Biology subjects

Scrutton, N.

Publications and source records attributed to Scrutton, N..

8 recordsLinked to original sources

bioRxiv2026

Biosensor-guided evolution of chalcone synthase enhances biosynthesis of natural and non-natural flavanones

Flavonoids constitute a large class of natural products widely investigated for their bioactive properties, with microbial production offering a potentially scalable alternative to plant extraction. However, achieving structural diversification of these compounds in microbial systems remains challenging, as modification of the flavonoid B-ring typically relies on downstream tailoring enzymes. An alternative strategy is to exploit the intrinsic promiscuity of the canonical flavanone biosynthesis pathway to introduce structural variation at an early stage. Here, we sought to improve microbial production of diverse flavanones by systematically leveraging pathway promiscuity. By constructing a combinatorial library of pathways comprising 4-coumarate-CoA ligase, chalcone synthase, and chalcone isomerase, we enabled the conversion of a panel of ring-substituted cinnamic acid precursors into ten natural and non-natural flavanones. In parallel, we established a genetically encoded biosensor based on the transcriptional regulator FdeR and demonstrated its responsiveness across all ten flavanones. Leveraging this biosensor for high-throughput screening, we performed directed evolution of chalcone synthases from Hordeum vulgare and Arabidopsis thaliana, identifying enzyme variants that led to improved production of O-methylated flavanones, including isosakuranetin, hesperetin, and homoeriodictyol, as well as fluoro-substituted flavanones. In addition, we demonstrated that specific variants of H. vulgare chalcone synthase promoted the formation of isoferuloyl-derived derailment products. Collectively, this work establishes the FdeR-based biosensor as a versatile platform for pathway and enzyme engineering, enabling efficient early-stage diversification of flavanones in microbial systems and providing insight into the mutational landscape of chalcone synthases.

Synthetic Biology↗

bioRxiv2026

The single nucleotide polymorphism rs1053230 modulates kynurenine 3-monooxygenase stability and is associated with cognitive and mood phenotypes

BackgroundThe single nucleotide polymorphism (SNP) rs1053230 within the kynurenine 3-monooxygenase (KMO) gene encodes either an arginine (CGC) or cysteine (TGC) at amino acid residue 452. The rs1053230 genotype is associated with alterations in KMO expression and activity, and impaired cognition. Additionally, KMO intronic SNP rs2275163 is associated with schizophrenia endophenotypes. However, the direct functional consequences of these SNPs on KMO function have never been investigated. MethodsHere we performed the first in vitro cell-based examination of the rs1053230 genotype on KMO expression, activity, cellular localisation and KMO-protein interactions, as well as examination of the effects of rs1053230 on schizophrenia-relevant clinical measures. We also examined the effects of rs2275163 genotype on KMO pre-mRNA stability and alternative splicing. ResultsHEK293T cells expressing KMO-Arg452 or KMO-Cys452 with a red fluorescent protein (RFP) tag produced equivalent levels of KMO mRNA, protein and enzymatic activity, and localised to mitochondria to the same extent. However, cycloheximide-mediated inhibition of protein translation revealed a striking reduction in protein stability of KMO-Arg452-RFP. KMO-RFP-trap pull-down followed by tandem liquid-chromatography-mass spectrometry (LC-MS/MS) identified dramatic differences in protein partners between KMO variants. Indeed, gene ontology-term enrichment analysis revealed that terms associated with synaptic function were more highly enriched amongst KMO-Cys452 interacting proteins. rs1053230 genotype was found to associate with chronic, trait-like depressive mood symptoms in patients. rs2275163 genotype had no effect on KMO pre-mRNA. ConclusionsDifferences in protein stability and protein-protein interactions may underlie the mechanisms by which the KMO rs1053230 genotype influences neuronal function, leading to cognitive differences in psychiatric conditions.

molecular biology↗

bioRxiv2025

From Discovery to Process Control: Positioning DIA Proteomics in Biomanufacturing Pipelines

Gaining control of existing biomanufacturing chassis organisms, such as Escherichia coli K12, and novel isolates, such as the salt tolerant Halomonas bluephagenesis sp TD01 studied here may be facilitated by the investigation and monitoring of their metabolic and regulatory processes, particularly through proteomics. Here we consider the performance of a range of typically available proteomics platforms across a range of price points to map chassis organisms metabolic pathways. A set of model bacterial samples was prepared from E. coli and H. bluephagenesis sp. TD01 in 1:1, 1:2 and 2:1 ratios and analyzed using five LC-MS systems. Of the 8,222 proteins identified across all samples analysed (4,401 proteins from E. coli; 3,821 from Halomonas sp. TD01), the TimsTOF and Exploris were able to achieve extensive proteome coverage quantifying 5.5k and 5k proteins respectively, with the ZenoTOF, Waters MRT and the legacy Waters Vion respectively quantifying 3.5k, 1.3k, and [~]850 proteins at 1% FDR. Proteins comprising core metabolic pathways critical to biomanufacturing in these chassis organisms can be quantified with all instruments. We characterize metabolic adaptation in H. bluephagenesis by showing that replacement of glucose with a carboxylic acid feed stock directs carbon flux towards potential butane precursors as well as how the acquired data permits monitoring of the cobalamin (vitamin B12) production pathway. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695173v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@148fd14org.highwire.dtl.DTLVardef@df4b7borg.highwire.dtl.DTLVardef@1d479b6org.highwire.dtl.DTLVardef@82a4df_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Structure-based analysis unveils co-origin of LPOR and nitrogenase-like proteins

Structures of nitrogenases, dark-operative protochlorophyllide oxidoreductases, and light-dependent protochlorophyllide oxidoreductases (LPOR) have been resolved. However, their evolutionary relatedness remains elusive. Here, we show, through structural alignment, that all subunits of nitrogenase-like proteins originated from a co-ancestral archaic one-subdomain precursor. LPOR evolved from the BchX/BchY subunits of nitrogenase-like chlorophyllide a oxidoreductase (COR), and the intermediary retinol dehydrogenase through possible genetic recombination. We thus establish previously unknown structural links among key enzymes involved in biological nitrogen-fixation (BNF) and photosynthesis, unraveling structure-guided functional evolution from a single-subunit iron protein to multi-subunit nitrogenase-like COR, and to the single-subunit LPOR for phototrophic metabolism via bacteriochlorophyll, retinal, and chlorophyll. This work also demonstrates structural similarities are imperative for inferring distant origins of functionally divergent proteins, particularly those lacking primary amino-acid sequence identity. Moreover, our findings coupled with AI may be exploited to design innovative light-driven CORs and/or light-utilizing nitrogenases with enhanced efficacy of photosynthesis and BNF. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/634645v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@9f9a2aorg.highwire.dtl.DTLVardef@4243ecorg.highwire.dtl.DTLVardef@796b4corg.highwire.dtl.DTLVardef@17373da_HPS_FORMAT_FIGEXP M_FIG C_FIG Video abstracthttps://www.youtube.com/watch?v=b0qBE1JMaH0 HighlightsO_LILPOR evolves from BchX and BchY subunits of the nitrogenase-like protein (NGLP) COR C_LIO_LIAll subunits of NGLPs can be traced back to the ancestral one-subdomain precursor C_LIO_LIRetinol dehydrogenase acts as a crucial intermediate in the COR to LPOR evolution C_LIO_LIStructure-based design of LUN is showcased to revolutionize protein engineering C_LI

evolutionary biology↗

bioRxiv2023

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

The photoenzyme protochlorophyliide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide (Pchlide) to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight about the light-driven reaction chemistry, a structural rationale for POR photocatalysis has proved more challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the Pchlide substrate and the architecture of the POR active site that have significant implications for the catalytic mechanism of Pchlide reduction. Here, we have used a combination of computational and experimental approaches to investigate the compatibility of each of these structural models with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR-Pchlide-NADPH ternary complex based on these findings. Through detailed site-directed mutagenesis studies we show that a strictly conserved Tyr residue, which has previously been proposed to act as the proton donor in POR photocatalysis, is not likely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site Cys residue is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved Gln residue is found to be important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal binding pose for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.

biochemistry↗

bioRxiv2020

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

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.

bioengineering↗

bioRxiv2020

Design and fabrication of recombinant reflectin-based Bragg reflectors: bio-design engineering and photoisomerism induced wavelength modulation

The remarkable camouflage capabilities of cephalopods have inspired many to develop dynamic optical materials which exploit certain design principles and/or material properties from cephalopod dermal cells. Here, the angle-dependent optical properties of various single-layer reflectin thin-films are characterized within the UV-Vis-NIR regions. Following this, the design and fabrication of the first bio-inspired reflectin-based Bragg reflector is described, which was found to conserve the optical properties of single layer films but exhibit a unique characteristic; reduced angle-dependent reflectivity. Finally, a novel method of controlling reflectin thin-film optical properties is introduced; visible light-induced photoisomerism, representing a new class of reflectin-based optical materials.

synthetic biology↗

bioRxiv2019

Distributed Biomanufacturing of Liquefied Petroleum Gas

Liquefied Petroleum Gas (LPG) is a major domestic and transport fuel. Its combustion lessens NOx, greenhouse gas and particulates emissions compared to other fuels. Propane - the major constituent of LPG - is a clean, high value drop-in fuel that can help governments develop integrated fuels and energy policies with low carbon burden, providing solutions to the multi-faceted challenges of future energy supply. We show that bio-LPG (bio-propane and bio-butane) can be produced by microbial conversion of waste volatile fatty acids that can be derived from anaerobic digestion, industrial waste, or CO2 via photosynthesis. Bio-LPG production was achieved photo-catalytically, using biomass propagated from bioengineered bacteria including E. coli, Halomonas (in non-sterile seawater), and Synechocystis (photosynthetic). These fuel generation routes could be implemented rapidly in advanced and developing nations of the world to meet energy needs, global carbon reduction targets and clean air directives.

bioengineering↗