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

Determinants of Product Outcome in Two Sesquiterpene Synthases from the Thermotolerant Bacterium Rubrobacter radiotolerans

Rubrobacter radiotolerans nerolidol synthase (NerS) and trans-α-bergamotene synthase (BerS) are among the first terpene synthases (TSs) discovered from thermotolerant bacteria, and, despite sharing the same substrate, make terpenoid products with different carbon scaffolds. Here, the potential thermostability of NerS and BerS was investigated, and NerS was found to retain activity up to 55 °C. A library of 22 NerS and BerS variants was designed to probe the differing reaction mechanisms of NerS and BerS, including residues putatively involved in substrate sequestration, cation-π stabilisation of reactive intermediates, and shaping of the active site contour. Two BerS variants showed improved in vivo titres vs the WT enzyme, and also yielded different ratios of the related sesquiterpenoids (E)-β-farnesene and trans-α-bergamotene. BerS-L86F was proposed to encourage substrate isomerisation by cation-π stabilisation of the first cationic intermediate, resulting in a greater proportion of trans-α-bergamotene. By contrast, BerS-S82L significantly preferred (E)-β-farnesene formation, attributed to steric blocking of the isomerisation step, consistent with what has been observed in several plant TSs. Our work highlights the importance of isomerisation as a key determinant of product outcome in TSs, and shows how a combined computational and experimental approach can characterise TSs and variants with improved and altered functionality

Laser-flash photolysis indicates that internal electron transfer is triggered by proton uptake by Alcaligenes xylosoxidans copper-dependent nitrite reductase

Enzyme-catalysed electron transfer reactions are often controlled by protein motions and coupled to chemical change such as proton transfer. We have investigated the nature of this control in the blue copper-dependent nitrite reductase from Alcaligenes xylosoxidans (AxNiR). Inter-Cu electron transfer from the T1Cu site to the T2Cu catalytic site in AxNiR occurs via a proton-coupled electron transfer mechanism. Here we have studied the kinetics of both electron and proton transfer independently using laser-flash photolysis for native AxNiR and its proton-channel mutant N90S. In native AxNiR, both inter-Cu electron transfer and proton transfer exhibit similar rates, and show an unusual dependence on the nitrite concentration. An initial decrease in the observed rates at low nitrite concentrations is followed by an increase in the observed rates at high nitrite concentrations (> 5 mm). In N90S, in which the T1Cu reduction potential is elevated by 60 mV, no inter-Cu electron transfer or proton transfer was observed in the absence of nitrite. Only in the presence of nitrite were both processes detected, with similar [nitrite] dependence, but the nitrite dependence was different compared with native enzyme. The substrate dependence in N90S was similar to that observed in steady-state assays, suggesting that this substitution resulted in proton-coupled electron transfer becoming rate-limiting. A pH perturbation experiment with native AxNiR revealed that protonation triggers inter-Cu electron transfer and generation of NO. Our results show a strong coupling of inter-Cu electron transfer and proton transfer for both native AxNiR and N90S, and provide novel insights into the controlled delivery of electrons and protons to the substrate-utilization T2Cu active site of AxNiR. © 2012 FEBS

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