H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions

The soluble hydrogenase from Ralstonia eutropha provides an atom efficient regeneration system for reduced flavin cofactors using H2 as an electron source. We demonstrated this system for highly selective ene-reductase-catalyzed C[double bond, length as m-dash]C-double bond reductions and monooxygenase-catalyzed epoxidation. Reactions were expanded to aerobic conditions to supply H2O2 for peroxygenase-catalyzed hydroxylations.DFG, 284111627, H2-basierende Kaskaden für die Biosynthese von N-HeterocyclenDFG, 405325648, ,Engineering von O2-toleranten Hydrogenasen und ihre physiologischen Auswirkungen in rekombinanten Bakterien im Hinblick auf die Hydrogenase-abhängige NAD(P)H-Regeneration und H2-ProduktionDFG, 390540038, EXC 2008: UniSysCatTU Berlin, Open-Access-Mittel - 202

Synthesis of N-heterocycles from diamines via H2-driven NADPH recycling in the presence of O2

Herein, we report an enzymatic cascade involving an oxidase, an imine reductase and a hydrogenase for the H2-driven synthesis of N-heterocycles. Variants of putrescine oxidase from Rhodococcus erythropolis with improved activity were identified. Substituted pyrrolidines and piperidines were obtained with up to 97% product formation in a one-pot reaction directly from the corresponding diamine substrates. The formation of up to 93% ee gave insights into the specificity and selectivity of the putrescine oxidase.DFG, 53182490, EXC 314: Unifying Concepts in CatalysisDFG, 284111627, H2-basierende Kaskaden für die Biosynthese von N-HeterocyclenTU Berlin, Open-Access-Mittel - 201

An engineered escherichia coli strain with synthetic metabolism for in‐cell production of translationally active methionine derivatives

In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re‐engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans‐sulfuration pathway. Our metabolic scheme operates in vivo, rescuing intermediates from core cell metabolism and combining them with small bio‐orthogonal compounds. Our reprogrammed E. coli strain is capable of the in‐cell production of L‐azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology‐based production

Powering Artificial Enzymatic Cascades with Electrical Energy

We have developed a scalable platform that employs electrolysis for an in vitro synthetic enzymatic cascade in a continuous flow reactor. Both H2 and O2 were produced by electrolysis and transferred through a gas‐permeable membrane into the flow system. The membrane enabled the separation of the electrolyte from the biocatalysts in the flow system, where H2 and O2 served as electron mediators for the biocatalysts. We demonstrate the production of methylated N‐heterocycles from diamines with up to 99 % product formation as well as excellent regioselective labeling with stable isotopes. Our platform can be applied for a broad panel of oxidoreductases to exploit electrical energy for the synthesis of fine chemicals.DFG, 284111627, H2-basierende Kaskaden für die Biosynthese von N-HeterocyclenTU Berlin, Open-Access-Mittel – 2020DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"DFG, 390677874, EXC 2033: RESOLV (Ruhr Explores Solvation

Biokatalytische Reaktionen angetrieben mittels Wasserstoff

H2 -driven cofactor regeneration is cheap, 100% atom-efficient, and has been applied in various biotechnological approaches both in vivo and in vitro. To date, H2-driven cofactor regeneration has been applied only in biocatalytic reactions involving NADH. H2-driven cofactor regeneration has also never been employed in multi-enzymatic cascades or for other cofactors besides NADH. In this work, the application of H2 to drive multi-enzymatic cascades and biocatalytical redox reactions involving cofactors other than NADH was demonstrated. The vital key for achieving this was the application of the O2-tolerant NAD+-hydrogenase (SH) from Ralstonia eutropha H16. Firstly, the efficiency of SH in reducing flavins was demonstrated for both FMN and FAD. The SH was then coupled with the “old yellow enzyme” (TsOYE) for the H2-driven reduction of enoates, yielding up to 22 mM of product. The O2-tolerance of SH was then demonstrated by coupling SH with styrene monooxygenase (StyA), which used O2 as a co-substrate to drive the epoxidation of styrene, yielding 1 mM of styrene oxide. The H2-driven flavin regeneration was used to generate H2O2 by uncoupling the reduced FMN with O2. The H2O2 produced was then used as a substrate for the unspecific peroxygenase to hydroxylate organic compounds, yielding up to 4.8 mM of hydroxylated product. Secondly, a novel multi-enzymatic cascade was designed to synthesize methylated N-heterocycles from diamines. N-heterocycles are valuable precursors in the synthesis of various pharmaceuticals. The cascade consisted of an engineered putrescine oxidase (PuOE203G), an imine reductase (IRED), and a NADP+-reducing hydrogenase (SHE341A/S342R). Methylated N-heterocycles were synthesized, with up to 97% conversion yield and >73% ee. The used H2-based regeneration system proved to be superior to other enzymatic-based systems. Moreover, this cascade avoided the use of toxic reducing agents and organic solvents. Finally, a new scalable electro-driven platform was developed to synthesize deuterated piperidines in vitro. This new platform used electrical energy to generate H2 and O2. Both gases were transferred via a gas-permeable membrane into a mini flow reactor with immobilized enzymes. H2 and O2 served as electron mediators to produce deuterated methylated piperidines from diamines, with up to 99% conversion yield and excellent regioselective labeling. The platform was scaled up to 300 mL, which resulted in a yield of up 68 mg of isolated product. This work opens the door for implementing electrical energy to drive redox-enzymatic reactions to access fine chemicals.Die H2-getriebene Cofaktor-Regeneration ist kostengünstig, 100% atomar effizient, und sie wurde in verschiedenen biotechnologischen Ansätzen sowohl in vivo als auch in vitro angewandt. Bislang wurde die H2-getriebene Cofaktor-Regeneration nur bei biokatalytischen Reaktionen mit NADH angewandt. Die H2-getriebene Kofaktor-Regeneration wurde auch nie in multi-enzymatischen Kaskaden oder unter Verwendung anderer Kofaktoren außer NADH eingesetzt. In dieser Arbeit wurde die Anwendung von molekularem H2 zur Steuerung multienzymatischer Kaskaden und biokatalytischer Redoxreaktionen mit anderen Kofaktoren als NADH demonstriert. Der entscheidende Schlüssel zum Erreichen dieses Ziels war die Anwendung der O2-toleranten NAD+-Hydrogenase aus Ralstonia eutropha H16 (SH). Zunächst wurde die Effizienz von SH bei der Reduktion von Flavinen sowohl für FMN als auch für FAD nachgewiesen. Die SH wurde dann mit dem alten gelben Enzym (TsOYE) für die H2-getriebene Reduktion von Enoaten gekoppelt, was bis zu 22 mM Produkt ergab. Die O2-Toleranz des SH wurde dann durch Kopplung des SH mit der Styrol-Monooxygenase (StyA), das O2 als Co-Substrate verwendet, nachgewiesen, um die Epoxidation von Styrol voranzutreiben, was 1 mM Styroloxid ergab. Die H2-getriebene Flavin-Regeneration wurde zur Erzeugung von H2O2 durch Entkopplung der reduzierten FMN mit molekularem O2 verwendet. Das erzeugte H2O2 wurde als Substrat für unspezifische Peroxygenase (UPO) verwendet, um organische Verbindungen zu hydroxylieren, was bis zu 4,8 mM an hydroxyliertem Produkt ergab. Zweitens wurde eine neuartige multi-enzymatische Kaskade entworfen, um methylierte N-Heterozyklen aus Diaminen zu synthetisieren. N-Heterozyklen sind wertvolle Bausteine bei der Synthese verschiedener Pharmazeutika. Die Kaskade bestand aus einer Putrescin-Oxidase Variante (PuOE203G), einer Imin-Reduktase (IRED) und einer NADP+-reduzierenden Hydrogenase (SHE341A/S342R). Methylierte N-Heterozyklen wurden mit bis zu 97% Umwandlungsausbeute und >73% ee synthetisiert. Das verwendete H2-basierte Regenerationssystem erwies sich als überlegen gegenüber anderen enzymatischen Systemen. Darüber hinaus konnte bei dieser Kaskade auf den Einsatz von toxischen Reduktionsmitteln und organischen Lösungsmitteln verzichtet werden. Schließlich wurde eine neue skalierbare elektrogetriebene Plattform entwickelt, um deuterierte methylierte Piperidinen in vitro zu synthetisieren. Die neue Plattform nutzte die elektrische Energie zur Erzeugung von H2 und O2. Beide Gase wurden über eine gasdurchlässige Membran in einen Durchfluss-Minireaktor mit immobilisierten Enzymen überführt. H2 und O2 dienten als Elektronenvermittler, um deuterierte methylierte Piperidinen aus Diaminen herzustellen, die eine bis zu 99% Umwandlungsausbeute und eine ausgezeichnete regioselektive Markierung ergaben. Die Plattform wurde auf bis zu 300 mL skaliert, was zu einer Ausbeute von bis zu 68 mg isoliertem Produkt führte. Diese Arbeit öffnet die Tür für die Implementierung der elektrischen Energie zur Durchführung von Redox-enzymatischen Reaktionen, um Feinchemikalien zu produzieren

H2 as Fuel for Flavin- and H2O2-Dependent Biocatalytic Reactions

Herein, we report a novel H2-driven regeneration system for reduced flavin cofactors. Using an O2-tolerant hydrogenase, we achieved NAD(P)H-independent, highly selective, ene-reductase-catalyzed C=C-double bond reductions, monooxygenase-catalyzed epoxidations and peroxygenase-catalyzed hydroxylations. The hydrogenase performed up to 46000 catalytic cycles and product titers of up to 22 mM have been accomplished. Overall, this system bears the promise to power flavin-dependent biocatalytic reactions with H2 in a highly atom efficient manner. </div

Hydrogenase-based oxidative biocatalysis without oxygen

Abstract Biocatalysis-based synthesis can provide a sustainable and clean platform for producing chemicals. Many oxidative biocatalytic routes require the cofactor NAD+ as an electron acceptor. To date, NADH oxidase (NOX) remains the most widely applied system for NAD+ regeneration. However, its dependence on O2 implies various technical challenges in terms of O2 supply, solubility, and mass transfer. Here, we present the suitability of a NAD+ regeneration system in vitro based on H2 evolution. The efficiency of the hydrogenase-based system is demonstrated by integrating it into a multi-enzymatic cascade to produce ketoacids from sugars. The total NAD+ recycled using the hydrogenase system outperforms NOX in all different setups reaching up to 44,000 mol per mol enzyme. This system proves to be scalable and superior to NOX in terms of technical simplicity, flexibility, and total output. Furthermore, the system produces only green H2 as a by-product even in the presence of O2

Erratum: Correction: H₂ as a fuel for flavin- and H₂O₂-dependent biocatalytic reactions (Chemical communications (Cambridge, England) (2020))

Correction for 'H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions' by Ammar Al-Shameri et al., Chem. Commun., 2020, DOI: 10.1039/d0cc03229h.BT/Biocatalysi

H₂as a fuel for flavin- And H₂O₂-dependent biocatalytic reactions

The soluble hydrogenase from Ralstonia eutropha provides an atom efficient regeneration system for reduced flavin cofactors using H2 as an electron source. We demonstrated this system for highly selective ene-reductase-catalyzed CC-double bond reductions and monooxygenase-catalyzed epoxidation. Reactions were expanded to aerobic conditions to supply H2O2 for peroxygenase-catalyzed hydroxylations.BT/Biocatalysi

An Engineered Escherichia coli Strain with Synthetic Metabolism for in‐Cell Production of Translationally Active Methionine Derivatives

In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re‐engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans‐sulfuration pathway. Our metabolic scheme operates in vivo, rescuing intermediates from core cell metabolism and combining them with small bio‐orthogonal compounds. Our reprogrammed E. coli strain is capable of the in‐cell production of l‐azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology‐based production.TU Berlin, Open-Access-Mittel – 2020DFG, 53182490, EXC 314: Unifying Concepts in Catalysi