Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo‐ and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone‐precursor premedrol by optimizing the CYP21A2‐based whole‐cell system on a laboratory scale. We successfully improved the whole‐cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N‐terminally truncated version of the bovine NADPH‐dependent cytochrome P450 reductase (bCPR−27) and coexpression of microsomal cytochrome b5; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed‐batch protocol. By overcoming the presented limitations in whole‐cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L−1·d−1 premedrol

A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 ◦C and its apparent half-life at 60 ◦C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale

Identifizierung und Charakterisierung neuer Proteine aus Bacillus megaterium für biotechnologische und pharmazeutische Anwendungen

The oxyfunctionalization of substrates (such as steroids and drugs) in a stereo- and regioselective manner is of great interest for the biotechnological and pharmaceutical industry. This study presents the identification and characterization of three enzymes from the genome of the Gram-positive bacterium Bacillus megaterium. The first enzyme is a novel NADPH-dependent diflavin reductase (BmCPR for Bacillus megaterium cytochrome P450 reductase), which was found to be able to transfer electrons efficiently to the microsomal P450s as well as to different ferredoxins. The second enzyme is a cytochrome P450 (CYP107DY1), which is the first plasmid-encoded cytochrome P450 identified so far in the Bacillus species. The recombinant CYP107DY1 exhibiting a characteristic P450 absolute and reduced CO-bound difference spectrum was found to possess activity towards mevastatin to produce pravastatin. In addition, this work presents also the identification of a novel short-chain dehydrogenase/reductase (SDR) with 17ß-hydroxy steroid dehydrogenase (17ß-HSD) activity. The new dehydrogenase was found to possess activity towards the sesquiterpene (trans)-nootkatol converting it to the industrial valuable (+)-nootkatone. Moreover and in view of the biotechnological applications, the new enzymes could be utilized in the establishment of whole-cell based biocatalyst systems for the biotransformation of biotechnological relevant substances.Die stereo- und regioselektive Oxyfunktionalisierung von Substraten, wie etwa Steroiden und Medikamenten, ist von großem Interesse für die biotechnologische und pharmazeutische Industrie. In der vorliegenden Arbeit wurden drei verschiedene Enzyme des Gram-positiven Bakteriums Bacillus megaterium identifiziert und charakterisiert. Bei dem ersten Enzym handelt es sich um eine neuartige NADPH abhängige Diflavin Reduktase (BmCPR für Bacillus megaterium Cytochrom P450 Reduktase), die in der Lage ist, Elektronen effektiv auf mikrosomale Cytochrome P450, ebenso wie auf verschiedene Ferredoxine zu übertragen. Bei dem zweiten Enzym handelt es sich um ein Cytochrom P450 (CYP107DY1), welches zur Biotransformation von Mevastatin zu Pravastatin verwendet werden kann. Das rekombinant exprimierte CYP107DY1 zeigt das für Cytochrome P450 charakteristische Absorptionsspektrum im reduzierten und CO gebundenen Zustand. Soweit bekannt, ist dieses Enzym das erste Plasmid kodierte Cytochrom P450 der Gattung Bacillus. Weiterhin umfasst die vorliegende Arbeit die Identifizierung eines neuen Vertreters aus der Superfamilie der Short-chain Dehydrogenasen/Reduktasen (SDR) mit Aktivität von 17ß-Hydroxysteroid Dehydrogenasen (17ß-HSD). Die Aktivität dieser Dehydrogenase erlaubt zudem die Produktion des industriell bedeutenden Sesquiterpens (+)-Nootkaton aus (trans)-Nootkatol. Hinsichtlich einer biotechnologischen Anwendung besitzen alle in dieser Arbeit charakterisierten Enzyme das Potenzial für die Etablierung von Ganzzell basierten Biokatalysatoren für die Biotransformation biotechnologisch relevanter Substanzen

Identifizierung und Charakterisierung neuer Proteine aus Bacillus megaterium für biotechnologische und pharmazeutische Anwendungen

The oxyfunctionalization of substrates (such as steroids and drugs) in a stereo- and regioselective manner is of great interest for the biotechnological and pharmaceutical industry. This study presents the identification and characterization of three enzymes from the genome of the Gram-positive bacterium Bacillus megaterium. The first enzyme is a novel NADPH-dependent diflavin reductase (BmCPR for Bacillus megaterium cytochrome P450 reductase), which was found to be able to transfer electrons efficiently to the microsomal P450s as well as to different ferredoxins. The second enzyme is a cytochrome P450 (CYP107DY1), which is the first plasmid-encoded cytochrome P450 identified so far in the Bacillus species. The recombinant CYP107DY1 exhibiting a characteristic P450 absolute and reduced CO-bound difference spectrum was found to possess activity towards mevastatin to produce pravastatin. In addition, this work presents also the identification of a novel short-chain dehydrogenase/reductase (SDR) with 17ß-hydroxy steroid dehydrogenase (17ß-HSD) activity. The new dehydrogenase was found to possess activity towards the sesquiterpene (trans)-nootkatol converting it to the industrial valuable (+)-nootkatone. Moreover and in view of the biotechnological applications, the new enzymes could be utilized in the establishment of whole-cell based biocatalyst systems for the biotransformation of biotechnological relevant substances.Die stereo- und regioselektive Oxyfunktionalisierung von Substraten, wie etwa Steroiden und Medikamenten, ist von großem Interesse für die biotechnologische und pharmazeutische Industrie. In der vorliegenden Arbeit wurden drei verschiedene Enzyme des Gram-positiven Bakteriums Bacillus megaterium identifiziert und charakterisiert. Bei dem ersten Enzym handelt es sich um eine neuartige NADPH abhängige Diflavin Reduktase (BmCPR für Bacillus megaterium Cytochrom P450 Reduktase), die in der Lage ist, Elektronen effektiv auf mikrosomale Cytochrome P450, ebenso wie auf verschiedene Ferredoxine zu übertragen. Bei dem zweiten Enzym handelt es sich um ein Cytochrom P450 (CYP107DY1), welches zur Biotransformation von Mevastatin zu Pravastatin verwendet werden kann. Das rekombinant exprimierte CYP107DY1 zeigt das für Cytochrome P450 charakteristische Absorptionsspektrum im reduzierten und CO gebundenen Zustand. Soweit bekannt, ist dieses Enzym das erste Plasmid kodierte Cytochrom P450 der Gattung Bacillus. Weiterhin umfasst die vorliegende Arbeit die Identifizierung eines neuen Vertreters aus der Superfamilie der Short-chain Dehydrogenasen/Reduktasen (SDR) mit Aktivität von 17ß-Hydroxysteroid Dehydrogenasen (17ß-HSD). Die Aktivität dieser Dehydrogenase erlaubt zudem die Produktion des industriell bedeutenden Sesquiterpens (+)-Nootkaton aus (trans)-Nootkatol. Hinsichtlich einer biotechnologischen Anwendung besitzen alle in dieser Arbeit charakterisierten Enzyme das Potenzial für die Etablierung von Ganzzell basierten Biokatalysatoren für die Biotransformation biotechnologisch relevanter Substanzen

Characterization of the stereoselective P450 enzyme BotCYP enables the in vitro biosynthesis of the Bottromycin Core Scaffold

Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development

Characterization of the Stereoselective P450 Enzyme BotCYP Enables the Biosynthesis of the Bottromycin Core Scaffold.

Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development

Applications and Potentials of Intelligent Swarms for magnetospheric studies

Earth's magnetosphere is vital for today's technologically dependent society. To date, numerous design studies have been conducted and over a dozen science missions have flown to study the magnetosphere. However, a majority of these solutions relied on large monolithic satellites, which limited the spatial resolution of these investigations, as did the technological limitations of the past. To counter these limitations, we propose the use of a satellite swarm carrying numerous and distributed payloads for magnetospheric measurements. Our mission is named APIS — Applications and Potentials of Intelligent Swarms. The APIS mission aims to characterize fundamental plasma processes in the Earth's magnetosphere and measure the effect of the solar wind on our magnetosphere. We propose a swarm of 40 CubeSats in two highly-elliptical orbits around the Earth, which perform radio tomography in the magnetotail at 8–12 Earth Radii (RE) downstream, and the subsolar magnetosphere at 8–12 RE upstream. These maps will be made at both low-resolutions (at 0.5 RE, 5 s cadence) and high-resolutions (at 0.025 RE, 2 s cadence). In addition, in-situ measurements of the magnetic and electric fields, plasma density will be performed by on-board instruments. In this article, we present an outline of previous missions and designs for magnetospheric studies, along with the science drivers and motivation for the APIS mission. Furthermore, preliminary design results are included to show the feasibility of such a mission. The science requirements drive the APIS mission design, the mission operation and the system requirements. In addition to the various science payloads, critical subsystems of the satellites are investigated e.g., navigation, communication, processing and power systems. Our preliminary investigation on the mass, power and link budgets indicate that the mission could be realized using Commercial Off-the-Shelf (COTS) technologies and with homogeneous CubeSats, each with a 12U form factor. We summarize our findings, along with the potential next steps to strengthen our design study.Circuits and System

Applications and Potentials of Intelligent Swarms for magnetospheric studies

Earth's magnetosphere is vital for today's technologically dependent society. To date, numerous design studies have been conducted and over a dozen science missions have flown to study the magnetosphere. However, a majority of these solutions relied on large monolithic satellites, which limited the spatial resolution of these investigations, as did the technological limitations of the past. To counter these limitations, we propose the use of a satellite swarm carrying numerous and distributed payloads for magnetospheric measurements. Our mission is named APIS — Applications and Potentials of Intelligent Swarms. The APIS mission aims to characterize fundamental plasma processes in the Earth's magnetosphere and measure the effect of the solar wind on our magnetosphere. We propose a swarm of 40 CubeSats in two highly-elliptical orbits around the Earth, which perform radio tomography in the magnetotail at 8–12 Earth Radii (RE) downstream, and the subsolar magnetosphere at 8–12 RE upstream. These maps will be made at both low-resolutions (at 0.5 RE, 5 s cadence) and high-resolutions (at 0.025 RE, 2 s cadence). In addition, in-situ measurements of the magnetic and electric fields, plasma density will be performed by on-board instruments. In this article, we present an outline of previous missions and designs for magnetospheric studies, along with the science drivers and motivation for the APIS mission. Furthermore, preliminary design results are included to show the feasibility of such a mission. The science requirements drive the APIS mission design, the mission operation and the system requirements. In addition to the various science payloads, critical subsystems of the satellites are investigated e.g., navigation, communication, processing and power systems. Our preliminary investigation on the mass, power and link budgets indicate that the mission could be realized using Commercial Off-the-Shelf (COTS) technologies and with homogeneous CubeSats, each with a 12U form factor. We summarize our findings, along with the potential next steps to strengthen our design study.</p

APIS : Applications and potentials of intelligent swarms for magnetospheric studies

Earth's magnetosphere is vital for today's technologically dependent society. The energy transferred from the solar wind to the magnetosphere triggers electromagnetic storms on Earth, knocking out power grids and infrastructure - e.g., communication and navigation systems. Despite occurring on our astrophysical doorstep, numerous physical processes connecting the solar wind and our magnetosphere remain poorly understood. To date, over a dozen science missions have flown to study the magnetosphere, and many more design studies have been conducted. However, the majority of these solutions relied on large monolithic satellites, which limited the spatial resolution of these investigations, in addition to the technological limitations of the past. To counter these limitations, we propose the use of a satellite swarm, carrying numerous payloads for magnetospheric measurements. Our mission is named APIS - Applications and Potentials of Intelligent Swarms. The APIS mission aims to characterize fundamental plasma processes in the magnetosphere and measure the effect of the solar wind on our magnetosphere. We propose a swarm of 40 CubeSats in two highly-elliptical orbits around the Earth, which perform radio tomography in the magnetotail at 8-12 Earth Radii (RE) downstream, and the subsolar magnetosphere at 8-12 RE upstream. These maps will be made at both low-resolutions (at 0.5 RE, 5 seconds cadence) and high-resolutions (at 0.025 RE, 2 seconds cadence). In addition, in-situ measurements of the magnetic and electric fields, and plasma density will be performed by on-board instruments. In this publication, we present a design study of the APIS mission, which includes the mission design, navigation, communication, processing, power systems, propulsion and other critical satellite subsystems. The science requirements of the APIS mission levy stringent system requirements, which are addressed using Commercial Off-the-Shelf (COTS) technologies. We show the feasibility of the APIS mission using COTS technologies using preliminary link, power, and mass budgets. In addition to the technological study, we also investigated the legal considerations of the APIS mission. The APIS mission design study was part of the International Space University Space Studies Program in 2019 (ISU-SSP19) Next Generation Space Systems: Swarms Team Project. The authors of this publication are the participants of this 9-week project, in addition to the Chairs and Support staff.</p