The combination of stable isotope labelling with direct infusion ion mobility mass spectrometry (IM-MS) enabled qualitative and quantitative monitoring of biocatalytic reactions with reduced analysis ...times, enhanced sensitivity and μL-level assay volumes. The new approach was demonstrated by applying to both lipase and monooxygenase enzymes, including multi-substrate screening.
Flavones and flavonols are important classes of flavonoids with nutraceutical and pharmacological value, and their production by fermentation with recombinant microorganisms promises to be a scalable ...and economically favorable alternative to extraction from plant sources. Flavones and flavonols have been produced recombinantly in a number of microorganisms, with
typically being a preferred production host for these compounds due to higher yields and titers of precursor compounds, as well as generally improved ability to functionally express cytochrome P450 enzymes without requiring modification to improve their solubility. Recently, a rapid prototyping platform has been developed for high-value compounds in
, and a number of gatekeeper (2
)-flavanones, from which flavones and flavonols can be derived, have been produced to high titers in
using this platform. In this study, we extended these metabolic pathways using the previously reported platform to produce apigenin, chrysin, luteolin and kaempferol from the gatekeeper flavonoids naringenin, pinocembrin and eriodictyol by the expression of either type-I flavone synthases (FNS-I) or type-II flavone synthases (FNS-II) for flavone biosynthesis, and by the expression of flavanone 3-dioxygenases (F3H) and flavonol synthases (FLS) for the production of the flavonol kaempferol. In our best-performing strains, titers of apigenin and kaempferol reached 128 mg L
and 151 mg L
in 96-DeepWell plates in cultures supplemented with an additional 3 mM tyrosine, though titers for chrysin (6.8 mg L
) from phenylalanine, and luteolin (5.0 mg L
) from caffeic acid were considerably lower. In strains with upregulated tyrosine production, apigenin and kaempferol titers reached 80.2 mg L
and 42.4 mg L
respectively, without the further supplementation of tyrosine beyond the amount present in the rich medium. Notably, the highest apigenin, chrysin and luteolin titers were achieved with FNS-II enzymes, suggesting that cytochrome P450s can show competitive performance compared with non-cytochrome P450 enzymes in prokaryotes for the production of flavones.
Synthetic biology utilizes the Design-Build-Test-Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA ...assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from
colonies to analyzed data). Given our procedure's low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.
The exploration and identification of new brassinosteroid (BR) compounds is critical to improve the biosynthetic research of BRs and expand the chemodiversity of active BRs. However, traditional ...methods are labor-intensive, time-consuming, and less sensitive. Here, we present a facile screening strategy for discovering and identifying novel BRs from plant tissues based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). A total of 14 potential BRs were discovered from only 1 g of rice tissues and structurally elucidated by following a MS-based clue, acquired through multiple reaction monitoring (MRM) data-dependent enhanced product ion (EPI) scan, high resolution MS, and MS survey-dependent MS/MS. One of the 14 candidates was identified as 6-deoxo-28-homotyphasterol, a brand new BR compound that is reported for the first time in the BRs biosynthesis pathway. Detailed comparison with reference standards and quantitative level analysis in rice BR mutants confirmed the availability of the other candidates. This effective, yet simple method provides an efficient way to find more and more chemically new BR biosynthetic intermediates in plants, which is significant for complementing the biosynthesis and metabolism network of BRs. This strategy may also be used to discover unknown compounds of other plant hormone species as well as their key metabolites.
•Five acidic phytohormones were directly separated by differential mobility spectrometry–mass spectrometry.•The field mobilities dependence was measured as the normalized α function.•Five acidic ...phytohormones behaved as type A ions.•The clustering separation mechanism dominated.
The direct separation of five acidic phytohormones including auxin (indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid), jasmonic acid, and abscisic acid by differential mobility spectrometry–mass spectrometry (DMS–MS) with the use of the polar gas-phase chemical modifier 2-propanol was demonstrated. The method was rapid and simple to operate with selectivity and repeatability. The influence of experimental conditions on the separation, such as modifier types and concentration, separation voltage, and temperature, was systematically investigated. The field mobilities dependence was measured as the normalized α function using the developed DMS–MS method. All the analytes behaved as type A ions and the clustering separation mechanism dominated. These results not only gave insights of ion properties but also contributed to the prediction of DMS separation by choosing targeted and appropriate conditions for the study of acidic phytohormones.
The effect of Liuwei Dihuang decoction (LW), a traditional Chinese medicine (TCM) prescription, on voltage-dependent currents and synaptic transmission were investigated in cultured hippocampal ...neurons of rat by whole-cell patch clamp recording technique. After application with serum from LW-treated rats, termed LW-containing serum (LWCS) for 48
h, the amplitude of delay rectifying K
+ current (
I
K) and voltage-gated Ca
2+ current (
I
Ca) decreased. While the frequency of spontaneous excitatory post-synaptic current (sEPSC) and miniature excitatory post-synaptic current (mEPSC) increased significantly. Yet the amplitude of voltage-depended Na
+ current (
I
Na) and transient outward K
+ current (
I
A), membrane capacitance and resistance remained unchanged. The results indicated that LWCS possessed the effect of modulating or improving neuronal and synaptic function, which possibly contribute to the cognition enhancing effect of LW.
High-resolution Sustained off resonance irradiation (SORI) CID was employed to distinguish four pairs of isomeric diglycosyl flavonoids in the negative mode using the electrospray ionization Fourier ...transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). All of these isomers can be distinguished via MS/MS data. For these diglycosyl flavones and flavanones, the deprotonated α1→6 linkage diglycosyl flavonoids produce fewer fragments than the α1→2 linkage type compounds and the Retro-Diels-Alder (RDA) reaction in MS/MS only takes place when the aglycone is a flavanone and glycosylated with an α1→2 intersaccharide linkage disaccharide. The deprotonation sites after collisional activation are discussed according to the high mass accuracy and high-resolution data of tandem spectrometry. Some of these high-resolution SORI CID product ions from α1→2 linkage diglycosyl flavonoids involve multibond cleavages; the possible mechanism is discussed based on the computer modeling using Gaussian 03 program package at the B3LYP/6-31G level of theory. Unambiguous elementary composition data provides fragmentation information that has not been reported previously.
The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly ...contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, L-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.
Based on the dual role of specific boronate affinity, making use of both novel self-synthesized boronate affinity-functionalized magnetic nanoparticles and a high-efficiency organic boronic acid-type ...derivatization reagent, we report a simple, convenient and highly-sensitive method for detection of endogenous brassinosteroids from real plant materials.
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