The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize.

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA.

Integration of targeted metabolome and transcript profiling of Pseudomonas syringae-triggered changes in defence-related phytochemicals in oat plants.

MAIN CONCLUSION: A gene-to-metabolite approach afforded new insights regarding defence mechanisms in oat plants that can be incorporated into plant breeding programmes for the selection of markers and genes related to disease resistance. Monitoring metabolite levels and changes therein can complement and corroborate transcriptome (mRNA) data on plant-pathogen interactions, thus revealing mechanisms involved in pathogen attack and host defence. A multi-omics approach thus adds new layers of information such as identifying metabolites with antimicrobial properties, elucidating metabolomic profiles of infected and non-infected plants, and reveals pathogenic requirements for infection and colonisation. In this study, two oat cultivars (Dunnart and SWK001) were inoculated with Pseudomonas syringae pathovars, pathogenic and non-pathogenic on oat. Following inoculation, metabolites were extracted with methanol from leaf tissues at 2, 4 and 6 days post-infection and analysed by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer system. Relatedly, mRNA was isolated at the same time points, and the cDNA analysed by quantitative PCR (RT-qPCR) for expression levels of selected gene transcripts associated with avenanthramide (Avn) biosynthesis. The targeted amino acids, hydroxycinnamic acids and Avns were successfully quantified. Distinct cultivar-specific differences in the metabolite responses were observed in response to pathogenic and non-pathogenic strains. Trends in aromatic amino acids and hydroxycinnamic acids seem to indicate stronger activation and flux through these pathways in Dunnart as compared to SWK001. A positive correlation between hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) gene expression and the abundance of Avn A in both cultivars was documented. However, transcript profiling of selected genes involved in Avn synthesis did not reveal a clear pattern to distinguish between the tolerant and susceptible cultivars.

Transcriptional heterogeneity of catabolic genes on the plasmid pCAR1 causes host-specific carbazole degradation.

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.IMPORTANCEHorizontally acquired genes increase the competitiveness of host bacteria under selective conditions, although unregulated expression of foreign genes may impose fitness costs. The "appropriate" host for a plasmid is empirically known to maximize the expression of plasmid-borne traits. In the case of pCAR1-harboring Pseudomonas strains, P. resinovorans CA10 exhibits strong carbazole-degrading capacity, whereas P. putida KT2440 harboring pCAR1 exhibits low degradation capacity. Our results suggest that a chromosomally encoded transcription factor affects transcriptional and metabolic fluctuations in host cells, resulting in different carbazole-degrading capacities as a population. This study may provide a clue for determining appropriate hosts for a plasmid and for regulating the expression of plasmid-borne traits, such as the degradation of xenobiotics and antibiotic resistance.

Role of Enterococcus mundtii in gut of the tomato leaf miner (Tuta absoluta) to detoxification of Chlorantraniliprole.

Chlorantraniliprole (CAP) is applied worldwide for the control of caterpillars (Lepidoptera). However, with the overuse of CAP, the resistance problem in pest control is becoming increasingly serious. Recent studies have indicated a central role of the gut symbiont in insect pest resistance to pesticides and these may apply to the tomato leaf miner Tuta absoluta, is one of the most destructive insects worldwide. Here, we successfully isolated seven strains of tolerant CAP bacterium from the CAP-resistant T. absoluta gut, of which Enterococcus mundtii E14 showed the highest CAP tolerance, with a minimum inhibitory concentration (MIC) of 1.6 g/L and CAP degradation rate of 42.4%. Through transcriptomics and metabolism analysis, we studied the detoxification process of CAP by the E. mundtii E14, and found that CAP can be degraded by E. mundtii E14 into non-toxic compounds, such as 3,4-dihydroxy-2-(5-hydroxy-3,7-dimethylocta-2,6-dien-1-yl) benzoic acid and 2-pyridylacetic acid. Additionally, 2-pyridylacetic acid was detected both intracellular and extracellular in E. mundtii E14 treated with CAP. Meanwhile, we identified 52 up-regulated genes, including those associated with CAP degradation, such as RS11670 and RS19130. Transcriptome results annotated using KEGG indicated significant enrichment in up-regulated genes related to the glyoxylate cycle, nitrogen metabolism, and biosynthesis of secondary metabolites. Additionally, we observed that reinfection with E. mundtii E14 may effectively enhance resistance of T. absoluta to CAP. The LC50 values of the antibiotic treatment population of T. absoluta reinfection with E. mundtii E14 is 0.6122 mg/L, which was 18.27 folds higher than before reinfection. These findings offer new insights into T. absoluta resistance to CAP and contribute to a better understanding of the relationship between insecticide resistance and gut symbionts of T. absoluta, which may play a pivotal role in pest management.

Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616.

Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real-time quantitative polymerase chain reaction (RT-qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N-(5'-phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid. This accumulation could in turn result in down-regulation of downstream tryptophan biosynthesis genes. Anthranilic acid is typically produced by chemical synthesis and has been used as a substrate for synthesising bioactive compounds including commercial drugs; our results could provide a new biological platform for production of this compound.

Dynamic flux regulation for high-titer anthranilate production by plasmid-free, conditionally-auxotrophic strains of Pseudomonas putida.

Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and pharmaceuticals. Traditional strategies adopted for microbial anthranilate production rely on the implementation of auxotrophic strains-which requires aromatic amino acids or complex additives to be supplemented in the culture medium, negatively impacting production costs. In this work, we engineered the soil bacterium Pseudomonas putida for high-titer, glucose-dependent anthranilate production by repurposing elements of the Esa quorum sensing (QS) system of Pantoea stewartii. The PesaS promoter mediated a self-regulated transcriptional response that effectively knocked-down the expression of the trpDC genes. Next, we harnessed the synthetic QS elements to engineer a growth-to-anthranilate production switch. The resulting plasmid-free P. putida strain produced the target compound at 3.8 ± 0.3 mM in shaken-flask cultures after 72 h-a titer >2-fold higher than anthranilate levels reported thus far. Our results highlight the value of dynamic flux regulation for the production of intermediate metabolites within highly-regulated routes (such as the shikimate pathway), thereby circumventing the need of expensive additives.

Structural basis for diamide modulation of ryanodine receptor.

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

Microbial production of methyl anthranilate, a grape flavor compound.

Methyl anthranilate (MANT) is a widely used compound to give grape scent and flavor, but is currently produced by petroleum-based processes. Here, we report the direct fermentative production of MANT from glucose by metabolically engineered Escherichia coli and Corynebacterium glutamicum strains harboring a synthetic plant-derived metabolic pathway. Optimizing the key enzyme anthranilic acid (ANT) methyltransferase1 (AAMT1) expression, increasing the direct precursor ANT supply, and enhancing the intracellular availability and salvage of the cofactor S-adenosyl-l-methionine required by AAMT1, results in improved MANT production in both engineered microorganisms. Furthermore, in situ two-phase extractive fermentation using tributyrin as an extractant is developed to overcome MANT toxicity. Fed-batch cultures of the final engineered E. coli and C. glutamicum strains in two-phase cultivation mode led to the production of 4.47 and 5.74 g/L MANT, respectively, in minimal media containing glucose. The metabolic engineering strategies developed here will be useful for the production of volatile aromatic esters including MANT.

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Combinatorial Assembly of Modular Glucosides via Carboxylesterases Regulates C. elegans Starvation Survival.

The recently discovered modular glucosides (MOGLs) form a large metabolite library derived from combinatorial assembly of moieties from amino acid, neurotransmitter, and lipid metabolism in the model organism C. elegans. Combining CRISPR-Cas9 genome editing, comparative metabolomics, and synthesis, we show that the carboxylesterase homologue Cel-CEST-1.2 is responsible for specific 2-O-acylation of diverse glucose scaffolds with a wide variety of building blocks, resulting in more than 150 different MOGLs. We further show that this biosynthetic role is conserved for the closest homologue of Cel-CEST-1.2 in the related nematode species C. briggsae, Cbr-CEST-2. Expression of Cel-cest-1.2 and MOGL biosynthesis are strongly induced by starvation conditions in C. elegans, one of the premier model systems for mechanisms connecting nutrition and physiology. Cel-cest-1.2-deletion results in early death of adult animals under starvation conditions, providing first insights into the biological functions of MOGLs.

Coordinated regulation of anthranilate metabolism and bacterial virulence by the GntR family regulator MpaR in Pseudomonas aeruginosa.

The GntR family regulators are widely distributed in bacteria and play critical roles in metabolic processes and bacterial pathogenicity. In this study, we describe a GntR family protein encoded by PA4132 that we named MpaR (MvfR-mediated PQS and anthranilate regulator) for its regulation of Pseudomonas quinolone signal (PQS) production and anthranilate metabolism in Pseudomonas aeruginosa. The deletion of mpaR increased biofilm formation and reduced pyocyanin production. RNA sequencing analysis revealed that the mRNA levels of antABC encoding enzymes for the synthesis of catechol from anthranilate, a precursor of the PQS, were most affected by mpaR deletion. Data showed that MpaR directly activates the expression of mvfR, a master regulator of pqs system, and subsequently promotes PQS production. Accordingly, deletion of mpaR activates the expression of antABC genes, and thus, increases catechol production. We also demonstrated that MpaR represses the rhl quorum-sensing (QS) system, which has been shown to control antABC activity. These results suggested that MpaR function is integrated into the QS regulatory network. Moreover, mutation of mpaR promotes bacterial survival in a mouse model of acute pneumonia infection. Collectively, this study identified a novel regulator of pqs system, which coordinately controls anthranilate metabolism and bacterial virulence in P. aeruginosa.

Avenanthramide Metabotype from Whole-Grain Oat Intake is Influenced by Faecalibacterium prausnitzii in Healthy Adults.

BACKGROUND: Oat has been widely accepted as a key food for human health. It is becoming increasingly evident that individual differences in metabolism determine how different individuals benefit from diet. Both host genetics and the gut microbiota play important roles on the metabolism and function of dietary compounds. OBJECTIVES: To investigate the mechanism of individual variations in response to whole-grain (WG) oat intake. METHODS: We used the combination of in vitro incubation assays with human gut microbiota, mouse and human S9 fractions, chemical analyses, germ-free (GF) mice, 16S rRNA sequencing, gnotobiotic techniques, and a human feeding study. RESULTS: Avenanthramides (AVAs), the signature bioactive polyphenols of WG oat, were not metabolized into their dihydro forms, dihydro-AVAs (DH-AVAs), by both human and mouse S9 fractions. DH-AVAs were detected in the colon and the distal regions but not in the proximal and middle regions of the perfused mouse intestine, and were in specific pathogen-free (SPF) mice but not in GF mice. A kinetic study of humans fed oat bran showed that DH-AVAs reached their maximal concentrations at much later time points than their corresponding AVAs (10.0-15.0 hours vs. 4.0-4.5 hours, respectively). We observed interindividual variations in the metabolism of AVAs to DH-AVAs in humans. Faecalibacterium prausnitzii was identified as the individual bacterium to metabolize AVAs to DH-AVAs by 16S rRNA sequencing analysis. Moreover, as opposed to GF mice, F. prausnitzii-monocolonized mice were able to metabolize AVAs to DH-AVAs. CONCLUSIONS: These findings demonstrate that the presence of intestinal F. prausnitzii is indispensable for proper metabolism of AVAs in both humans and mice. We propose that the abundance of F. prausnitzii can be used to subcategorize individuals into AVA metabolizers and nonmetabolizers after WG oat intake. This study was registered at clinicaltrials.gov as NCT04335435.

The role of anthranilic acid in the increase of depressive symptoms and major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a and oral ribavirin.

Background: Tryptophan metabolism via the kynurenine pathway is considered the link between the immune and endocrine systems. Dysregulation of serotonergic transmission can stem from the direct influence of interferon-α on the activity of serotonergic receptors 5-HT1A and 5-HT2A, and from its indirect effect on tryptophan metabolism. Induction of the kynurenine pathway increases the concentration of neurotoxic kynurenine metabolites, and the activity of kynurenine derivatives is linked to the onset of depression. The aim of our study was to evaluate the relationships between depressive symptoms and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, indolamine 2,3-dioxygenase (IDO) activity and tryptophan availability to the brain. Methods: The study followed a prospective longitudinal cohort design. We evaluated 101 patients with chronic hepatitis C who were treated with pegylated interferon-α2a, and 40 controls who were awaiting treatment. We evaluated the relationships between total score on the Montgomery-Åsberg Depression Rating Scale and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, IDO activity and tryptophan availability to the brain. A logistic regression model was adapted for the diagnosis of major depressive disorder at each time point, taking into account changes in parameters of the kynurenine pathway between a given time point and the baseline measurement. Results: Of the treated patients, 44% fulfilled the criteria for major depressive disorder at least once during the 24 weeks of treatment. Anthranilic acid concentrations were significantly increased compared to baseline for all time points except week 2. Tryptophan availability showed a significant decrease (ß = -0.09, p = 0.01) only in week 12 of treatment. Over time, kynurenine, tryptophan and anthranilic acid concentrations, as well as IDO activity and tryptophan availability to the brain, were significantly associated with total score on the Montgomery-Åsberg Depression Rating Scale. A logistic regression model revealed that participants with decreased tryptophan availability to the brain at 12 weeks of treatment and participants with increased anthranilic acid concentrations at week 24 of treatment were at increased risk for diagnosis of major depressive disorder (odds ratios 2.92 and 3.59, respectively). Limitations: This study had an open-label design in a population receiving naturalistic treatment. Conclusion: The present study provides the first direct evidence of the role of anthranilic acid in the pathogenesis of inflammation-induced major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a.

Engineering of Saccharomyces cerevisiae for anthranilate and methyl anthranilate production.

BACKGROUND: Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products. Microorganisms produce anthranilate as an intermediate in the tryptophan biosynthetic pathway. Several prokaryotic microorganisms have been engineered to overproduce anthranilate but attempts to engineer eukaryotic microorganisms for anthranilate production are scarce. RESULTS: We subjected Saccharomyces cerevisiae, a widely used eukaryotic production host organism, to metabolic engineering for anthranilate production. A single gene knockout was sufficient to trigger anthranilate accumulation both in minimal and SCD media and the titer could be further improved by subsequent genomic alterations. The effects of the modifications on anthranilate production depended heavily on the growth medium used. By growing an engineered strain in SCD medium an anthranilate titer of 567.9 mg l-1 was obtained, which is the highest reported with an eukaryotic microorganism. Furthermore, the anthranilate biosynthetic pathway was extended by expression of anthranilic acid methyltransferase 1 from Medicago truncatula. When cultivated in YPD medium, this pathway extension enabled production of the grape flavor compound methyl anthranilate in S. cerevisiae at 414 mg l-1. CONCLUSIONS: In this study we have engineered metabolism of S. cerevisiae for improved anthranilate production. The resulting strains may serve as a basis for development of efficient production host organisms for anthranilate-derived compounds. In order to demonstrate suitability of the engineered S. cerevisiae strains for production of such compounds, we successfully extended the anthranilate biosynthesis pathway to synthesis of methyl anthranilate.

The ATPase mechanism of UvrA2 reveals the distinct roles of proximal and distal ATPase sites in nucleotide excision repair.

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.

Synergistic Effect of Methyl Jasmonate and Abscisic Acid Co-Treatment on Avenanthramide Production in Germinating Oats.

The oat (Avena sativa L.) is a grain of the Poaceae grass family and contains many powerful anti-oxidants, including avenanthramides as phenolic alkaloids with anti-inflammatory, anti-oxidant, anti-itch, anti-irritant, and anti-atherogenic activities. Here, the treatment of germinating oats with methyl jasmonate (MeJA) or abscisic acid (ABA) resulted in 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increase in avenanthramide content, respectively, relative to untreated controls (232.6 mg/kg FW). Moreover, MeJA and ABA co-treatment synergistically increased avenanthramide production in germinating oats to 1505 mg/kg FW. Individual or combined MeJA and ABA treatment increased the expression of genes encoding key catalytic enzymes in the avenanthramide-biosynthesis pathway, including hydroxycinnamoyl-CoA:hydrocyanthranilate N-hydroxycinnamoyl transferase (HHT). Further analyses showed that six AsHHT genes were effectively upregulated by MeJA or ABA treatment, especially AsHHT4 for MeJA and AsHHT5 for ABA, thereby enhancing the production of all three avenanthramides in germinating oats. Specifically, AsHHT5 exhibited the highest expression following MeJA and ABA co-treatment, indicating that AsHHT5 played a more crucial role in avenanthramide biosynthesis in response to MeJA and ABA co-treatment of germinating oats. These findings suggest that elicitor-mediated metabolite farming using MeJA and ABA could be a valuable method for avenanthramide production in germinating oats.

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

The MYB transcription factor Emission of Methyl Anthranilate 1 stimulates emission of methyl anthranilate from Medicago truncatula hairy roots.

Plants respond to herbivore or pathogen attacks by activating specific defense programs that include the production of bioactive specialized metabolites to eliminate or deter the attackers. Volatiles play an important role in the interaction of a plant with its environment. Through transcript profiling of jasmonate-elicited Medicago truncatula cells, we identified Emission of Methyl Anthranilate (EMA) 1, a MYB transcription factor that is involved in the emission of the volatile compound methyl anthranilate when expressed in M. truncatula hairy roots, giving them a fruity scent. RNA sequencing (RNA-Seq) analysis of the fragrant roots revealed the upregulation of a methyltransferase that was subsequently characterized to catalyze the O-methylation of anthranilic acid and was hence named M. truncatula anthranilic acid methyl transferase (MtAAMT) 1. Given that direct activation of the MtAAMT1 promoter by EMA1 could not be unambiguously demonstrated, we further probed the RNA-Seq data and identified the repressor protein M. truncatula plant AT-rich sequence and zinc-binding (MtPLATZ) 1. Emission of Methyl Anthranilate 1 binds a tandem repeat of the ACCTAAC motif in the MtPLATZ1 promoter to transactivate gene expression. Overexpression of MtPLATZ1 in transgenic M. truncatula hairy roots led to transcriptional silencing of EMA1, indicating that MtPLATZ1 may be part of a negative feedback loop to control the expression of EMA1. Finally, application of exogenous methyl anthranilate boosted EMA1 and MtAAMT1 expression dramatically, thus also revealing a positive amplification loop. Such positive and negative feedback loops seem to be the norm rather than the exception in the regulation of plant specialized metabolism.

Adaptive laboratory evolution of Pseudomonas putida and Corynebacterium glutamicum to enhance anthranilate tolerance.

Microbial bioproduction of the aromatic acid anthranilate (ortho-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition. The microorganisms Corynebacterium glutamicum and Pseudomonas putida are known for high tolerance towards a variety of chemicals and could serve as promising platform strains. In this study, the resistance of both wild-type strains towards anthranilate was assessed. To further enhance their native tolerance, adaptive laboratory evolution (ALE) was applied. Sequential batch fermentation processes were developed, adapted to the cultivation demands for C. glutamicum and P. putida, to enable long-term cultivation in the presence of anthranilate. Isolation and analysis of single mutants revealed phenotypes with improved growth behaviour in the presence of anthranilate for both strains. The characterization and improvement of both potential hosts provide an important basis for further process optimization and will aid the establishment of an industrially competitive method for microbial synthesis of anthranilate.