13 research outputs found
Isopentenol utilization pathway for the production of linalool in Escherichia coli using an improved bacterial linalool/nerolidol synthase
Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches
From Wiley via Jisc Publications RouterHistory: received 2021-09-14, rev-recd 2021-10-15, pub-electronic 2021-11-03Article version: VoRPublication status: PublishedFunder: Future Biomanufacturing Research Hub; Grant(s): EP/S01778X/1Funder: Engineering and Physical Sciences Research Council (EPSRC)Funder: Biotechnology and Biological Sciences Research Council (BBSRC)Funder: UK Research and Innovation; Id: http://dx.doi.org/10.13039/100014013Abstract: Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence‐function relationship within the TS family, making product‐outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high‐throughput screening, and machine‐learning that will allow more predictive engineering of these fascinating enzymes in the near future
Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase
From Wiley via Jisc Publications RouterHistory: received 2021-03-10, rev-recd 2021-05-02, pub-electronic 2021-05-25Article version: VoRPublication status: PublishedFunder: Future Biomanufacturing Research Hub; Grant(s): EP/S01778X/1Funder: Engineering and Physical Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000266Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268Funder: Office of Naval Research Global; Id: http://dx.doi.org/10.13039/100007297Abstract: Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non‐canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes
Structural basis of catalysis in the bacterial monoterpene synthases linalool synthase and 1,8-cineole synthase
Terpenoids form the
largest and stereochemically most diverse class
of natural products, and there is considerable interest in producing
these by biocatalysis with whole cells or purified enzymes, and by
metabolic engineering. The monoterpenes are an important class of
terpenes and are industrially important as flavors and fragrances.
We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool
synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that
these are active biocatalysts for monoterpene production using biocatalysis
and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher
linalool production compared with the corresponding plant monoterpene
synthase. With bCinS, 1,8-cineole is produced with 96% purity compared
to 67% from plant species. Structures of bLinS and bCinS, and their
complexes with fluorinated substrate analogues, show that these bacterial
monoterpene synthases are similar to previously characterized sesquiterpene
synthases. Molecular dynamics simulations suggest that these monoterpene
synthases do not undergo large-scale conformational changes during
the reaction cycle, making them attractive targets for structured-based
protein engineering to expand the catalytic scope of these enzymes
toward alternative monoterpene scaffolds. Comparison of the bLinS
and bCinS structures indicates how their active sites steer reactive
carbocation intermediates to the desired acyclic linalool (bLinS)
or bicyclic 1,8-cineole (bCinS) products. The work reported here provides
the analysis of structures for this important class of monoterpene
synthase. This should now guide exploitation of the bacterial enzymes
as gateway biocatalysts for the production of other monoterpenes and
monoterpenoids
Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures
There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30°C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30°C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of ∼1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30°C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183
A Lifetime of Native American Architecture: Building Towards the Indigenous Millennium
Carbohydrate oxidases are found in all kingdoms of life but are mostly found in fungi. Their natural role is not always clear. Usage of molecular oxygen as electron acceptor is not a logical choice when the enzyme is part of a catabolic pathway. This chapter provides an overview of the occurrence and properties of carbohydrate oxidases. The physiological role of the different enzymes is discussed in relation to their origin, and the catalytic and structural properties are discussed in relation to their family background. It also provides a summary of the biocatalytic applications of carbohydrate oxidases. Carbohydrate oxidases are valuable enzymes for several applications. They are relatively stable and do not need expensive coenzymes. Carbohydrate oxidases are widely used in diagnostic applications, in the food and drinks industry, and for carbohydrate synthesis. They are also used for bleaching (production of H2O2) and as oxygen scavenger
Strålande tider, härliga tider? Konsumtion av pilsnerfilmer och äldre svenska komiska filmer i en nutida kontext
In Sweden there has been a surge of interest in the films classified as pilsnerfilms, i.e. comical
films produced for little money in the 1930's and containing a more working class and/or rural
setting then the so called "champagne comedies" from the same era. The concept
"pilsnerfilm" is how ever a little vague and although the main focus of this paper is
pilsnerfilms, it will also cover other types of comical films made in Sweden between the
1930's and 1940's.
This paper examines the now-existing network of people interested in these types of movies.
It will do so by interviewing seven of the most prominent fans of movies made during the
1930's and 1940's. Most of them are living in Stockholm. The paper discuss how through the
consumption of these types of films, a nostalgic gaze is created. With a nostalgic gaze the
enthusiasts for pilsnerfilms can in the present create a history of a bygone Sweden and they
can also try to establish a form of contact with the actors in the movies from the 1930's and
1940's. The contact can be by collecting the movies, but more prominently the contact is even
more grounded in a physical world, by having contact with still living actors who were active
during that time or the now deceased actors relatives or by visiting the departed actors grave.
The paper also discuss questions regarding cultural heritage and how the network thinks about
the treatment the pilsnerfilms and other comical films from 1930's and 1940's have received
from the owner's of the movies. Particularly the main company the owns the large majority of
Swedish films; SF. It evokes questions regarding how to present movies as cultural heritage
and which people should have to power to do that.
The aspects brings the question of knowledge and feelings to mind. The network of
enthusiasts can be viewed as a affective alliance, meaning it exists a emotional connection
between cultural expression and individuals. It is the networks affective alliance that the
connoisseur can organize it's knowledge. Emotions and knowledge can't be separated. To both
have the right knowledge and the right emotions is crucial for the network. And through the
networks emotions and knowledge, the fragments that create the story of pilsnerfilm is put
together
Exploring novel bacterial terpene synthases.
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
How a 10-epi-cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity
[Image: see text] Terpenes are the largest class of natural products and are attractive targets in the fuel, fragrance, pharmaceutical, and flavor industries. Harvesting terpenes from natural sources is environmentally intensive and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity among TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid 10-epi-cubebol from farnesyl pyrophosphate (FPP) requires many steps and some of Nature’s most difficult chemistry. 10-epi-Cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching and ensuring correct stereochemistry at every stage. The cyclizations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here, we present the high-resolution crystal structure of ScCubS in complex with both a trinuclear magnesium cluster and pyrophosphate. Computational modeling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10-epi-cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations and whether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we reengineered ScCubS so that its main product was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasizes that mechanistic understanding of cation stabilization in TSs can be used to guide catalytic outcomes
Energy landscapes and catalysis in nitric-oxide synthase
Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape