Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering.

Gastrodin, 4-hydroxybenzyl alcohol-4-O-ß-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica.

The Peroxisomal ß-Oxidative Pathway and Benzyl Alcohol O-Benzoyltransferase HSR201 Cooperatively Contribute to the Biosynthesis of Salicylic Acid.

The phytohormone salicylic acid (SA) regulates plant defense responses against pathogens. Previous studies have suggested that SA is mainly produced from trans-cinnamic acid (CA) in tobacco, but the underlying mechanisms remain largely unknown. SA synthesis is activated by wounding in tobacco plants in which the expression of WIPK and SIPK, two stress-related mitogen-activated protein kinases, is suppressed. Using this phenomenon, we previously revealed that HSR201 encoding benzyl alcohol O-benzoyltransferase is required for pathogen signal-induced SA synthesis. In this study, we further analyzed the transcriptomes of wounded WIPK-/SIPK-suppressed plants and found that the expression of NtCNL, NtCHD and NtKAT1, homologous to cinnamate-coenzyme A (CoA) ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD) and 3-ketoacyl-CoA thiolase (KAT), respectively, is associated with SA biosynthesis. CNL, CHD and KAT constitute a ß-oxidative pathway in the peroxisomes and produce benzoyl-CoA, a precursor of benzenoid compounds in petunia flowers. Subcellular localization analysis showed that NtCNL, NtCHD and NtKAT1 localize in the peroxisomes. Recombinant NtCNL catalyzed the formation of CoA esters of CA, whereas recombinant NtCHD and NtKAT1 proteins converted cinnamoyl-CoA to benzoyl-CoA, a substrate of HSR201. Virus-induced gene silencing of any one of NtCNL, NtCHD and NtKAT1 homologs compromised SA accumulation induced by a pathogen-derived elicitor in Nicotiana benthamiana leaves. Transient overexpression of NtCNL in N. benthamiana leaves resulted in SA accumulation, which was enhanced by co-expression of HSR201, although overexpression of HSR201 alone did not cause SA accumulation. These results suggested that the peroxisomal ß-oxidative pathway and HSR201 cooperatively contribute to SA biosynthesis in tobacco and N. benthamiana.

The Effect of Scale Insects on Growth Parameters of cv. Chardonnay and cv. Sauvignon Blanc Grapevines Grown in a Greenhouse.

Plants can respond to insects that feed with stylet mouthparts using various processes that are initiated via the salicylic acid metabolic pathway. In Australia, scale insects of the genus Parthenolecanium can cause economic damage to grapevines as they feed on the vines and produce honeydew as a waste by-product, which supports the growth of black sooty mould on fruit and leaves, potentially affecting the plant growth and yield. Using rootlings of Sauvignon Blanc (SB, resistant) and Chardonnay (Char, susceptible), the growth and production of volatile organic compounds (VOCs) following exposure to scale insect infestations were measured under controlled greenhouse conditions. At harvest, the numbers of scale insects per five leaves were higher on plants infested at the start of the study compared with the control plants. Infested SB had increased dry root and shoot mass compared with the SB control, which was also the case with Char (control and infested). Leaf volatiles differed between cultivars in response to scale infestation. Benzyl alcohol decreased among infested SB plants compared with the other treatments. A change in the salicylic acid pathway as indicated by the change in benzyl alcohol may cause the increased growth in SB associated with the increased scale insect infestation.

Dependence of crystallographic atomic displacement parameters on temperature (25-150†K) for complexes of horse liver alcohol dehydrogenase.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Šresolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Šand is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Improved furfural tolerance in Escherichia coli mediated by heterologous NADH-dependent benzyl alcohol dehydrogenases.

While lignocellulose is a promising source of renewable sugars for microbial fermentations, the presence of inhibitory compounds in typical lignocellulosic feedstocks, such as furfural, has hindered their utilisation. In Escherichia coli, a major route of furfural toxicity is the depletion of NADPH pools due to its use as a substrate by the YqhD enzyme that reduces furfural to its less toxic alcohol form. Here, we examine the potential of exploiting benzyl alcohol dehydrogenases as an alternative means to provide this same catalytic function but using the more abundant reductant NADH, as a strategy to increase the capacity for furfural removal. We determine the biochemical properties of three of these enzymes, from Pseudomonas putida, Acinetobacter calcoaceticus, and Burkholderia ambifaria, which all demonstrate furfural reductase activity. Furthermore, we show that the P. putida and B. ambifaria enzymes are able to provide substantial increases in furfural tolerance in vivo, by allowing more rapid conversion to furfuryl alcohol and resumption of growth. The study demonstrates that methods to seek alternative cofactor dependent enzymes can improve the intrinsic robustness of microbial chassis to feedstock inhibitors.

Elucidation of the Transport Mechanism of Puerarin and Gastrodin and Their Interaction on the Absorption in a Caco-2 Cell Monolayer Model.

Puerarin (PUR) and gastrodin (GAS) are often used in combined way for treating diseases caused by microcirculation disorders. The current study aimed to investigate the absorption and transportation mechanism of PUR and GAS and their interaction via Caco-2 monolayer cell model. In this work, the concentration in Caco-2 cell of PUR and GAS was determined by HPLC method. The bidirectional transport of PUR and GAS and the inhibition of drug efflux including verapamil and cyclosporine on the transport of these two components were studied. The mutual influence between PUR and GAS, especially the effect of the latter on the former of the bidirectional transport were also investigated. The transport of 50 µg·mL-1 PUR in Caco-2 cells has no obvious directionality. While the transport of 100 and 200 µg·mL-1 PUR presents a strong directionality, and this directionality can be inhibited by verapamil and cyclosporine. When PUR and GAS were used in combination, GAS could increase the absorption of PUR while PUR had no obvious influence on GAS. Therefore, the compatibility of PUR and GAS is reasonable, and GAS can promote the transmembrane transport of PUR, the effect of which is similar to that of verapamil.

Comparative Anti-Inflammatory Effects of Salix Cortex Extracts and Acetylsalicylic Acid in SARS-CoV-2 Peptide and LPS-Activated Human In Vitro Systems.

The usefulness of anti-inflammatory drugs as an adjunct therapy to improve outcomes in COVID-19 patients is intensely discussed in this paper. Willow bark (Salix cortex) has been used for centuries to relieve pain, inflammation, and fever. Its main active ingredient, salicin, is metabolized in the human body into salicylic acid, the precursor of the commonly used pain drug acetylsalicylic acid (ASA). Here, we report on the in vitro anti-inflammatory efficacy of two methanolic Salix extracts, standardized to phenolic compounds, in comparison to ASA in the context of a SARS-CoV-2 peptide challenge. Using SARS-CoV-2 peptide/IL-1ß- or LPS-activated human PBMCs and an inflammatory intestinal Caco-2/HT29-MTX co-culture, Salix extracts, and ASA concentration-dependently suppressed prostaglandin E2 (PGE2), a principal mediator of inflammation. The inhibition of COX-2 enzyme activity, but not protein expression was observed for ASA and one Salix extract. In activated PBMCs, the suppression of relevant cytokines (i.e., IL-6, IL-1ß, and IL-10) was seen for both Salix extracts. The anti-inflammatory capacity of Salix extracts was still retained after transepithelial passage and liver cell metabolism in an advanced co-culture model system consisting of intestinal Caco-2/HT29-MTX cells and differentiated hepatocyte-like HepaRG cells. Taken together, our in vitro data suggest that Salix extracts might present an additional anti-inflammatory treatment option in the context of SARS-CoV-2 peptides challenge; however, more confirmatory data are needed.

Antioxidant Function and Metabolomics Study in Mice after Dietary Supplementation with Methionine.

The antioxidant function and metabolic profiles in mice after dietary supplementation with methionine were investigated. The results showed that methionine supplementation enhanced liver GSH-Px activity and upregulated Gpx1 expression in the liver and SOD1 and Gpx4 expressions in the jejunum. Nrf2/Keap1 is involved in oxidative stress, and the western blotting data exhibited that dietary methionine markedly increased Keap1 abundance, while failed to influence the Nrf2 signal. Metabolomics investigation showed that methionine administration increased 2-hydroxypyridine, salicin, and asparagine and reduced D-Talose, maltose, aminoisobutyric acid, and inosine 5'-monophosphate in the liver, which are widely reported to involve in oxidative stress, lipid metabolism, and nucleotides generation. In conclusion, our study provides insights into antioxidant function and liver metabolic profiles in response to dietary supplementation with methionine.

Identification, repair and characterization of a benzyl alcohol-inducible promoter for recombinant proteins overexpression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial organism for the production of a variety of biological commodities. We discovered a promoter encoded by the gene NCgl2319 in C. glutamicum, which could be induced by benzyl alcohol, could be used as an efficient tunable expression system. In initial attempts, this promoter failed to function in a recombinant expression system. This was remedied by extending the original genetic context of the promoter, generating a new version Pcat-B. The Pcat-B transcription initiation site, its critical active regions, and its effect of inducers were fully characterized resulting in tunable expression. This approach proved to be very efficient in producing a pharmaceutical protein, N-terminal pro-brain natriuretic peptide (NT-proBNP). Production of approximately 440.43 mg/L NT-proBNP was achieved with the Pcat-B expression system demonstrating its application for controllable pharmaceutical protein production in C. glutamicum.

Suppression of hTAS2R16 Signaling by Umami Substances.

Interaction between umami and bitter taste has long been observed in human sensory studies and in neural responses in animal models, however, the molecular mechanism for their action has not been delineated. Humans detect diverse bitter compounds using 25-30 members of the type 2 taste receptor (TAS2R) family of G protein-coupled receptor. In this study, we investigated the putative mechanism of antagonism by umami substances using HEK293T cells expressing hTAS2R16 and two known probenecid-insensitive mutant receptors, hTAS2R16 N96T and P44T. In wild type receptor, Glu-Glu, inosine monophosphate (IMP), and l-theanine behave as partial insurmountable antagonists, and monosodium glutamate (MSG) acts as a surmountable antagonist in comparison with probenecid as a full insurmountable antagonist. The synergism with IMP of umami substances still stands in the suppression of hTAS2R16 signaling. In mutagenesis analysis, we found that Glu-Glu, MSG, and l-theanine share at least one critical binding site on N96 and P44 with probenecid. These results provide the first evidence for a direct binding of umami substances to the hTAS2R16 through the probenecid binding pocket on the receptor, resulting in the suppression of bitterness.

Phosphotransferase System Uptake and Metabolism of the ß-Glucoside Salicin Impact Group A Streptococcal Bloodstream Survival and Soft Tissue Infection.

Streptococcus pyogenes (group A Streptococcus [GAS]), a major human-specific pathogen, relies on efficient nutrient acquisition for successful infection within its host. The phosphotransferase system (PTS) couples the import of carbohydrates with their phosphorylation prior to metabolism and has been linked to GAS pathogenesis. In a screen of an insertional mutant library of all 14 annotated PTS permease (EIIC) genes in MGAS5005, the annotated ß-glucoside PTS transporter (bglP) was found to be crucial for GAS growth and survival in human blood and was validated in another M1T1 GAS strain, 5448. In 5448, bglP was shown to be in an operon with a putative phospho-ß-glucosidase (bglB) downstream and a predicted antiterminator (licT) upstream. Using defined nonpolar mutants of the ß-glucoside permease (bglP) and ß-glucosidase enzyme (bglB) in 5448, we showed that bglB, not bglP, was important for growth in blood. Furthermore, transcription of the licT-blgPB operon was found to be repressed by glucose and induced by the ß-glucoside salicin as the sole carbon source. Investigation of the individual bglP and bglB mutants determined that they influence in vitro growth in the ß-glucoside salicin; however, only bglP was necessary for growth in other non-ß-glucoside PTS sugars, such as fructose and mannose. Additionally, loss of BglP and BglB suggests that they are important for the regulation of virulence-related genes that control biofilm formation, streptolysin S (SLS)-mediated hemolysis, and localized ulcerative lesion progression during subcutaneous infections in mice. Thus, our results indicate that the ß-glucoside PTS transports salicin and its metabolism can differentially influence GAS pathophysiology during soft tissue infection.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

RIFM fragrance ingredient safety assessment, p-isopropylbenzyl alcohol, CAS Registry Number 536-60-7.

The existing information supports the use of this material as described in this safety assessment. p-Isopropylbenzyl alcohol was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from the read-across analog benzyl alcohol (CAS # 100-51-6) show that p-isopropylbenzyl alcohol is not expected to be genotoxic. Data from the read-across analog benzyl alcohol (CAS # 100-51-6) provide a calculated MOE >100 for the repeated dose, developmental, and local respiratory toxicity endpoints. The reproductive toxicity endpoint was evaluated using the TTC for a Cramer Class I material, and the exposure is below the TTC (0.03 mg/kg/day). Data from read-across analog benzyl alcohol (CAS # 100-51-6) provided p-isopropylbenzyl alcohol a NESIL of 5900 µg/cm2 for the skin sensitization endpoint. The phototoxicity and photoallergenicity endpoints were evaluated based on UV spectra; p-isopropylbenzyl alcohol is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; p-isopropylbenzyl alcohol was found not to be a PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC) are <1.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Catabolism of aromatic ß-glucosides by bacteria can lead to antibiotics resistance.

Antimicrobial resistance is a serious public health threat worldwide today. Escherichia coli is known to resist low doses of antibiotics in the presence of sodium salicylate and related compounds by mounting non-heritable transient phenotypic antibiotic resistance (PAR). In the present study, we demonstrate that Bgl+ bacterial strains harboring a functional copy of the ß-glucoside (bgl) operon and are actively hydrolyzing plant-derived aromatic ß-glucosides such as salicin show PAR to low doses of antibiotics. The aglycone released during metabolism of aromatic ß-glucosides is responsible for conferring this phenotype by de-repressing the multiple antibiotics resistance (mar) operon. We also show that prolonged exposure of Bgl+ bacteria to aromatic ß-glucosides in the presence of sub-lethal doses of antibiotics can lead to a significant increase in the frequency of mutants that show heritable resistance to higher doses of antibiotics. Although heritable drug resistance in many cases is known to reduce the fitness of the carrier strain, we did not see a cost associated with resistance in the mutants, most of which carry clinically relevant mutations. These findings indicate that the presence of the activated form of the bgl operon in the genome facilitates the survival of bacteria in environments in which both aromatic ß-glucosides and antibiotics are present.

Biosynthesis of the Prenylated Salicylaldehyde Flavoglaucin Requires Temporary Reduction to Salicyl Alcohol for Decoration before Reoxidation to the Final Product.

The biosynthetic pathway of the prenylated salicylaldehyde flavoglaucin and congeners in Aspergillus ruber was elucidated by genome mining, heterologous expression, precursor feeding, and biochemical characterization. The polyketide skeleton was released as alkylated salicyl alcohols, which is a prerequisite for consecutive hydroxylation and prenylation, before reoxidation to the final aldehyde products. Our results provide an excellent example for a highly programmed machinery in natural product biosynthesis.

The Occurrence of Sulfated Salicinoids in Poplar and Their Formation by Sulfotransferase1.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

Lipase-mediated synthesis of ricinoleic acid vanillyl ester and evaluation of antioxidant and antibacterial activity.

Castor oil extracted from castor bean has antibacterial property, and has been used in various folk remedies. The major structural component of castor oil, ricinoleic acid, has actual antibacterial activity. Some phenolic compounds derived from plants have antioxidant property. Among them, vanillyl alcohol from vanilla bean has strong antioxidant activity. As vanillyl alcohol has low solubility in hydrophobic solvents and castor oil has low solubility in hydrophilic solvents, there is practical difficulty in using them. We performed lipase-mediated transesterification using vanillyl alcohol and castor oil, and synthesized ricinoleic acid vanillyl ester (RAVE). 2,2-Diphenyl-1-picrylhydrazyl assay and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assay revealed that RAVE had a strong antioxidant activity in various organic solvents. RAVE also had antibacterial activity against some food spoilage bacteria. It showed more powerful antibacterial activity for gram positive bacteria than for gram negative bacteria. The critical micelle concentration of RAVE was measured at 7.36 µM and it partitioned exclusively into emulsion phase in water-emulsion system. Zeta potential measurement, membrane release test, and fluorescent microscopy showed that RAVE inserted itself into the bacterial cell membrane, destroyed membrane permeability, and induced cell death. As such, RAVE is a novel multi-functional compound with antioxidant and antibacterial activity, so it can be used as a functional material in the food and cosmetic industries.

Effects of poplar secondary metabolites on performance and detoxification enzyme activity of Lymantria dispar.

To identify the effects of poplar secondary metabolites on Lymantria dispar, six poplar secondary metabolites (i.e., caffeic acid, salicin, rutin, quercetin, flavone, and catechol) and three mixtures containing characteristic secondary metabolites in poplar were selected. Mixture 1 contained flavone and salicin, mixture 2 contained salicin, caffeic acid, and catechol, and mixture 3 contained flavone, catechol, and caffeic acid. Mixtures were added to artificial diets used to feed 2nd instar L. dispar larvae. The effects of different secondary metabolites on larval growth and development, antifeedant activity, nutrient utilization, and detoxifying enzymatic activity were investigated. Results revealed that there were different influences on L. dispar larvae. The maximum antifeedant rate of flavone was 87.58%. Larvae treated with mixture 2 had a significantly longer development time of 5.61 d with a survival rate of 38.75% for 15 d, which is lower than a single secondary metabolite. No L. dispar larvae survived on feeding diets containing flavone for 7 d. An increase in GST and P450 activities in larvae was significantly induced during the 72 h feeding on artificial diets containing experimental secondary metabolites. After treatment containing salicin and flavone for 24-72 h, P450 activity increased at first then decreased. These results provide a foundation for further investigation on the host selection and underlying adaptation mechanisms in L. dispar.

Positional differences of intronic transposons in pAMT affect the pungency level in chili pepper through altered splicing efficiency.

Capsaicinoids are unique compounds that give chili pepper fruits their pungent taste. Capsaicinoid levels vary widely among pungent cultivars, which range from low pungency to extremely pungent. However, the molecular mechanisms underlying this quantitative variation have not been elucidated. Our previous study identified various loss-of-function alleles of the pAMT gene which led to low pungency. The mutations in these alleles are commonly defined by Tcc transposon insertion and its footprint. In this study, we identified two leaky pamt alleles (pamtL1 and pamtL2 ) with different levels of putative aminotransferase (pAMT) activity. Notably, both alleles had a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron were different. Genetic analysis revealed that pamtL1 , pamtL2 and a loss-of-function pamt allele reduced capsaicinoid levels to about 50%, 10% and less than 1%, respectively. pamtL1 and pamtL2 encoded functional pAMT proteins, but they exhibited lower transcript levels than the functional type. RNA sequencing analysis showed that intronic transposons disrupted splicing in intron 3, which resulted in simultaneous expression of functional pAMT mRNA and non-functional splice variants containing partial sequences of Tcc. The non-functional splice variants were more dominant in pamtL2 than in pamtL1 . This suggested that the difference in position of the intronic transposons could alter splicing efficiency, leading to different pAMT activities and reducing capsaicinoid content to different levels. Our results provide a striking example of allelic variations caused by intronic transposons; these variations contribute to quantitative differences in secondary metabolite contents.