Synthetic biotechnology to study and engineer natural product biosynthesis in actinomycetes

Bacterial natural products like from the well-studied actinomycetes display diverse structural features and interesting bioactivities making these secondary metabolites attractive targets for scientific research and clinical application. The present thesis deals with the identification, elucidation and engineering of biosynthetic pathways of the natural products cinnabaramides from Streptomyces sp. JS360 and bottromycins from Streptomyces sp. BC16019. Synthetic biotechnology tools were employed to improve production yields and to produce novel derivatives via biosynthetic engineering. Precursor-directed biosynthesis and mutasynthesis approaches allowed the generation of novel cinnabaramide derivatives exhibiting intriguing bioactivities. For bottromycins, a heterologous production platform was established, optimized for higher production yields and novel analogues were identified after rational pathway modification. Moreover, detailed biochemical and structural characterization of a key enzyme involved in the formation of an unusual polyketide extender unit during cinnabaramide biosynthesis was performed. By active site mutations it was possible to change the substrate specificity of the enzyme, which generates dicarboxylic acids by reductive carboxylation of enolate derivatives. The obtained results provided deeper insights into the biochemistry of this enzyme family and set the stage for generation of deliberately altered polyketides with improved pharmaceutical properties.Bakterielle Naturstoffe wie von den gut studierten Aktinomyceten weisen eine hohe strukturelle Diversität und interessante Bioaktivitäten auf und stellen daher attraktive Zielstrukturen für Forschung und klinische Anwendung dar. Die vorliegende Arbeit beschäftigt sich mit der Identifizierung, Aufklärung und Manipulation der Biosynthesewege zu Cinnabaramiden aus Streptomyces sp. JS360 und Bottromycinen aus Streptomyces sp. BC16019. Über Synthetische Biotechnologie wurde die Produktion verbessert und neue Derivate dieser Naturstoffe erzeugt. Mittels Vorläufer-dirigierter Biosynthese und Mutasynthese wurden neue Cinnabaramid-Derivate mit interessanten Bioaktivitäten hergestellt. Für die Bottromycine wurde eine heterologe Produktionsplattform etabliert, die Produktionsausbeuten optimiert und nach rationaler Modifikation des Biosyntheseweges neue Bottromycin-Analoga identifiziert. Zudem wurde ein Enzym, welches entscheidend an der Bildung einer ungewöhnlichen Polyketid-Verlängerungseinheit für die Cinnabaramid-Biosynthese beteiligt ist, detailliert biochemisch und strukturell charakterisiert. Über Mutationen in der Bindetasche gelang es, die Substratspezifität des Enzyms zu verändern, welches ausgehend von Enolat-Derivaten über reduktive Carboxylierung Dicarbonsäuren generiert. Die erhaltenen Resultate lieferten tiefere Einblicke in die Biochemie dieser Enzymfamilie und stellen eine Grundlage zur Erzeugung veränderter Polyketide mit verbesserten pharmazeutischen Eigenschaften dar

Synthetic Biotechnology to Study and Engineer Ribosomal Bottromycin Biosynthesis

SummaryBottromycins represent a promising class of antibiotics binding to the therapeutically unexploited A-site of the bacterial ribosome. By inhibiting translation they are active against clinically important pathogens, such as vancomycin-resistant Enterococci. Structurally, bottromycins are heavily modified peptides exhibiting various unusual biosynthetic features. To set the stage for compound modification and yield optimization, we identified the biosynthetic gene cluster, used synthetic biotechnology approaches to establish and improve heterologous production, and generated analogs by pathway genetic engineering. We unambiguously identified three radical SAM methyltransferase-encoding genes required for various methylations at unactivated carbons yielding tert-butyl valine, methyl-proline, and β-methyl-phenylalanine residues, plus a gene involved in aspartate methyl-ester formation. Evidence for the formation of the exo-thiazole unit and for a macrocyclodehydration mechanism leading to amidine ring formation is provided

Bringing Telepresence to Every Desk

In this paper, we work to bring telepresence to every desktop. Unlike commercial systems, personal 3D video conferencing systems must render high-quality videos while remaining financially and computationally viable for the average consumer. To this end, we introduce a capturing and rendering system that only requires 4 consumer-grade RGBD cameras and synthesizes high-quality free-viewpoint videos of users as well as their environments. Experimental results show that our system renders high-quality free-viewpoint videos without using object templates or heavy pre-processing. While not real-time, our system is fast and does not require per-video optimizations. Moreover, our system is robust to complex hand gestures and clothing, and it can generalize to new users. This work provides a strong basis for further optimization, and it will help bring telepresence to every desk in the near future. The code and dataset will be made available on our website https://mcmvmc.github.io/PersonalTelepresence/

Structure and substrate recognition of the Bottromycin maturation enzyme BotP

JK would like to thank the University of St Andrews, which is supported by a Wellcome Trust Capital Award (086036) and the Deutsche Forschungsgemeinschaft for an Emmy Noether fellowship (KO4116/3-1). BN would like to thank the European Research Council (339367).The bottromycins are a family of highly modified peptide natural products displaying potent antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphyloccoccus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N- terminal "leader" sequence. We report the structural and biochemical characterization of BotP, a leucyl-aminopeptidase like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of apo BotP and of BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottromycin.PostprintPeer reviewe

The Designing Acceleration of Driving Motor of Theodolite in the Tracking System

Abstract: In this study, the laser tracking system was introduced. In order to capture the target in a finite area, the laser tracking system was first given a moving speed in the horizontal direction to make that the target and laser tracking system were stillness in horizontal orientation relatively. The scanning pattern was raster scanning. The smallest acceleration of the driving motor of the theodolite must be 8.5º/s 2 in order to make the target to be still on the area of pointing beam spot in the course of acquisition and tracking. When the numerical value of eccentricity that the target departed from the center of laser beam spot was bigger than a planning one, the motor compensated the eccentricity and pointed the target again. The driving motor of theodolite compensated the eccentricity by an acceleration, uniform motion and speed-down process. The minimum demanding acceleration was 5.72º/s 2 . So the acceleration of the motor of laser tracking system was 8.5º/s 2

Comprehensive analysis of the extreme short-time heavy rain event on 26 June 2022 in Jinsi Gorge, Shangluo

Affected by the Hetao trough and low vortex, an extreme short-term heavy rainfall (referred to as the "6.26" event) occurred on 26 June 2022 from 12∶00 BT to 13∶00 BT in the western part of Shangnan County in Shaanxi province. During this process, an hourly rainfall of up to 108.3 mm was observed. This event was a typical local heavy rainstorm event caused by a newly-born mesoscale thunderstorm, which was characterized by its localized, sudden, short duration, and high-intensity precipitation. All numerical models failed to predict this event. Based on the data from conventional meteorological observations, encrypted automatic weather stations, Doppler weather radar, and the fifth-generation atmospheric reanalysis data of the European Center for Medium-Range Weather Forecasting (ERA5), this study analyzed in detail the environmental field characteristics and convective triggering mechanisms of this short-time heavy precipitation event. The results are as follows: (1) This event occurred under a combined circulation background of the 500 hPa low trough in Hetao and 700 hPa low vortex shear slowly moving eastward. The dynamic uplift and energy conditions over Shangnan were favorable, and the K-index reached 42.83 ℃ before the convection occurred. (2) The local abundant water vapor and the strong vertical upward movement at the lower level could rapidly transport the water vapor upward, which was conducive to the formation of short-term heavy rain over Jinsi Gorge. (3) The small and medium-scale surface convergence line formed between the local circulation caused by the thermal heterogeneity underlying surface near Jinsi Gorge and the local flow field caused by variable pressure wind is the main reason for the extremely short-term heavy precipitation in Jinsi Gorge.In this process, the special valley terrain of Jinsi Gorge played a significant role. On the one hand, it triggered the generation of convection. On the other hand, because of its block, the low-level southeast airflow rose along the mountain slope. This further strengthened the convergence of the atmosphere over Jinsi Gorge, which finally enhanced the precipitation of this rainstorm

シロイヌナズナのアルミニウム誘導性リンゴ酸放出の分子機構

博士(農学)岐阜大学(Gifu University

Interaction Between Spire and Rab GTPases and its Effect on the Developing Drosophila Oocyte

Spire, an actin nucleator, is critical for proper oocyte development in D. melanogaster via the establishment of an actin mesh network. Knowledge of its interaction with Drosophila Rab GTPases, a group of master regulators of membrane trafficking dynamics, would contribute to our understanding of Spire’s role in the developing oocyte. Drosophila Spire has a putative Rab-binding sequence, the Spir-box, located in the C-terminal half of the protein. Flies lacking the Spir-box have about a 50% decrease in fertility, which supports the idea that the interaction between Drosophila Spire and Drosophila Rabs is functionally significant. However, the specific Rab(s) involved in the interaction remains unknown. Thus, we set out to identify which specific Drosophila Rabs interact with Drosophila Spire and whether it is through direct or indirect interactions. Based on previous data showing binding interaction between mammalian Spire1 and mammalian Rabs 6 and 11 as well as Drosophila Rab expression patterns in the oocyte, we chose to start with Drosophila Rabs 5, 6, and 11. However, we did not detect a direct interaction in GST pulldown assays with GST tagged Rab 5, 6 and 11 and C-terminal constructs of Spire. This raises the possibility of indirect interactions or the requirement of additional components to stabilize the complex of Drosophila Rab(s) and Drosophila Spire. Co-IP experiments from ovary lysate will be performed to probe for indirect binding between Spire and Rab 5, 6, and 11. Once specific Rabs have been identified, the complex will be further characterized biochemically with the long-term goal of modifying the binding sites in vitro and determining the functional consequences of these changes in vivo