Showing papers by "Rainer Breitling published in 2020"

Engineering Escherichia coli towards de novo production of gatekeeper (2S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol.

Abstract: Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.

Exploring novel bacterial terpene synthases.

Abstract: Terpenes are the largest class of natural products with extensive structural diversity and are widely used as pharmaceuticals, herbicides, flavourings, fragrances, and biofuels. While they have mostly been isolated from plants and fungi, the availability and analysis of bacterial genome sequence data indicates that bacteria also possess many putative terpene synthase genes. In this study, we further explore this potential for terpene synthase activity in bacteria. Twenty two potential class I terpene synthase genes (TSs) were selected to represent the full sequence diversity of bacterial synthase candidates and recombinantly expressed in E. coli. Terpene synthase activity was detected for 15 of these enzymes, and included mono-, sesqui- and diterpene synthase activities. A number of confirmed sesquiterpene synthases also exhibited promiscuous monoterpene synthase activity, suggesting that bacteria are potentially a richer source of monoterpene synthase activity then previously assumed. Several terpenoid products not previously detected in bacteria were identified, including aromandendrene, acora-3,7(14)-diene and longiborneol. Overall, we have identified promiscuous terpene synthases in bacteria and demonstrated that terpene synthases with substrate promiscuity are widely distributed in nature, forming a rich resource for engineering terpene biosynthetic pathways for biotechnology.

Non-covalent protein-based adhesives for transparent substrates-bovine serum albumin vs. recombinant spider silk

Abstract: Protein-based adhesives could have several advantages over petroleum-derived alternatives, including substantially lower toxicity, smaller environmental footprint and renewable sourcing. Here, we report that non-covalently crosslinked bovine serum albumin (BSA) and recombinant spider silk proteins have high adhesive strength on glass (8.53 and 6.28 MPa, respectively) and other transparent substrates. Moreover, the adhesives have high visible transparency and showed no apparent degradation over a period of several months. The mechanism of adhesion was investigated and primarily attributed to dehydration-induced reorganisation of protein secondary structure, resulting in the supramolecular association of β-sheets into a densely hydrogen-bonded network.

The effect of terminal globular domains on the response of recombinant mini-spidroins to fiber spinning triggers.

Abstract: Spider silk spidroins consist of long repetitive protein strands, flanked by globular terminal domains The globular domains are often omitted in recombinant spidroins, but are thought to be essential for the spiders' natural spinning process Mimicking this spinning process could be an essential step towards producing strong synthetic spider silk Here we describe the production of a range of mini-spidroins with both terminal domains, and characterize their response to a number of biomimetic spinning triggers Our results suggest that mini-spidroins which are able to form protein micelles due to the addition of both terminal domains exhibit shear-thinning, a property which native spidroins also show Furthermore, our data also suggest that a pH drop alone is insufficient to trigger assembly in a wet-spinning process, and must be combined with salting-out for effective fiber formation With these insights, we applied these assembly triggers for relatively biomimetic wet spinning This work adds to the foundation of literature for developing improved biomimetic spinning techniques, which ought to result in synthetic silk that more closely approximates the unique properties of native spider silk

Bioengineering horizon scan 2020

Abstract: Horizon scanning is intended to identify the opportunities and threats associated with technological, regulatory and social change. In 2017 some of the present authors conducted a horizon scan for bioengineering (Wintle et al., 2017). Here we report the results of a new horizon scan that is based on inputs from a larger and more international group of 38 participants. The final list of 20 issues includes topics spanning from the political (the regulation of genomic data, increased philanthropic funding and malicious uses of neurochemicals) to the environmental (crops for changing climates and agricultural gene drives). The early identification of such issues is relevant to researchers, policy-makers and the wider public.

Unravelling the γ-butyrolactone network in Streptomyces coelicolor by computational ensemble modelling.

Abstract: Antibiotic production is coordinated in the Streptomyces coelicolor population through the use of diffusible signaling molecules of the γ-butyrolactone (GBL) family. The GBL regulatory system involves a small, and not completely defined two-gene network which governs a potentially bi-stable switch between the "on" and "off" states of antibiotic production. The use of this circuit as a tool for synthetic biology has been hampered by a lack of mechanistic understanding of its functionality. We here present the creation and analysis of a versatile and adaptable ensemble model of the Streptomyces GBL system (detailed information on all model mechanisms and parameters is documented in http://www.systemsbiology.ls.manchester.ac.uk/wiki/index.php/Main_Page). We use the model to explore a range of previously proposed mechanistic hypotheses, including transcriptional interference, antisense RNA interactions between the mRNAs of the two genes, and various alternative regulatory activities. Our results suggest that transcriptional interference alone is not sufficient to explain the system's behavior. Instead, antisense RNA interactions seem to be the system's driving force, combined with an aggressive scbR promoter. The computational model can be used to further challenge and refine our understanding of the system's activity and guide future experimentation.

Multi-Omics Analysis of the Effect of cAMP on Actinorhodin Production in Streptomyces coelicolor.

Abstract: Cyclic adenosine monophosphate (cAMP) has been known to play an important role in regulating morphological development and antibiotic production in Streptomyces coelicolor. However, the functional connection between cAMP levels and antibiotic production and the mechanism by which cAMP regulates antibiotic production remain unclear. In this study, metabolomics- and transcriptomics-based multi-omics analysis was applied to S. coelicolor strains that either produce the secondary metabolite actinorhodin (Act) or lack most secondary metabolite biosynthesis pathways including Act. Comparative multi-omics analysis of the two strains revealed that intracellular and extracellular cAMP abundance was strongly correlated with actinorhodin production. Notably, supplementation of cAMP improved cell growth and antibiotic production. Further multi-omics analysis of cAMP-supplemented S. coelicolor cultures showed an increase of guanine and the expression level of purine metabolism genes. Based on this phenomenon, supplementation with 7-methylguanine, a competitive inhibitor of reactions utilizing guanine, with or without additional cAMP supplementation, was performed. This experiment revealed that the reactions inhibited by 7-methylguanine are mediating the positive effect on growth and antibiotic production, which may occur downstream of cAMP supplementation.

Towards engineering and production of artificial spider silk using tools of synthetic biology

Abstract: Spider silk is one of the strongest biomaterials available in nature. Its mechanical properties make it a good candidate for applications in various fields ranging from protective armour to bandages for wound dressing to coatings for medical implants. Spider silk is formed by an intricate arrangement of spidroins, which are extremely large proteins containing long stretches of repeating segments rich in alanine and glycine. A large amount of research has been directed towards harnessing the spectacular potential of spider silks and using them for different applications. The interdisciplinary approach of synthetic biology is an ideal tool to study these spider silk proteins and work towards the engineering and production of synthetic spider silk. This review aims to highlight the recent progress that has been made in the study of spider silk proteins using different branches of synthetic biology. Here, the authors discuss the different computational approaches, directed evolution techniques and various expression platforms that have been tested for the successful production of spider silk. Future challenges facing the field and possible solutions offered by synthetic biology are also discussed.

The effect of terminal globular domains on the response of recombinant mini-spidroins to fiber spinning triggers

Abstract: Spider silk spidroins consist of long repetitive protein strands, flanked by globular terminal domains. The globular domains are often omitted in recombinant spidroins, but are thought to be essential for the spiders’ natural spinning process. Mimicking this spinning process could be an essential step towards producing strong synthetic spider silk. Here we describe the production of a range of mini-spidroins with both terminal domains, and characterize their response to a number of biomimetic spinning triggers. Our results suggest that the inclusion of the terminal domains is needed to match the response to shear that native spidroins exhibit. Our results also suggest that a pH drop alone is insufficient to trigger assembly in a wet-spinning process, and must be combined with salting-out for effective fiber formation. With these insights, we applied these assembly triggers for relatively biomimetic wet spinning. This work adds to the foundation of literature for developing improved biomimetic spinning techniques, which ought to result in synthetic silk that more closely approximates the unique properties of native spider silk.

South European spiders from the Duffey collection in the Manchester Museum (Arachnida: Araneae)

Abstract: Eric Duffey's spider collection in the Manchester Museum, accumulated over more than 40 years, contains more than 300 samples from a diverse range of biotopes in most southern European countries. Most of this material was previously unsorted. It has now been sorted and identified to species level. It contains more than 2500 specimens of more than 500 species in 240 genera and 42 families. The collection details of all of these samples are listed here, and a number of selected specimens are illustrated.

List of German names for the spiders of Germany (Araneae).

Abstract: Common names play an important role in the efficient communication of scientific results in biology, for instance in the area of applied conservation science, in popular articles and in Citizen Science projects. Common names can increase the readability of texts aimed at the general public and increase the relatability of the described species. For many groups of invertebrates such names are not generally available and often there are no (published) lists of common names, resulting in different names being used for the same species, thus subverting the aims stated above. Here, we propose common German names for all spider genera and species recorded from Germany, and some whose occurrence is expected in this country. Many of these names are coined here for the first time.