (C
Publikation

Knorrscheidt, A.; Soler, J.; Hünecke, N.; Püllmann, P.; Garcia-Borràs, M.; Weissenborn, M. J.; Simultaneous screening of multiple substrates with an unspecific peroxygenase enabled modified alkane and alkene oxyfunctionalisations Catal. Sci. Technol. 11, 6058, (2021) DOI: 10.1039/d0cy02457k

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 peroxygenase MthUPO. 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.
Publikation

Knorrscheidt, A.; Soler, J.; Hünecke, N.; Püllmann, P.; Garcia-Borràs, M.; Weissenborn, M. J.; Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase ACS Catal. 11, 7327-7338, (2021) DOI: 10.1021/acscatal.1c00847

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.
Publikation

Püllmann, P.; Weissenborn, M. J.; Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System ACS Synth. Biol. 10, 1360-1372, (2021) DOI: 10.1021/acssynbio.0c00641

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.
Publikation

Püllmann, P.; Knorrscheidt, A.; Münch, J.; Palme, P. R.; Hoehenwarter, W.; Marillonnet, S.; Alcalde, M.; Westermann, B.; Weissenborn, M. J.; A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases Commun. Biol. 4, 562, (2021) DOI: 10.1038/s42003-021-02076-3

AbstractFungal 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.
Publikation

Münch, J.; Püllmann, P.; Zhang, W.; Weissenborn, M. J.; Enzymatic Hydroxylations of sp3-Carbons ACS Catal. 11, 9168-9203, (2021) DOI: 10.1021/acscatal.1c00759

Enzymatic hydroxylation of activated and nonactivated sp3-carbons attracts keen interest from the chemistry community as it is one of the most challenging tasks in organic synthesis. Nature provides a vast number of enzymes with an enormous catalytic versatility to fulfill this task. Given that those very different enzymes have a distinct specificity in substrate scope, selectivity, activity, stability, and catalytic cycle, it is interesting to outline similarities and differences. In this Review, we intend to delineate which enzymes possess considerable advantages within specific issues. Heterologous production, crystal structure availability, enzyme engineering potential, and substrate promiscuity are essential factors for the applicability of these biocatalysts.
Publikation

Adjedje, V. K. B.; Schell, E.; Wolf, Y. L.; Laub, A.; Weissenborn, M. J.; Binder, W. H.; Enzymatic degradation of synthetic polyisoprenes via surfactant-free polymer emulsification Green Chemistry 23, 9433-9438, (2021) DOI: 10.1039/d1gc03515k

We report the enzymatic degradation of a synthetic polyisoprene with a cis : trans ratio of 56 : 27 for the first time. Utilizing a bioinspired surfactant-free emulsification strategy in water resulted in substantially increased enzymatic activities with the latex clearing protein LcpK30.
Preprints

Püllmann, P.; Knorrscheidt, A.; Münch, J.; Palme, P. R.; Hoehenwarter, W.; Marillonnet, S.; Alcalde, M.; Westermann, B.; Weissenborn, M. J.; A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases bioRxiv (2020) DOI: 10.1101/2020.07.22.216432

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.
Publikation

Knorrscheidt, A.; Püllmann, P.; Schell, E.; Homann, D.; Freier, E.; Weissenborn, M. J.; Identification of novel unspecific peroxygenase chimeras and unusual YfeX axial heme ligand by a versatile high‐throughput GC‐MS approach ChemCatChem 12, 4788-4795, (2020) DOI: 10.1002/cctc.202000618

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.
Publikation

Weissenborn, M. J.; Koenigs, R. M.; Iron‐porphyrin Catalyzed Carbene Transfer Reactions – an Evolution from Biomimetic Catalysis towards Chemistry‐inspired Non‐natural Reactivities of Enzymes ChemCatChem 12, 2171-2179, (2020) DOI: 10.1002/cctc.201901565

Bioinspired, synthetic porphyrin complexes are important catalysts in organic synthesis and play a pivotal role in efficient carbene transfer reactions. The advances in this research area stimulated recent, “chemo‐inspired” developments in biocatalysis. Today, both synthetic iron complexes and enzymes play an important role to conduct carbene transfer reactions. The advances and potential developments in both research areas are discussed in this concept article.
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