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1. The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

   https://doi.org/10.1093/plcell/koaa042  

   Yokoyama, Ryo; de Oliveira, Marcos V; Kleven, Bailey; Maeda, Hiroshi A (January 2021, The Plant Cell)

   null (Ed.)

   Abstract The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants. 

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2. Role of cytosolic, tyrosine‐insensitive prephenate dehydrogenase in Medicago truncatula

   https://doi.org/10.1002/pld3.218  

   Schenck, Craig A.; Westphal, Josh; Jayaraman, Dhileepkumar; Garcia, Kevin; Wen, Jiangqi; Mysore, Kirankumar S.; Ané, Jean‐Michel; Sumner, Lloyd W.; Maeda, Hiroshi A. (May 2020, Plant Direct)

   Abstract l‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrA_a), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrA_p), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterizedTnt1‐transposon mutants ofMtPDH1(pdh1) inMedicago truncatulato investigate PDH function. The pdh1mutants lackedPDHtranscript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence thatMtPDH1is responsible for the PDH activity detected inM. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity ofpdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism. 

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3. Xylose and shikimate transporters facilitates microbial consortium as a chassis for benzylisoquinoline alkaloid production

   https://doi.org/10.1038/s41467-023-43049-w  

   Gao, Meirong; Zhao, Yuxin; Yao, Zhanyi; Su, Qianhe; Van Beek, Payton; Shao, Zengyi (November 2023, Nature Communications)

   Abstract Plant-sourced aromatic amino acid (AAA) derivatives are a vast group of compounds with broad applications. Here, we present the development of a yeast consortium for efficient production of (S)-norcoclaurine, the key precursor for benzylisoquinoline alkaloid biosynthesis. A xylose transporter enables the concurrent mixed-sugar utilization inScheffersomyces stipitis, which plays a crucial role in enhancing the flux entering the highly regulated shikimate pathway located upstream of AAA biosynthesis. Two quinate permeases isolated fromAspergillus nigerfacilitates shikimate translocation to the co-culturedSaccharomyces cerevisiaethat converts shikimate to (S)-norcoclaurine, resulting in the maximal titer (11.5 mg/L), nearly 110-fold higher than the titer reported for anS. cerevisiaemonoculture. Our findings magnify the potential of microbial consortium platforms for the economical de novo synthesis of complex compounds, where pathway modularization and compartmentalization in distinct specialty strains enable effective fine-tuning of long biosynthetic pathways and diminish intermediate buildup, thereby leading to increases in production. 

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4. A simplified liquid chromatography‐mass spectrometry methodology to probe the shikimate and aromatic amino acid biosynthetic pathways in plants

   https://doi.org/10.1111/tpj.17105  

   El‐Azaz, Jorge; Maeda, Hiroshi A (December 2024, The Plant Journal)

   SUMMARY Plants direct substantial amounts of carbon toward the biosynthesis of aromatic amino acids (AAAs), particularly phenylalanine to produce lignin and other phenylpropanoids. Yet, we have a limited understanding of how plants regulate AAA metabolism, partially because of a scarcity of robust analytical methods. Here, we established a simplified workflow for simultaneous quantification of AAAs and their pathway intermediates from plant tissues, based on extraction at two alternative pH and analysis by Zwitterionic hydrophilic interaction liquid chromatography coupled to mass spectrometry. This workflow was then used to analyze metabolic responses to elevated or reduced carbon flow through the shikimate pathway in plants. Increased flow upon expression of a feedback‐insensitive isoform of the first shikimate pathway enzyme elevated all AAAs and pathway intermediates, especially arogenate, the last common precursor within the post‐chorismate pathway of tyrosine and phenylalanine biosynthesis. Additional overexpression of an arogenate dehydrogenase enzyme increased tyrosine levels and depleted phenylalanine and arogenate pools; however, the upstream shikimate pathway intermediates remained accumulated at high levels. Glyphosate treatment, which restricts carbon flow through the shikimate pathway by inhibiting its penultimate step, led to a predictable accumulation of shikimate and other precursors upstream of its target enzyme but also caused an unexpected accumulation of downstream metabolites, including arogenate. These findings highlight that the shikimate pathway and the downstream post‐chorismate AAA pathways function as independently regulated modules in plants. The method developed here paves the way for a deeper understanding of the shikimate and AAA biosynthetic pathways in plants. 

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5. Optimized small‐molecule pull‐downs define MLBP 1 as an acyl‐lipid‐binding protein

   https://doi.org/10.1111/tpj.14272  

   Sterlin, Yelena; Pri‐Tal, Oded; Zimran, Gil; Park, Sang‐Youl; Ben‐Ari, Julius; Kourelis, Jiorgos; Verstraeten, Inge; Gal, Maayan; Cutler, Sean R.; Mosquna, Assaf (March 2019, The Plant Journal)

   Summary Abscisic acid (ABA) receptors belong to theSTARTdomain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life.STARTdomain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterizedSTARTdomain proteins are the 14PYR/PYL/RCAR ABAreceptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently inNicotiana benthamianacoupled to untargetedLC‐MSto identify candidate binding ligands. We optimized this method usingABA–PYLinteractions and show thatABAco‐purifies with wild‐typePYL5 but not a binding site mutant. TheK_dofPYL5 forABAis 1.1 μm, which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37STARTdomain‐related proteins, which resulted in the identification of ligands that co‐purified withMLBP1 (At4G01883) orMLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed thatMLBP1 binds to monolinolenin, which we confirmed using recombinantMLBP1. Monolinolenin also co‐purified withMLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants. 

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