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Harnessing evolutionary diversification of primary metabolism for plant synthetic biology

  • Hiroshi A. Maeda
    Correspondence
    To whom correspondence should be addressed:Dept. of Botany, University of Wisconsin-Madison, 430 Lincoln Dr., Madison, WI 53706
    Affiliations
    Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Author Footnotes
    2 The abbreviations used are:IPPisopentenyl diphosphateMVAmevalonateDMAPPdimethylallyl diphosphateDMAPdimethylallyl phosphateMEP2-C-methyl-d-erythritol 4-phosphateMPDCmevalonate diphosphate decarboxylasePMKphosphomevalonate kinaseERendoplasmic reticulumIDIIPP:DMAPP isomeraseDXP1-deoxy-d-xylulose 5-phosphateDXRDXP reductaseDXSDXP synthaseHMBPP4-hydroxy-3-methyl-butenyl 1-diphosphateMEcPP2-C-methyl-d-erythritol-2,4-cyclodiphosphateHMG–CoA3-hydroxy-3-methylglutaryl–CoAHMGRHMG–CoA reductaseMVPmevalonate 5-phosphatePMDphosphomevalonate decarboxylaseIPisopentenyl phosphateIPKisopentenyl phosphate kinaseCMchorismate mutaseHDSHMBPP synthaseHDRHMBPP reductasePPA-ATprephenate aminotransferaseADTarogenate dehydratasePDTprephenate dehydratasePPY-ATphenylpyruvate aminotransferaseASanthranilate synthaseTyrAaarogenate TyrA dehydrogenasencTyrAanoncanonical TyrAaPheHPhe hydroxylaseTyrApprephenate TyrA dehydrogenaseIPMSisopropylmalate synthase3MOB3-methyl-2-oxobutanoate4MOP4-methyl-2-oxopropanoate.
Open AccessPublished:September 26, 2019DOI:https://doi.org/10.1074/jbc.REV119.006132
      Plants produce numerous natural products that are essential to both plant and human physiology. Recent identification of genes and enzymes involved in their biosynthesis now provides exciting opportunities to reconstruct plant natural product pathways in heterologous systems through synthetic biology. The use of plant chassis, although still in infancy, can take advantage of plant cells' inherent capacity to synthesize and store various phytochemicals. Also, large-scale plant biomass production systems, driven by photosynthetic energy production and carbon fixation, could be harnessed for industrial-scale production of natural products. However, little is known about which plants could serve as ideal hosts and how to optimize plant primary metabolism to efficiently provide precursors for the synthesis of desirable downstream natural products or specialized (secondary) metabolites. Although primary metabolism is generally assumed to be conserved, unlike the highly-diversified specialized metabolism, primary metabolic pathways and enzymes can differ between microbes and plants and also among different plants, especially at the interface between primary and specialized metabolisms. This review highlights examples of the diversity in plant primary metabolism and discusses how we can utilize these variations in plant synthetic biology. I propose that understanding the evolutionary, biochemical, genetic, and molecular bases of primary metabolic diversity could provide rational strategies for identifying suitable plant hosts and for further optimizing primary metabolism for sizable production of natural and bio-based products in plants.

      Opportunities to produce plant natural products in plant hosts

      Plants produce diverse and often abundant chemical compounds, which play critical roles in these sessile and multicellular organisms to habitat in various environmental niches. Many of these phytochemicals are produced in a lineage-specific manner and thus are often referred to as specialized or secondary metabolites. Many of these plant natural products also provide essential nutrients and valuable resources for the production of pharmaceuticals and biomaterials to the human society (
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      ) and are providing exciting opportunities to produce plant natural products in heterologous systems through synthetic biology (Fig. 1A). Microbial hosts, having well-developed genetic tools and industrial-scale culture methods (e.g. yeast), have been engineered to build chemical production platforms that are optimized for a certain primary metabolic branch on which various downstream pathways, including plant specialized metabolic pathways, have been introduced (
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      ). Although significant success has been made in industrial-scale terpenoid production in microbes (
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      • Wang Y.
      • Simeon F.
      • Leonard E.
      • Mucha O.
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      • Pfeifer B.
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      Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli.
      ,
      • Paddon C.J.
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      High-level semi-synthetic production of the potent antimalarial artemisinin.
      ), microbial production of certain classes of plant natural products, such as alkaloids and phenolics, appear to be more challenging, likely due to their toxicity, pathway complexity, and inefficiency of plant-derived enzymes (
      • Galanie S.
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      • Filsinger Interrante M.
      • Smolke C.D.
      Complete biosynthesis of opioids in yeast.
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      Strategies for microbial synthesis of high-value phytochemicals.
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      Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.
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      Recent advances in microbial production of aromatic chemicals and derivatives.
      ).
      Figure thumbnail gr1
      Figure 1Producing natural products in plants through synthetic biology and primary metabolic pathway engineering. A, Tremendous chemical diversity has evolved in different plant lineages (left). The underlying specialized metabolic pathways can be identified and reconstructed in a heterologous host, or chassis, through synthetic biology (green, right) for efficient production of target compounds (e.g. nutraceuticals, pharmaceuticals, and bio-based materials). Additionally, the upstream primary metabolic pathways can be engineered in the host to optimize the supply of a specific precursor(s) (blue, right). B, Besides microbial hosts, plants can provide alternative chassis to produce natural plant products in sustainable and potentially efficient manners, if their pros and cons (table) are carefully evaluated and addressed. See Appendix S1 for image credits.
      The use of heterologous plant hosts, although still in early stages, provides alternative and sustainable means to produce plant natural products, which take advantage of global cultivation systems that are propelled by endogenous photosynthetic energy production and carbon fixation (Fig. 1B) (
      • Owen C.
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      Harnessing plant metabolic diversity.
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      Metabolic engineering with plants for a sustainable biobased economy.
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      Combinatorial biosynthesis of small molecules in plants: engineering strategies and tools.
      ). The past decade of investments and efforts in developing bioenergy crops (e.g. perennial grasses, fast-growing trees) have further advanced opportunities to grow high-yielding plants in marginal lands, which can avoid direct competition with food crop production and minimize environmental impacts (
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      Plants to power: bioenergy to fuel the future.
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      Towards a sustainable bio-based economy: redirecting primary metabolism to new products with plant synthetic biology.
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      Harnessing energy from plant biomass.
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      Sweet sorghum as a model system for bioenergy crops.
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      Biofuel and energy crops: high-yield Saccharinae take center stage in the post-genomics era.
      ). Plant hosts may also have better storage capacity and toxicity resistance for phytochemical production compared with microbial hosts (Fig. 1B). Thus, plant chassis potentially provide promising alternative platforms to produce some of these metabolites that are difficult to produce in microbes, especially if tailored plant hosts (or chassis) are carefully selected and generated depending on downstream target compounds.

      Challenges to build plant chassis for synthetic biology

      Many specialized metabolic pathways have been successfully introduced to heterologous plants (
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      The seco-iridoid pathway from Catharanthus roseus.
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      • Runguphan W.
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      Integrating carbon–halogen bond formation into medicinal plant metabolism.
      ). However, relatively little effort has been made in plants to optimize the supply of their primary metabolite precursors (e.g. amino acids, sugars, nucleotides, and fatty acids), from which specialized metabolites are produced (Fig. 1A) (
      • Shih P.M.
      Towards a sustainable bio-based economy: redirecting primary metabolism to new products with plant synthetic biology.
      ). Microbial metabolic engineering and synthetic biology studies demonstrated that redirection of carbon flux and efficient supply of a specific primary precursor(s) are critical to achieve efficient production of downstream target products (Fig. 1A) (
      • Li S.
      • Li Y.
      • Smolke C.D.
      Strategies for microbial synthesis of high-value phytochemicals.
      ,
      • Huccetogullari D.
      • Luo Z.W.
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      Metabolic engineering of microorganisms for production of aromatic compounds.
      • Kirby J.
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      Biosynthesis of plant isoprenoids: perspectives for microbial engineering.
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      Engineering cellular metabolism.
      • Vickers C.E.
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      Recent advances in synthetic biology for engineering isoprenoid production in yeast.
      ). Thus, holistic understanding and engineering of both primary and specialized metabolisms are crucial for efficient and sizable production of natural products in plants.
      Unlike in microbes, engineering of plant primary metabolism poses several major challenges (Fig. 1B). (i) There is a much more limited capacity to conduct genetic engineering and mutagenesis screening in plants than in microbes, due to low transformation efficiency and long generation cycles of most plants (months to years versus hours to days). (ii) Plant metabolism is likely more constrained due to almost exclusive reliance on the carbon input from photosynthetic CO2 fixation, unlike microbes that can utilize multiple carbon sources. (iii) Plant primary metabolic pathways are tightly integrated with each other and directly linked to the growth and development of these complex multicellular organisms, and their manipulation often compromises overall growth and yield (
      • Shaul O.
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      Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase.
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      ).
      One way to overcome these challenges is to carefully choose host plants, which are naturally tailored toward production of certain classes of compounds, and then to conduct rational and precise engineering of primary metabolism to optimize a certain precursor supply. Here, I discuss one promising approach to achieve this goal by learning from millions of years of experimentations that nature has done. Although primary metabolism is generally assumed to be conserved across the plant kingdom, unlike highly-diversified specialized metabolism (
      • Moghe G.D.
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      Something old, something new: conserved enzymes and the evolution of novelty in plant specialized metabolism.
      • Pichersky E.
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      Convergent evolution in plant specialized metabolism.
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      • Weng J.-K.
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      The rise of chemodiversity in plants.
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      Cytochrome P450 enzymes: a driving force of plant diterpene diversity.
      ), there are some examples of evolutionary diversification of primary metabolic pathways, especially at the interface between primary and specialized metabolism (
      • Maeda H.A.
      Evolutionary diversification of primary metabolism and its contribution to plant chemical diversity.
      ). Exploring and harnessing such relatively-rare but key evolutionary innovations of plant metabolism will provide useful tools and strategies to optimize plant primary metabolism in coordination with downstream specialized metabolic pathways, in order to achieve efficient production of plant natural products in carefully-selected plant hosts.

      Ancient diversifications of primary metabolism in plants from other kingdoms

      Despite the general conservation of primary metabolic pathways among different kingdoms of life, some of them are unique in plants, which likely contributed to the tremendous chemical diversity seen in the plant kingdom today. Understanding such fundamental differences provides a critical basis for constructing plant chemical production platforms through metabolic engineering. Here, I highlight prominent examples found in primary metabolic pathways that support two major classes of plant natural products, terpenoid (isoprenoid) and phenylpropanoid compounds.

      Two alternative isopentenyl diphosphate biosynthetic pathways to support diverse terpenoid formation in plants

      Isopentenyl diphosphate (IPP),
      The abbreviations used are: IPP
      isopentenyl diphosphate
      MVA
      mevalonate
      DMAPP
      dimethylallyl diphosphate
      DMAP
      dimethylallyl phosphate
      MEP
      2-C-methyl-d-erythritol 4-phosphate
      MPDC
      mevalonate diphosphate decarboxylase
      PMK
      phosphomevalonate kinase
      ER
      endoplasmic reticulum
      IDI
      IPP:DMAPP isomerase
      DXP
      1-deoxy-d-xylulose 5-phosphate
      DXR
      DXP reductase
      DXS
      DXP synthase
      HMBPP
      4-hydroxy-3-methyl-butenyl 1-diphosphate
      MEcPP
      2-C-methyl-d-erythritol-2,4-cyclodiphosphate
      HMG–CoA
      3-hydroxy-3-methylglutaryl–CoA
      HMGR
      HMG–CoA reductase
      MVP
      mevalonate 5-phosphate
      PMD
      phosphomevalonate decarboxylase
      IP
      isopentenyl phosphate
      IPK
      isopentenyl phosphate kinase
      CM
      chorismate mutase
      HDS
      HMBPP synthase
      HDR
      HMBPP reductase
      PPA-AT
      prephenate aminotransferase
      ADT
      arogenate dehydratase
      PDT
      prephenate dehydratase
      PPY-AT
      phenylpyruvate aminotransferase
      AS
      anthranilate synthase
      TyrAa
      arogenate TyrA dehydrogenase
      ncTyrAa
      noncanonical TyrAa
      PheH
      Phe hydroxylase
      TyrAp
      prephenate TyrA dehydrogenase
      IPMS
      isopropylmalate synthase
      3MOB
      3-methyl-2-oxobutanoate
      4MOP
      4-methyl-2-oxopropanoate.
      and its allylic isomer dimethylallyl diphosphate (DMAPP), is the precursor and building blocks of diverse isoprenoid compounds, such as sterols (e.g. cholesterols), dolichol, and quinones (e.g. ubiquinone). In plants, IPP and DMAPP are also used to synthesize photosynthetic pigments (i.e. chlorophylls and carotenoids) and quinones (i.e. plastoquinone and phylloquinone), plant hormones (e.g. gibberellins, brassinosteroids, and abscisic acid), rubbers, isoprene, mono- and sesquiterpene volatiles, and diverse di- and tri-terpenoids (
      • Rodríguez-Concepción M.
      • Boronat A.
      Breaking new ground in the regulation of the early steps of plant isoprenoid biosynthesis.
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      Biosynthesis and biological functions of terpenoids in plants.
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      To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism.
      ). IPP (and DMAPP) can be synthesized via two different routes, the mevalonate (MVA) and 2-C-methyl-d-erythritol 4-phosphate (MEP) pathways (Fig. 2) (
      • Kirby J.
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      Biosynthesis of plant isoprenoids: perspectives for microbial engineering.
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      ). Most organisms have either one of the two pathways: for example, the MVA pathway is present in animals, fungi, and archaea, and the MEP pathway is found in many bacteria, including Escherichia coli and cyanobacteria (
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      ). Notably, however, plants and many algae have both MVA and MEP pathways to synthesize IPP and DMAPP, which support the formation of these diverse isoprenoid compounds in different subcellular compartments (Fig. 2). These two pathways appear to have some but limited metabolic cross-talks (
      • Vranová E.
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      Network analysis of the MVA and MEP pathways for isoprenoid synthesis.
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      ). Although various isoprenoids, including the plant-derived sesquiterpene artemisinin, have been successfully produced through microbial synthetic biology (
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      • et al.
      High-level semi-synthetic production of the potent antimalarial artemisinin.
      ,
      • Chang M.C.
      • Keasling J.D.
      Production of isoprenoid pharmaceuticals by engineered microbes.
      ), the natural capacity of plants to produce abundant IPP can also be utilized for production of various isoprenoid compounds using plant hosts (
      • Kirby J.
      • Keasling J.D.
      Biosynthesis of plant isoprenoids: perspectives for microbial engineering.
      ,
      • Ye X.
      • Al-Babili S.
      • Klöti A.
      • Zhang J.
      • Lucca P.
      • Beyer P.
      • Potrykus I.
      Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm.
      ,
      • Mahmoud S.S.
      • Croteau R.B.
      Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase.
      • Wu S.
      • Schalk M.
      • Clark A.
      • Miles R.B.
      • Coates R.
      • Chappell J.
      Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants.
      ). However, tight and complex regulation of IPP (and DMAPP) biosynthesis has been a major bottleneck in efficient production of isoprenoid compounds in plants (
      • Schaller H.
      • Grausem B.
      • Benveniste P.
      • Chye M.L.
      • Tan C.T.
      • Song Y.H.
      • Chua N.H.
      Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 in tobacco results in sterol overproduction.
      • Chappell J.
      • Wolf F.
      • Proulx J.
      • Cuellar R.
      • Saunders C.
      Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants?.
      ,
      • Zhao L.
      • Chang W.C.
      • Xiao Y.
      • Liu H.W.
      • Liu P.
      Methylerythritol phosphate pathway of isoprenoid biosynthesis.
      ,
      • Bach T.J.
      • Rogers D.H.
      • Rudney H.
      Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings.
      ,
      • Brooker J.D.
      • Russell D.W.
      Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings.
      ,
      • Nagegowda D.A.
      • Bach T.J.
      • Chye M.-L.
      Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes.
      • Banerjee A.
      • Wu Y.
      • Banerjee R.
      • Li Y.
      • Yan H.
      • Sharkey T.D.
      Feedback inhibition of deoxy-d-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.
      ), and it is critical to understand how plants regulate IPP and DMAPP production through the MVA and MEP pathways.
      Figure thumbnail gr2
      Figure 2Plants have two alternative pathways to synthesize IPP precursor for production of diverse terpenoid compounds. The MVA pathway occurs outside of the plastids and provides IPP and DMAPP precursors for downstream specialized metabolism to synthesize diverse sterols, sesquiterpenes, and triterpenes, for example. The MEP pathway is localized in the plastids and supports biosynthesis of isoprene and monoterpene volatiles, various diterpenes, and photosynthetic isoprenoids (e.g. chlorophylls and plastoquinone). The compartment in light blue depicts ER. Although it is not shown here, some of the MVA pathway enzymes, PMK, MPDC, and IDI, appear to be localized in the peroxisome, in addition to the cytosol. The alternative MVA pathway enzymes of archaea is shown in gray. MVPP, mevalonate-5-diphosphate. Enzymes abbreviated in boxes include: Fd, ferredoxin; MK, MVA kinase; Nudix, Nudix hydrolase. See the footnotes for other abbreviations introduced in the text.
      The MVA pathway starts from acetyl coenzyme A (CoA), three of which are condensed to 3-hydroxy-3-methylglutaryl–CoA (HMG–CoA) and then reduced to MVA, followed by ATP-dependent phosphorylation and decarboxylation to IPP (Fig. 2). IPP and DMAPP are then interconverted by IPP:DMAPP isomerase (IDI). In plants, the MVA pathway operates mainly in the cytosol, but the later steps catalyzed by phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MPDC), and IDI appear to be localized also in the peroxisomes, based on fluorescence protein-tagged subcellular localization studies (
      • Guirimand G.
      • Guihur A.
      • Phillips M.A.
      • Oudin A.
      • Glévarec G.
      • Melin C.
      • Papon N.
      • Clastre M.
      • St-Pierre B.
      • Rodríguez-Concepción M.
      • Burlat V.
      • Courdavault V.
      A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus.
      ,
      • Simkin A.J.
      • Guirimand G.
      • Papon N.
      • Courdavault V.
      • Thabet I.
      • Ginis O.
      • Bouzid S.
      • Giglioli-Guivarc'h N.
      • Clastre M.
      Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta.
      • Sapir-Mir M.
      • Mett A.
      • Belausov E.
      • Tal-Meshulam S.
      • Frydman A.
      • Gidoni D.
      • Eyal Y.
      Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes.
      ). HMG–CoA reductase (HMGR), which converts HMG–CoA into MVA in an irreversible manner and is anchored to endoplasmic reticulum (ER), appears to be the key regulatory enzyme of the MVA pathway in plants (
      • Vranová E.
      • Coman D.
      • Gruissem W.
      Network analysis of the MVA and MEP pathways for isoprenoid synthesis.
      ,
      • Hemmerlin A.
      Post-translational events and modifications regulating plant enzymes involved in isoprenoid precursor biosynthesis.
      ), like in bacteria, fungi, and animals (
      • Burg J.S.
      • Espenshade P.J.
      Regulation of HMG–CoA reductase in mammals and yeast.
      ). Besides transcriptional regulation of different plant HMGR isoforms (
      • Enjuto M.
      • Lumbreras V.
      • Marín C.
      • Boronat A.
      Expression of the Arabidopsis HMG2 gene, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, is restricted to meristematic and floral tissues.
      ,
      • Stermer B.A.
      • Bianchini G.M.
      • Korth K.L.
      Regulation of HMG–CoA reductase activity in plants.
      ), plant HMGR activity is regulated by free CoA, HMG, and NADP+ (
      • Bach T.J.
      • Rogers D.H.
      • Rudney H.
      Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings.
      ,
      • Brooker J.D.
      • Russell D.W.
      Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings.
      ). Also, some plant HMGR proteins are post-translationally modified by interacting with various regulators, such as some kinases (
      • Kevei Z.
      • Lougnon G.
      • Mergaert P.
      • Horváth G.V.
      • Kereszt A.
      • Jayaraman D.
      • Zaman N.
      • Marcel F.
      • Regulski K.
      • Kiss G.B.
      • Kondorosi A.
      • Endre G.
      • Kondorosi E.
      • Ané J.-M.
      3-Hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula.
      ,
      • Robertlee J.
      • Kobayashi K.
      • Suzuki M.
      • Muranaka T.
      AKIN10, a representative Arabidopsis SNF1-related protein kinase 1 (SnRK1), phosphorylates and downregulates plant HMG–CoA reductase.
      ), protein phosphatase 2A (PP2A) (
      • Leivar P.
      • Antolín-Llovera M.
      • Ferrero S.
      • Closa M.
      • Arró M.
      • Ferrer A.
      • Boronat A.
      • Campos N.
      Multilevel control of Arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A.
      ), and ER-associated degradation–type RING membrane-anchor E3 ubiquitin ligase (
      • Pollier J.
      • Moses T.
      • González-Guzmán M.
      • De Geyter N.
      • Lippens S.
      • Vanden Bossche R.
      • Marhavý P.
      • Kremer A.
      • Morreel K.
      • Guérin C.J.
      • Tava A.
      • Oleszek W.
      • Thevelein J.M.
      • Campos N.
      • Goormachtig S.
      • Goossens A.
      The protein quality control system manages plant defence compound synthesis.
      ,
      • Doblas V.G.
      • Amorim-Silva V.
      • Posé D.
      • Rosado A.
      • Esteban A.
      • Arró M.
      • Azevedo H.
      • Bombarely A.
      • Borsani O.
      • Valpuesta V.
      • Ferrer A.
      • Tavares R.M.
      • Botella M.A.
      The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.
      ).
      Recent studies revealed further complexity of the plant MVA pathway and its regulation. The last two steps of the MVA pathway appear to be flipped in archaea and Chloroflexi bacteria: mevalonate 5-phosphate (MVP) is converted by phosphomevalonate decarboxylase (PMD) to isopentenyl phosphate (IP), which is then further phosphorylated to IPP by ATP-dependent isopentenyl phosphate kinases (gray in Fig. 2) (IPKs) (
      • Vannice J.C.
      • Skaff D.A.
      • Keightley A.
      • Addo J.K.
      • Wyckoff G.J.
      • Miziorko H.M.
      Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.
      • Dellas N.
      • Thomas S.T.
      • Manning G.
      • Noel J.P.
      Discovery of a metabolic alternative to the classical mevalonate pathway.
      ,
      • Grochowski L.L.
      • Xu H.
      • White R.H.
      Methanocaldococcus jannaschii uses a modified mevalonate pathway for biosynthesis of isopentenyl diphosphate.
      • Dellas N.
      • Noel J.P.
      Mutation of archaeal isopentenyl phosphate kinase highlights mechanism and guides phosphorylation of additional isoprenoid monophosphates.
      ). All sequenced plant genomes also encode the IPK enzymes, which can phosphorylate both IP and dimethylallyl phosphate (DMAP) to IPP and DMAPP, respectively (
      • Dellas N.
      • Thomas S.T.
      • Manning G.
      • Noel J.P.
      Discovery of a metabolic alternative to the classical mevalonate pathway.
      ,
      • Henry L.K.
      • Gutensohn M.
      • Thomas S.T.
      • Noel J.P.
      • Dudareva N.
      Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants.
      ). Unlike archaea, however, plants appear to lack the PMD orthologs and instead produce IP and DMAP by Nudix hydrolases through dephosphorylation of IPP and DMAPP, respectively (Fig. 2) (
      • Henry L.K.
      • Thomas S.T.
      • Widhalm J.R.
      • Lynch J.H.
      • Davis T.C.
      • Kessler S.A.
      • Bohlmann J.
      • Noel J.P.
      • Dudareva N.
      Contribution of isopentenyl phosphate to plant terpenoid metabolism.
      ). Further genetic studies demonstrated that reducing the formation of IP and DMAP by either down-regulating Nudix hydroxylase or up-regulating IPK led to elevated accumulation of both sesquiterpenes and monoterpenes produced in the cytosol and plastids, respectively (
      • Henry L.K.
      • Gutensohn M.
      • Thomas S.T.
      • Noel J.P.
      • Dudareva N.
      Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants.
      ,
      • Henry L.K.
      • Thomas S.T.
      • Widhalm J.R.
      • Lynch J.H.
      • Davis T.C.
      • Kessler S.A.
      • Bohlmann J.
      • Noel J.P.
      • Dudareva N.
      Contribution of isopentenyl phosphate to plant terpenoid metabolism.
      ). These results suggest that IP and DMAP negatively regulate terpenoid production in plants. Therefore, the reactivation of IP and DMAP through phosphorylation provides a promising approach to enhance terpenoid productions in plants, especially when combined with up-regulation of other rate-limiting enzymes of the MVA pathway, such as HMGR and PMK (
      • Henry L.K.
      • Thomas S.T.
      • Widhalm J.R.
      • Lynch J.H.
      • Davis T.C.
      • Kessler S.A.
      • Bohlmann J.
      • Noel J.P.
      • Dudareva N.
      Contribution of isopentenyl phosphate to plant terpenoid metabolism.
      ).
      The alternative MEP pathway takes place in the plastids and starts from the thiamine diphosphate–dependent condensation of glyceraldehyde 3-phosphate and pyruvate to the 1-deoxy-d-xylulose 5-phosphate (DXP), which is then reductively isomerized to MEP (Fig. 2). MEP is activated by coupling to cytidine triphosphate (CTP) and ATP-dependent phosphorylation, followed by cyclization to 2-C-methyl-d-erythritol-2,4-cyclodiphosphate (MEcPP). MEcPP undergoes ring opening and reductive dehydration to 4-hydroxy-3-methyl-butenyl 1-diphosphate (HMBPP), which is then converted to both IPP and DMAPP (Fig. 2) (
      • Vranová E.
      • Coman D.
      • Gruissem W.
      Network analysis of the MVA and MEP pathways for isoprenoid synthesis.
      ,
      • Zhao L.
      • Chang W.C.
      • Xiao Y.
      • Liu H.W.
      • Liu P.
      Methylerythritol phosphate pathway of isoprenoid biosynthesis.
      ). Given that one of the MEP precursors, glyceraldehyde 3-phosphate, is the primary product of the Calvin-Benson cycle, the plastidic MEP pathway likely provides a robust IPP precursor supply for synthesis of abundant photosynthetic isoprenoids, including chlorophylls, carotenoids, and prenylquinones, as well as isoprene, which can account for 99% of de novo synthesized isoprenoids in poplar leaves (
      • Ghirardo A.
      • Wright L.P.
      • Bi Z.
      • Rosenkranz M.
      • Pulido P.
      • Rodríguez-Concepción M.
      • Niinemets Ϝ.
      • Brüggemann N.
      • Gershenzon J.
      • Schnitzler J.-P.
      Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene.
      ). Indeed, stable isotope-labeled 13CO2 is rapidly incorporated into the intermediates of the MEP but not MVA pathway in illuminated Arabidopsis leaves (
      • Wright L.P.
      • Rohwer J.M.
      • Ghirardo A.
      • Hammerbacher A.
      • Ortiz-Alcaide M.
      • Raguschke B.
      • Schnitzler J.-P.
      • Gershenzon J.
      • Phillips M.A.
      Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis.
      ). The last two enzymes, HMBPP synthase (HDS) and reductase (HDR), are iron–sulfur cluster proteins and can accept electrons directly from ferredoxin, the final donor of the photosynthetic electron transport chain, under light (
      • Okada K.
      • Hase T.
      Cyanobacterial nonmevalonate pathway: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase interacts with ferredoxin in Thermosynechococcus elongatus BP-1.
      ,
      • Seemann M.
      • Tse Sum Bui B.
      • Wolff M.
      • Miginiac-Maslow M.
      • Rohmer M.
      Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG.
      • Seemann M.
      • Wegner P.
      • Schünemann V.
      • Bui B.T.
      • Wolff M.
      • Marquet A.
      • Trautwein A.X.
      • Rohmer M.
      Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe-4S] protein.
      ). This likely provides an additional mechanism of coordination between photosynthesis and the MEP pathway in the chloroplasts (Fig. 2).
      One might speculate that the plastidic MEP pathway of plants and algae is derived from endosymbiosis of cyanobacteria, which also synthesize IPP and DMAPP by the MEP pathway. However, evolutionary analyses of individual MEP pathway enzymes of plants and algae revealed that these enzymes have mosaic evolutionary origins and share last common ancestors with either cyanobacteria, α-proteobacteria, or Chlamydia; some of these genes were horizontally transferred to a common ancestor of plastid-bearing eukaryotes (
      • Lange B.M.
      • Rujan T.
      • Martin W.
      • Croteau R.
      Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes.
      ,
      • Matsuzaki M.
      • Kuroiwa H.
      • Kuroiwa T.
      • Kita K.
      • Nozaki H.
      A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus.
      ). Because of its complex evolutionary history and the high and diverse demand for synthesizing numerous and abundant isoprenoid compounds, the plant MEP pathway is likely regulated differently from that of bacteria. The initial reaction, catalyzed by DXP synthase (DXS), is irreversible and commits carbon to the MEP pathway. The DXS enzyme hence plays the major role in controlling the flux through the MEP pathway, with a flux control coefficient of 0.82 in Arabidopsis leaves—the coefficient of 0 or 1 indicates that an individual enzyme (i.e. DXS) exerts no control or complete control, respectively, over the flux through an entire pathway (i.e. the MEP pathway) (
      • Wright L.P.
      • Rohwer J.M.
      • Ghirardo A.
      • Hammerbacher A.
      • Ortiz-Alcaide M.
      • Raguschke B.
      • Schnitzler J.-P.
      • Gershenzon J.
      • Phillips M.A.
      Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis.
      ). However, DXS overexpression had only a modest increase in isoprenoid accumulation, partly due to the export of the downstream MEcPP intermediate to a nonplastidic pool (Fig. 2) (
      • Wright L.P.
      • Rohwer J.M.
      • Ghirardo A.
      • Hammerbacher A.
      • Ortiz-Alcaide M.
      • Raguschke B.
      • Schnitzler J.-P.
      • Gershenzon J.
      • Phillips M.A.
      Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis.
      ), which, interestingly, can participate in the plastid–to–nucleus retrograde signaling (
      • Xiao Y.
      • Savchenko T.
      • Baidoo E.E.
      • Chehab W.E.
      • Hayden D.M.
      • Tolstikov V.
      • Corwin J.A.
      • Kliebenstein D.J.
      • Keasling J.D.
      • Dehesh K.
      Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes.
      ,
      • Benn G.
      • Bjornson M.
      • Ke H.
      • De Souza A.
      • Balmond E.I.
      • Shaw J.T.
      • Dehesh K.
      Plastidial metabolite MEcPP induces a transcriptionally centered stress-response hub via the transcription factor CAMTA3.
      ). Also, DXS protein level and activity are regulated through the stromal protein quality control system mediated by concerted actions of Hsp chaperons and Clp proteases (
      • Llamas E.
      • Pulido P.
      • Rodriguez-Concepcion M.
      Interference with plastome gene expression and Clp protease activity in Arabidopsis triggers a chloroplast unfolded protein response to restore protein homeostasis.
      ,
      • Pulido P.
      • Llamas E.
      • Llorente B.
      • Ventura S.
      • Wright L.P.
      • Rodríguez-Concepción M.
      Specific Hsp100 chaperones determine the fate of the first enzyme of the plastidial isoprenoid pathway for either refolding or degradation by the stromal Clp protease in Arabidopsis.
      • Pulido P.
      • Toledo-Ortiz G.
      • Phillips M.A.
      • Wright L.P.
      • Rodríguez-Concepción M.
      Arabidopsis J-protein J20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control.
      ). DXS from poplar is feedback-inhibited by IPP and DMAPP in a noncompetitive manner (
      • Banerjee A.
      • Wu Y.
      • Banerjee R.
      • Li Y.
      • Yan H.
      • Sharkey T.D.
      Feedback inhibition of deoxy-d-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.
      ,
      • Ghirardo A.
      • Wright L.P.
      • Bi Z.
      • Rosenkranz M.
      • Pulido P.
      • Rodríguez-Concepción M.
      • Niinemets Ϝ.
      • Brüggemann N.
      • Gershenzon J.
      • Schnitzler J.-P.
      Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene.
      ), which may set the upper limit of IPP and DMAPP accumulation in the plastids. Furthermore, like many other plastidic enzymes (e.g. glyceraldehyde 3-phosphate dehydrogenase and glutamine synthetase), the downstream enzymes, DXP reductase (DXR), HDS, and HDR are targets of thioredoxins and likely subjected to redox regulation (
      • Hemmerlin A.
      Post-translational events and modifications regulating plant enzymes involved in isoprenoid precursor biosynthesis.
      ,
      • Balmer Y.
      • Koller A.
      • del Val G.
      • Manieri W.
      • Schürmann P.
      • Buchanan B.B.
      Proteomics gives insight into the regulatory function of chloroplast thioredoxins.
      ,
      • Lemaire S.D.
      • Guillon B.
      • Le Maréchal P.
      • Keryer E.
      • Miginiac-Maslow M.
      • Decottignies P.
      New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii.
      • Montrichard F.
      • Alkhalfioui F.
      • Yano H.
      • Vensel W.H.
      • Hurkman W.J.
      • Buchanan B.B.
      Thioredoxin targets in plants: the first 30 years.
      ), although their physiological significance remains to be tested. Thus, modulating both transcriptional and post-transcriptional regulation, along with the MEcPP-mediated signaling pathway, will likely lead to enhanced supply of IPP and DMAPP in the plastids and increased production of MEP pathway-derived isoprenoid compounds in plants. It remains to be explored, however, whether some of these MVA and MEP pathway regulations are different in certain plant lineages. Such variations in this key plant metabolic branch, if any, can provide useful tools to further improve IPP and/or DMAPP supply and downstream terpenoid production.

      Alternative phenylalanine biosynthetic pathways for phenolic compound production in plants

      l-Phenylalanine (Phe) is an aromatic amino acid required for protein synthesis in all organisms and is produced in microbes and plants but not in animals (
      • Tzin V.
      • Galili G.
      New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants.
      • Maeda H.
      • Dudareva N.
      The shikimate pathway and aromatic amino acid biosynthesis in plants.
      ,
      • Bentley R.
      The shikimate pathway–a metabolic tree with many branches.
      • Herrmann K.M.
      • Weaver L.M.
      The shikimate pathway.
      ). Plants also use Phe as the precursor to synthesize various phenolic natural products, including diverse and abundant phenylpropanoids, such as lignin, lignans, condensed tannin, flavonoids, anthocyanin pigments, coumarins, stilbenes, and more (
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ,
      • Tohge T.
      • Watanabe M.
      • Hoefgen R.
      • Fernie A.R.
      The evolution of phenylpropanoid metabolism in the green lineage.
      ). Some of these phenolic compounds likely played critical roles during plant evolution, such as UV-absorbing phenolic compounds (e.g. sinapoyl derivatives), lignin, and sporopollenin during the evolution of land, vascular, and seed plants, respectively (
      • Weng J.-K.
      • Chapple C.
      The origin and evolution of lignin biosynthesis.
      ). A defense hormone salicylic acid and an electron carrier ubiquinone can be also synthesized from Phe in plants (
      • Soubeyrand E.
      • Johnson T.S.
      • Latimer S.
      • Block A.
      • Kim J.
      • Colquhoun T.A.
      • Butelli E.
      • Martin C.
      • Wilson M.A.
      • Basset G.J.
      The peroxidative cleavage of kaempferol contributes to the biosynthesis of the benzenoid moiety of ubiquinone in plants.
      • Block A.
      • Widhalm J.R.
      • Fatihi A.
      • Cahoon R.E.
      • Wamboldt Y.
      • Elowsky C.
      • Mackenzie S.A.
      • Cahoon E.B.
      • Chapple C.
      • Dudareva N.
      • Basset G.J.
      The origin and biosynthesis of the benzenoid moiety of ubiquinone (coenzyme Q) in Arabidopsis.
      ,
      • Shine M.B.
      • Yang J.-W.
      • El-Habbak M.
      • Nagyabhyru P.
      • Fu D.-Q.
      • Navarre D.
      • Ghabrial S.
      • Kachroo P.
      • Kachroo A.
      Cooperative functioning between phenylalanine ammonia lyase and isochorismate synthase activities contributes to salicylic acid biosynthesis in soybean.
      • Chen Z.
      • Zheng Z.
      • Huang J.
      • Lai Z.
      • Fan B.
      Biosynthesis of salicylic acid in plants.
      ). Significantly, up to 30% of total deposited carbon in plants can be directed toward Phe biosynthesis in vascular plants for the production of lignin and tannin (
      • Weiss U.
      ,
      • Boerjan W.
      • Ralph J.
      • Baucher M.
      Lignin biosynthesis.
      ). Thus, most plants have inherent capacity to produce a large quantity of phenolic natural products, and it is important to understand biochemical and genetic mechanisms underlying and controlling the production of the Phe precursor. Although efforts have been made to reduce content or modify composition of lignin, which impedes bio-ethanol production by microbial fermentation of cellulosic plant biomass (
      • Mottiar Y.
      • Vanholme R.
      • Boerjan W.
      • Ralph J.
      • Mansfield S.D.
      Designer lignins: harnessing the plasticity of lignification.
      ,
      • Wang P.
      • Dudareva N.
      • Morgan J.A.
      • Chapple C.
      Genetic manipulation of lignocellulosic biomass for bioenergy.
      • Loqué D.
      • Scheller H.V.
      • Pauly M.
      Engineering of plant cell walls for enhanced biofuel production.
      ), increased synthesis of Phe will enable production of a variety of Phe-derived natural products and other phenolic compounds (
      • Gottardi M.
      • Reifenrath M.
      • Boles E.
      • Tripp J.
      Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.
      ,
      • Noda S.
      • Kondo A.
      Recent advances in microbial production of aromatic chemicals and derivatives.
      ,
      • Shih P.M.
      Towards a sustainable bio-based economy: redirecting primary metabolism to new products with plant synthetic biology.
      ,
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ).
      Phe biosynthesis starts from the shikimate pathway, which converts erythrose 4-phosphate and phosphoenolpyruvate, derived from the pentose phosphate pathways and glycolysis, respectively, into chorismate, the last common precursor of all three aromatic amino acids—Phe, l-tyrosine, and l-tryptophan (Fig. 3) (
      • Tzin V.
      • Galili G.
      New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants.
      ,
      • Maeda H.
      • Dudareva N.
      The shikimate pathway and aromatic amino acid biosynthesis in plants.
      ). Although plants and microbes have a very similar tryptophan biosynthetic pathway (
      • Crawford I.P.
      Evolution of a biosynthetic pathway: the tryptophan paradigm.
      ,
      • Radwanski E.R.
      • Last R.L.
      Tryptophan biosynthesis and metabolism: biochemical and molecular genetics.
      ), plants have different biosynthetic routes for Phe and tyrosine from most microbes. In model microbes such as E. coli and yeast, chorismate is converted by chorismate mutase (CM) into prephenate, which undergoes dehydration or NAD(P)+-dependent oxidative decarboxylation into phenylpyruvate or 4-hydroxyphenylpyruvate, followed by transamination into Phe or tyrosine, respectively (gray pathways in Fig. 3) (
      • Bentley R.
      The shikimate pathway–a metabolic tree with many branches.
      ). In most plants, Phe and tyrosine biosynthesis predominantly proceeds via a different, nonproteogenic amino acid intermediate, l-arogenate, in the plastids. In the arogenate pathway, prephenate is first transaminated to arogenate (
      • Siehl D.L.
      • Connelly J.A.
      • Conn E.E.
      Tyrosine biosynthesis in Sorghum bicolor: characteristics of prephenate aminotransferase.
      • Bonner C.A.
      • Jensen R.A.
      Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris.
      ,
      • Maeda H.
      • Yoo H.
      • Dudareva N.
      Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate.
      • Graindorge M.
      • Giustini C.
      • Jacomin A.C.
      • Kraut A.
      • Curien G.
      • Matringe M.
      Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis.
      ), which then undergoes dehydration or NADP+-dependent oxidative decarboxylation into Phe and tyrosine, respectively (Fig. 3) (
      • Cho M.H.
      • Corea O.R.
      • Yang H.
      • Bedgar D.L.
      • Laskar D.D.
      • Anterola A.M.
      • Moog-Anterola F.A.
      • Hood R.L.
      • Kohalmi S.E.
      • Bernards M.A.
      • Kang C.
      • Davin L.B.
      • Lewis N.G.
      Phenylalanine biosynthesis in Arabidopsis thaliana–identification and characterization of arogenate dehydratases.
      • Maeda H.
      • Shasany A.K.
      • Schnepp J.
      • Orlova I.
      • Taguchi G.
      • Cooper B.R.
      • Rhodes D.
      • Pichersky E.
      • Dudareva N.
      RNAi suppression of arogenate dehydratase 1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.
      ,
      • Siehl D.L.
      • Conn E.E.
      Kinetic and regulatory properties of arogenate dehydratase in seedlings of Sorghum bicolor (L.) Moench.
      ,
      • Connelly J.A.
      • Conn E.E.
      Tyrosine biosynthesis in Sorghum bicolor: isolation and regulatory properties of arogenate dehydrogenase.
      • Rippert P.
      • Matringe M.
      Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.
      ).
      Figure thumbnail gr3
      Figure 3Evolutionary diversification of the aromatic amino acid biosynthetic pathways in plants. These aromatic amino acids, l-phenylalanine (Phe), l-tyrosine, and l-tryptophan, are required for protein synthesis in all organisms, but they are also used to synthesize diverse natural products (green) in plants. Plants synthesize Phe and tyrosine predominantly via the arogenate intermediate, unlike many microbes that make them via phenylpyruvate and 4-hydroxyphenylpyruvate intermediates, respectively (gray). Plants have an additional pathway to synthesize Phe in the cytosol. In certain plant lineages, the tyrosine and tryptophan pathways and their regulation have diversified: arogenate TyrA dehydrogenase (TyrAa) and anthranilate synthase û subunit (ASα) are typically strongly feedback-inhibited by tyrosine and tryptophan, respectively (red lines); however, their lineage-specific noncanonical counterparts (blue) are not and provide abundant tyrosine or anthranilate precursors for synthesis of downstream specialized metabolites (green). Dotted lines denote hypothesized but uncharacterized transport processes. Abbreviations: cCM, cytosolic chorismate mutase; pCM, plastidic CM; ncTyrAa, noncanonical TyrAa found in some dicots; TyrAaα, Caryophyllales-specific TyrAa. See the footnotes for other abbreviations introduced in the text.
      Some cyanobacteria also have the arogenate Phe and tyrosine biosynthetic pathways (
      • Legrand P.
      • Dumas R.
      • Seux M.
      • Rippert P.
      • Ravelli R.
      • Ferrer J.-L.
      • Matringe M.
      Biochemical characterization and crystal structure of Synechocystis arogenate dehydrogenase provide insights into catalytic reaction.
      • Bonner C.A.
      • Jensen R.A.
      • Gander J.E.
      • Keyhani N.O.
      A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis.
      ,
      • Graindorge M.
      • Giustini C.
      • Kraut A.
      • Moyet L.
      • Curien G.
      • Matringe M.
      Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms.
      • Hall G.C.
      • Flick M.B.
      • Gherna R.L.
      • Jensen R.A.
      Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.
      ); however, the plant pathways are not simply derived from cyanobacteria endosymbiosis, but are likely acquired through horizontal gene transfer from other bacterial lineages (
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ,
      • Schenck C.A.
      • Men Y.
      • Maeda H.A.
      Conserved molecular mechanism of TyrA dehydrogenase substrate specificity underlying alternative tyrosine biosynthetic pathways in plants and microbes.
      ). Prephenate aminotransferase (PPA-AT), which directs carbon flux toward the arogenate Phe and tyrosine pathways (Fig. 3) (
      • Maeda H.
      • Yoo H.
      • Dudareva N.
      Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate.
      ,
      • Graindorge M.
      • Giustini C.
      • Jacomin A.C.
      • Kraut A.
      • Curien G.
      • Matringe M.
      Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis.
      ,
      • Dal Cin V.
      • Tieman D.M.
      • Tohge T.
      • McQuinn R.
      • de Vos R.C.
      • Osorio S.
      • Schmelz E.A.
      • Taylor M.G.
      • Smits-Kroon M.T.
      • Schuurink R.C.
      • Haring M.A.
      • Giovannoni J.
      • Fernie A.R.
      • Klee H.J.
      Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit.
      ), evolved convergently in different microbial lineages from at least three distinct transaminase classes: Ib aspartate aminotransferase (e.g. in Chlorobi/Bacteroidetes, α-proteobacteria); N-succinyl-l,l-diaminopimelate aminotransferase (e.g. in actinobacteria); and branched-chain aminotransferase (e.g. in cyanobacteria) (
      • Graindorge M.
      • Giustini C.
      • Kraut A.
      • Moyet L.
      • Curien G.
      • Matringe M.
      Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms.
      ,
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ,
      • Giustini C.
      • Graindorge M.
      • Cobessi D.
      • Crouzy S.
      • Robin A.
      • Curien G.
      • Matringe M.
      Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase.
      ). Notably, plant PPA-ATs are most closely related to the Ib aspartate aminotransferase-type of Chlorobi/Bacteroidetes (
      • Graindorge M.
      • Giustini C.
      • Kraut A.
      • Moyet L.
      • Curien G.
      • Matringe M.
      Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms.
      ,
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ). Arogenate dehydratase (ADT) and dehydrogenase (TyrAa) enzymes catalyze subsequent reactions of PPA-AT and produce Phe and tyrosine, respectively, from arogenate (Fig. 3). Although model microbes, such as E. coli and yeast, only have prephenate dehydratase (PDT) and dehydrogenase (TyrAp), some bacteria have ADT and TyrAa enzymes, which likely evolved through enzyme neofunctionalization of PDT and TyrAp, respectively, and switch in their substrate specificity from prephenate to arogenate (
      • Hall G.C.
      • Flick M.B.
      • Gherna R.L.
      • Jensen R.A.
      Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.
      ,
      • Schenck C.A.
      • Men Y.
      • Maeda H.A.
      Conserved molecular mechanism of TyrA dehydrogenase substrate specificity underlying alternative tyrosine biosynthetic pathways in plants and microbes.
      ,
      • Bonner C.A.
      • Disz T.
      • Hwang K.
      • Song J.
      • Vonstein V.
      • Overbeek R.
      • Jensen R.A.
      Cohesion group approach for evolutionary analysis of TyrA, a protein family with wide-ranging substrate specificities.
      • Schenck C.A.
      • Chen S.
      • Siehl D.L.
      • Maeda H.A.
      Nonplastidic, tyrosine-insensitive prephenate dehydrogenases from legumes.
      ,
      • Kleeb A.C.
      • Kast P.
      • Hilvert D.
      A monofunctional and thermostable prephenate dehydratase from the archaeon Methanocaldococcus jannaschii.
      ,
      • El-Azaz J.
      • de la Torre F.
      • Ávila C.
      • Cánovas F.M.
      Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity.
      ,
      • Schenck C.A.
      • Holland C.K.
      • Schneider M.R.
      • Men Y.
      • Lee S.G.
      • Jez J.M.
      • Maeda H.A.
      Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.
      • Bross C.D.
      • Corea O.R.
      • Kaldis A.
      • Menassa R.
      • Bernards M.A.
      • Kohalmi S.E.
      Complementation of the pha2 yeast mutant suggests functional differences for arogenate dehydratases from Arabidopsis thaliana.
      ). Interestingly, all known plant ADTs are most closely related to those of Chlorobi/Bacteroidetes (
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ), suggesting that both PPA-AT and ADT enzymes required for the arogenate Phe pathway were transferred from Chlorobi/Bacteroidetes to the common ancestor of green algae and land plants. For tyrosine biosynthesis, plant TyrAa enzymes are most closely related to TyrAa enzymes of Spirochaetes and δ-proteobacteria (
      • Schenck C.A.
      • Holland C.K.
      • Schneider M.R.
      • Men Y.
      • Lee S.G.
      • Jez J.M.
      • Maeda H.A.
      Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.
      ), suggesting that yet another horizontal gene transfer contributed to the formation of the arogenate tyrosine biosynthetic pathway in plants.
      More recent studies further revealed that some plants have an additional microbial-like Phe biosynthetic pathway that operates in the cytosol (Fig. 3) (
      • Yoo H.
      • Widhalm J.R.
      • Qian Y.
      • Maeda H.
      • Cooper B.R.
      • Jannasch A.S.
      • Gonda I.
      • Lewinsohn E.
      • Rhodes D.
      • Dudareva N.
      An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase.
      ,
      • Qian Y.
      • Lynch J.H.
      • Guo L.
      • Rhodes D.
      • Morgan J.A.
      • Dudareva N.
      Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants.
      ), which might have provided robust production and homeostasis of Phe and diverse plant natural products derived from Phe. It has been known for a long time that many plants have both plastidic and cytosolic CM enzymes, the latter are not feedback-regulated by AAAs (
      • Eberhard J.
      • Ehrler T.T.
      • Epple P.
      • Felix G.
      • Raesecke H.R.
      • Amrhein N.
      • Schmid J.
      Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties.
      ,
      • Westfall C.S.
      • Xu A.
      • Jez J.M.
      Structural evolution of differential amino acid effector regulation in plant chorismate mutases.
      • Romero R.M.
      • Roberts M.F.
      • Phillipson J.D.
      Chorismate mutase in microorganisms and plants.
      ). However, in planta functions of the cytosolic isoforms had been enigmatic. Genetic down-regulation of the cytosolic CM gene in petunia flowers and wounded Arabidopsis leaves led to reduced production of Phe-derived compounds, e.g. phenylacetaldehyde. The cytosolic prephenate is further converted to phenylpyruvate by a partial PDT activity of some ADT isoforms having dual localization to the plastids and cytosol due to an alternative transcription start site (
      • Qian Y.
      • Lynch J.H.
      • Guo L.
      • Rhodes D.
      • Morgan J.A.
      • Dudareva N.
      Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants.
      ). Phenylpyruvate is then transaminated to Phe via cytosolic phenylpyruvate aminotransferase (PPY-AT) (Fig. 3) (
      • Yoo H.
      • Widhalm J.R.
      • Qian Y.
      • Maeda H.
      • Cooper B.R.
      • Jannasch A.S.
      • Gonda I.
      • Lewinsohn E.
      • Rhodes D.
      • Dudareva N.
      An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase.
      ). The cytosolic CM orthologs are present in all angiosperms, but appear to be absent in gymnosperms, ferns, mosses, and Amborella trichopoda, an early diverged flowering plant (
      • Qian Y.
      • Lynch J.H.
      • Guo L.
      • Rhodes D.
      • Morgan J.A.
      • Dudareva N.
      Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants.
      ,
      • Westfall C.S.
      • Xu A.
      • Jez J.M.
      Structural evolution of differential amino acid effector regulation in plant chorismate mutases.
      ,
      • Schenck C.A.
      • Maeda H.A.
      Tyrosine biosynthesis, metabolism, and catabolism in plants.
      ). Because plastidic ADT isoforms having PDT activity were found in Pinus pinaster (
      • El-Azaz J.
      • de la Torre F.
      • Ávila C.
      • Cánovas F.M.
      Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity.
      ), a part of the alternative phenylpyruvate Phe pathway may take place also in the plastids in nonflowering plants (
      • Pascual M.B.
      • El-Azaz J.
      • de la Torre F.N.
      • Cañas R.A.
      • Avila C.
      • Cánovas F.M.
      Biosynthesis and metabolic fate of phenylalanine in conifers.
      ). Thus, some variations exist in the phenylpyruvate Phe pathway at least for its enzyme subcellular localization among different plant groups. Future studies can explore potential variations of the Phe biosynthetic pathways among different plant lineages in both the arogenate and phenylpyruvate routes. Such variations, if any, will not only advance our understanding of the evolutionary history of this highly-active amino acid pathway in plants, but also provide useful tools to further optimize the supply of Phe precursor and the production of various phenolic compounds in plants.

      Recent and lineage-specific diversification of primary metabolism within the plant kingdom

      Besides the above ancient diversification of primary metabolism in the ancestor of Plantae, more recent diversifications of primary metabolism have been reported in specific lineages within the plant kingdom.

      Diversification of the tyrosine biosynthetic pathways and their regulation

      Besides serving as a protein building block, l-tyrosine is utilized in plants to synthesize various natural products, such as tocopherols, plastoquinone, betalain pigments, cyanogenic glycosides, catecholamines, and various alkaloids (
      • Schenck C.A.
      • Maeda H.A.
      Tyrosine biosynthesis, metabolism, and catabolism in plants.
      ). Unlike Phe-derived phenylpropanoids (e.g. lignin and flavonoids), these tyrosine-derived plant natural products are typically produced in specific plant lineages (
      • Schenck C.A.
      • Maeda H.A.
      Tyrosine biosynthesis, metabolism, and catabolism in plants.
      ), with the exceptions of tocopherols and plastoquinone ubiquitously found in plants and other photosynthetic microbes (
      • Norris S.R.
      • Barrette T.R.
      • DellaPenna D.
      Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation.
      • Cheng Z.
      • Sattler S.
      • Maeda H.
      • Sakuragi Y.
      • Bryant D.A.
      • DellaPenna D.
      Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes.
      ,
      • Soll J.
      • Kemmerling M.
      • Schultz G.
      Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions.
      • Maeda H.
      • DellaPenna D.
      Tocopherol functions in photosynthetic organisms.
      ). Also, in most plants, tyrosine biosynthesis is less active than Phe biosynthesis and is strictly feedback-inhibited by tyrosine at the TyrAa enzymes (Fig. 3) (
      • Maeda H.
      • Dudareva N.
      The shikimate pathway and aromatic amino acid biosynthesis in plants.
      ,
      • Connelly J.A.
      • Conn E.E.
      Tyrosine biosynthesis in Sorghum bicolor: isolation and regulatory properties of arogenate dehydrogenase.
      ,
      • Rippert P.
      • Matringe M.
      Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.
      ,
      • Schenck C.A.
      • Maeda H.A.
      Tyrosine biosynthesis, metabolism, and catabolism in plants.
      ,
      • Gaines C.G.
      • Byng G.S.
      • Whitaker R.J.
      • Jensen R.A.
      l-Tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes.
      ). A recent study, however, identified TyrAa enzymes having relaxed sensitivity to the tyrosine-mediated feedback inhibition in the plant order Caryophyllales (
      • Lopez-Nieves S.
      • Yang Y.
      • Timoneda A.
      • Wang M.
      • Feng T.
      • Smith S.A.
      • Brockington S.F.
      • Maeda H.A.
      Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales.
      ). Some Caryophyllales species uniquely produce red to yellow betalain pigments that replaced more ubiquitous Phe-derived anthocyanin pigments (
      • Polturak G.
      • Aharoni A.
      “La Vie en Rose”: biosynthesis, sources, and applications of betalain pigments.
      ,
      • Tanaka Y.
      • Sasaki N.
      • Ohmiya A.
      Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids.
      • Brockington S.F.
      • Walker R.H.
      • Glover B.J.
      • Soltis P.S.
      • Soltis D.E.
      Complex pigment evolution in the Caryophyllales.
      ). Betalain-producing species, such as beets, quinoa, spinach, and cacti, have at least two copies of recently-duplicated TyrAa enzymes, TyrAaα and TyrAaβ. The TyrAaα enzymes exhibit substantially reduced sensitivity to tyrosine inhibition with IC50 values of >1 mm as compared with ∼50 μm of the other TyrAaβ copies (
      • Lopez-Nieves S.
      • Yang Y.
      • Timoneda A.
      • Wang M.
      • Feng T.
      • Smith S.A.
      • Brockington S.F.
      • Maeda H.A.
      Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales.
      ) and typical TyrAaα enzymes of plants (
      • Connelly J.A.
      • Conn E.E.
      Tyrosine biosynthesis in Sorghum bicolor: isolation and regulatory properties of arogenate dehydrogenase.
      ,
      • Rippert P.
      • Matringe M.
      Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.
      ,
      • Gaines C.G.
      • Byng G.S.
      • Whitaker R.J.
      • Jensen R.A.
      l-Tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes.
      ). Some Caryophyllales lineages, such as the Caryophyllaceae family that includes carnation, reverted back to anthocyanin pigmentation (
      • Brockington S.F.
      • Walker R.H.
      • Glover B.J.
      • Soltis P.S.
      • Soltis D.E.
      Complex pigment evolution in the Caryophyllales.
      ,
      • Brockington S.F.
      • Yang Y.
      • Gandia-Herrero F.
      • Covshoff S.
      • Hibberd J.M.
      • Sage R.F.
      • Wong G.K.
      • Moore M.J.
      • Smith S.A.
      Lineage-specific gene radiations underlie the evolution of novel betalain pigmentation in Caryophyllales.
      ) and also down-regulated or lost the TyrAaα gene (
      • Lopez-Nieves S.
      • Yang Y.
      • Timoneda A.
      • Wang M.
      • Feng T.
      • Smith S.A.
      • Brockington S.F.
      • Maeda H.A.
      Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales.
      ). Further evolutionary analyses utilizing transcriptome data of over 100 Caryophyllales species, combined with their TyrA enzyme characterization, revealed that the de-regulated TyrAaα evolved prior to the emergence of betalain pigmentation (
      • Lopez-Nieves S.
      • Yang Y.
      • Timoneda A.
      • Wang M.
      • Feng T.
      • Smith S.A.
      • Brockington S.F.
      • Maeda H.A.
      Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales.
      ). The results suggest that a lineage-specific de-regulation of tyrosine biosynthesis contributed to the evolution of a downstream natural product pathway, betalain biosynthesis. The finding also suggests that enhanced supply of the tyrosine precursor is important for efficient production of tyrosine-derived natural products in plants.
      A further diversification of the tyrosine biosynthetic pathway was found in other plant lineages. Earlier biochemical studies detected microbial-like prephenate-specific TyrAp activity in some plants, all belonging to the legume family (
      • Rubin J.L.
      • Jensen R.A.
      Enzymology of l-tyrosine biosynthesis in mung bean (Vigna radiata [L.] Wilczek).
      ,
      • Gamborg O.L.
      • Keeley F.W.
      Aromatic metabolism in plants. I. A study of the prephenate dehydrogenase from bean plants.
      • Siehl D.L.
      ). More recently, the genes and enzymes responsible for the TyrAp activity were identified from soybean and Medicago and found to be highly specific to prephenate than arogenate substrate (kcat/Km of 100–200 versus 0.05–0.5 mm−1 s−1) (
      • Schenck C.A.
      • Chen S.
      • Siehl D.L.
      • Maeda H.A.
      Nonplastidic, tyrosine-insensitive prephenate dehydrogenases from legumes.
      ,
      • Schenck C.A.
      • Holland C.K.
      • Schneider M.R.
      • Men Y.
      • Lee S.G.
      • Jez J.M.
      • Maeda H.A.
      Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.
      ). Unlike TyrAa enzymes (
      • Rippert P.
      • Puyaubert J.
      • Grisollet D.
      • Derrier L.
      • Matringe M.
      Tyrosine and phenylalanine are synthesized within the plastids in Arabidopsis.
      ), these legume TyrAp enzymes are localized outside of the plastids and are completely insensitive to tyrosine feedback inhibition (Fig. 3) (
      • Schenck C.A.
      • Chen S.
      • Siehl D.L.
      • Maeda H.A.
      Nonplastidic, tyrosine-insensitive prephenate dehydrogenases from legumes.
      ). The phylogenetic analyses of these legume TyrAp genes as well as other plant and microbial TyrA genes revealed that legume TyrAp orthologs were derived from two events of gene duplication within the plant kingdom, with the recent one specifically occurring in the legume family and giving rise to the prephenate-specific TyrAp (
      • Schenck C.A.
      • Chen S.
      • Siehl D.L.
      • Maeda H.A.
      Nonplastidic, tyrosine-insensitive prephenate dehydrogenases from legumes.
      ). Interestingly, the first duplication event at the base of angiosperms (flowering plants) led to noncanonical TyrAa enzymes that are found in legumes and some other eudicots, but not in all plants. This third type of plant TyrA enzymes prefers arogenate substrate (and thus noncanonical, ncTyrAa, Fig. 3), is partially insensitive to tyrosine inhibition, and is likely localized outside of the plastids, judging from the lack of a plastid transit peptide (
      • Schenck C.A.
      • Holland C.K.
      • Schneider M.R.
      • Men Y.
      • Lee S.G.
      • Jez J.M.
      • Maeda H.A.
      Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.
      ). Thus, legumes have at least three pathways to synthesize tyrosine. Additionally, a fourth pathway of tyrosine biosynthesis exists in lycophytes (mosses) and gymnosperms, which have Phe hydroxylase (PheH) enzymes that are localized in the plastids and can convert Phe into tyrosine in a 10-formyltetrahydrofolate-dependent manner (Fig. 3) (
      • Pribat A.
      • Noiriel A.
      • Morse A.M.
      • Davis J.M.
      • Fouquet R.
      • Loizeau K.
      • Ravanel S.
      • Frank W.
      • Haas R.
      • Reski R.
      • Bedair M.
      • Sumner L.W.
      • Hanson A.D.
      Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts.
      ). Although the physiological functions of these alternative tyrosine biosynthetic pathways are largely unknown, the PheH genes are up-regulated together with tyrosine degradation pathway genes under drought stress. Thus, PheH may allow catabolism of Phe via tyrosine in nonflowering plants (
      • Frelin O.
      • Dervinis C.
      • Wegrzyn J.L.
      • Davis J.M.
      • Hanson A.D.
      Drought stress in Pinus taeda L. induces coordinated transcript accumulation of genes involved in the homogentisate pathway.
      ). Also, genes encoding the tyrosine-insensitive TyrAp enzymes were found to be highly expressed in several Inga species, tropical legume trees, that accumulate extremely high levels of tyrosine and/or tyrosine-derived natural products (e.g. tyrosine-gallate conjugates) at >10% of dry weight (
      • Lokvam J.
      • Brenes-Arguedas T.
      • Lee J.S.
      • Coley P.D.
      • Kursar T.A.
      Allelochemic function for a primary metabolite: the case of l-tyrosine hyper-production in Inga umbellifera (Fabaceae).
      ,
      • Coley P.D.
      • Endara M.-J.
      • Ghabash G.
      • Kidner C.A.
      • Nicholls J.A.
      • Pennington R.T.
      • Mills A.G.
      • Soule A.J.
      • Lemes M.R.
      • Stone G.N.
      • Kursar T.A.
      Macroevolutionary patterns in overexpression of tyrosine: an anti-herbivore defence in a speciose tropical tree genus, Inga (Fabaceae).
      ). Therefore, these lineage-specific alternative tyrosine biosynthetic pathways and their regulation likely play important roles in the production and evolution of downstream specialized metabolites in plants.

      Lineage-specific de-regulation of anthranilate biosynthesis

      The tryptophan branch of the aromatic amino acid pathways also provides precursors to synthesize various plant-specialized metabolites, such as tryptophan-derived indole alkaloids and glucosinolates (
      • Kutchan T.M.
      Alkaloid biosynthesis: the basis for metabolic engineering of medicinal plants.
      • De Luca V.
      • Salim V.
      • Levac D.
      • Atsumi S.M.
      • Yu F.
      Discovery and functional analysis of monoterpenoid indole alkaloid pathways in plants.
      ,
      • Hansen B.G.
      • Halkier B.A.
      New insight into the biosynthesis and regulation of indole compounds in Arabidopsis thaliana.
      • Glawischnig E.
      Camalexin.
      ), anthranilate-derived anthranilamide phytoalexins (
      • Niemann G.J.
      The anthranilamide phytoalexins of the Caryophyllaceae and related compounds.
      ), and indole-derived benzoxazinones (Fig. 3) (
      • Frey M.
      • Schullehner K.
      • Dick R.
      • Fiesselmann A.
      • Gierl A.
      Benzoxazinoid biosynthesis, a model for evolution of secondary metabolic pathways in plants.
      ,
      • Frey M.
      • Chomet P.
      • Glawischnig E.
      • Stettner C.
      • Grün S.
      • Winklmair A.
      • Eisenreich W.
      • Bacher A.
      • Meeley R.B.
      • Briggs S.P.
      • Simcox K.
      • Gierl A.
      Analysis of a chemical plant defense mechanism in grasses.
      ). Some species of the Rutaceae family produce anthranilate-derived acridone and furoquinoline alkaloids, some of which have antimicrobial activities and are strongly induced upon elicitor treatment (
      • Eilert U.
      • Wolters B.
      Elicitor induction of S-adenosyl-l-methionine: anthranilic acid N-methyltransferase activity in cell suspension and organ cultures of Ruta graveolens L.
      ). In Ruta graveolens, the induction of acridone alkaloid accumulation correlates with increased activity of anthranilate synthase (AS) (
      • Bohlmann J.
      • Eilert U.
      Elicitor-induced secondary metabolism in Ruta graveolens L–Role of chorismate utilizing enzymes.
      ), which catalyzes the first step of tryptophan biosynthesis and converts chorismate into anthranilate (Fig. 3) (
      • Romero R.M.
      • Roberts M.F.
      • Phillipson J.D.
      Anthranilate synthase in microorganisms and plants.
      ). AS is composed of two distinct subunits, ASα and ASβ, the former is usually strictly regulated by the pathway product, tryptophan (
      • Romero R.M.
      • Roberts M.F.
      • Phillipson J.D.
      Anthranilate synthase in microorganisms and plants.
      ,
      • Poulsen C.
      • Bongaerts R.J.
      • Verpoorte R.
      Purification and characterization of anthranilate synthase from Catharanthus roseus.
      • Bernasconi P.
      • Walters E.W.
      • Woodworth A.R.
      • Siehl D.L.
      • Stone T.E.
      • Subramanian M.V.
      Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.
      ). It was found that R. graveolens has two ASα copies, one of which is induced under pathogen infection and is not inhibited by tryptophan, whereas the other copy is noninducible and inhibited by tryptophan (
      • Bohlmann J.
      • Lins T.
      • Martin W.
      • Eilert U.
      Anthranilate synthase from Ruta graveolens. Duplicated AS α genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.
      ,
      • Bohlmann J.
      • DeLuca V.
      • Eilert U.
      • Martin W.
      Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes.
      ). Thus, the lineage-specific duplication and neofunctionalization gave rise to the inducible and feedback-insensitive ASα enzyme, which diverts carbon flow away from tryptophan biosynthesis and provides the anthranilate precursor for the formation of acridone alkaloids in this plant (Fig. 3). Furthermore, the distinct temporal and possibly spatial expression patterns of ASα1 and ASα2 (
      • Bohlmann J.
      • Lins T.
      • Martin W.
      • Eilert U.
      Anthranilate synthase from Ruta graveolens. Duplicated AS α genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.
      ,
      • Bohlmann J.
      • DeLuca V.
      • Eilert U.
      • Martin W.
      Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes.
      ) likely allow fine regulation of carbon allocation between biosynthesis of tryptophan- and anthranilate-derived plant natural products. Although the phylogenetic distribution and evolutionary history of the feedback-insensitive ASα enzyme are currently unknown, the emergence of the lineage-specific ASα likely provided a unique opportunity in some Rutaceae lineages to produce anthranilate-derived plant natural products.

      Impacts of altered branched-chain amino acid biosynthesis on acylsugar specialized metabolism

      Branched-chain amino acid biosynthesis has been also altered in a specific plant lineage, which impacted its downstream specialized metabolic pathways. Isopropylmalate synthase (IPMS) catalyzes the committed reaction of l-leucine biosynthesis, the conversion of 3-methyl-2-oxobutanoate (3MOB) into 2-isopropylmalate (Fig. 4) (
      • de Kraker J.-W.
      • Luck K.
      • Textor S.
      • Tokuhisa J.G.
      • Gershenzon J.
      Two Arabidopsis genes (IPMS1 and IPMS2) encode isopropylmalate synthase, the branchpoint step in the biosynthesis of leucine.
      ). Because 3MOB is also used for l-valine biosynthesis, IPMS is usually feedback inhibited by leucine, controlling carbon allocation between the leucine and valine biosynthetic pathways (
      • de Kraker J.-W.
      • Gershenzon J.
      From amino acid to glucosinolate biosynthesis: protein sequence changes in the evolution of methylthioalkylmalate synthase in Arabidopsis.
      ,
      • Koon N.
      • Squire C.J.
      • Baker E.N.
      Crystal structure of LeuA from Mycobacterium tuberculosis, a key enzyme in leucine biosynthesis.
      ). Interestingly, the IPMS3 isoform has been altered in wild and cultivated tomatoes, Solanum pennellii and Solanum lycopersicum, respectively, at its C-terminal regulatory domain. The IPMS3 isoform of S. lycopersicum is truncated and hence insensitive to leucine-mediated feedback inhibition (green, Fig. 4), whereas that of S. pennellii is further truncated into its catalytic domain and has lost its enzyme activity (blue, Fig. 4) (
      • Ning J.
      • Moghe G.D.
      • Leong B.
      • Kim J.
      • Ofner I.
      • Wang Z.
      • Adams C.
      • Jones A.D.
      • Zamir D.
      • Last R.L.
      A feedback-insensitive isopropylmalate synthase affects acylsugar composition in cultivated and wild tomato.
      ). As a result, more carbon flows toward leucine and valine biosynthesis in S. pennellii and S. lycopersicum, respectively. Notably, the changes in leucine and valine biosynthesis at IPMS3 likely underlie the structural differences in their acylsugar-specialized metabolites (
      • Ning J.
      • Moghe G.D.
      • Leong B.
      • Kim J.
      • Ofner I.
      • Wang Z.
      • Adams C.
      • Jones A.D.
      • Zamir D.
      • Last R.L.
      A feedback-insensitive isopropylmalate synthase affects acylsugar composition in cultivated and wild tomato.
      ), which accumulate in the glandular trichomes of Solanaceae plants as insecticides (
      • Fan P.
      • Leong B.J.
      • Last R.L.
      Tip of the trichome: evolution of acylsugar metabolic diversity in Solanaceae.
      ). The acylsugars of S. pennellii and S. lycopersicum have 2-methylpropanoic and 3-methylbutanoic acid (iC4 and iC5) acyl chains, which are derived from the corresponding branched-chain keto acids of valine and leucine, 3MOB and 4-methyl-2-oxopropanoate (4MOP), respectively (Fig. 4). These examples highlight the role of primary metabolite precursor supply in the formation and potentially the evolution of their downstream specialized metabolites in specific plant lineages.
      Figure thumbnail gr4
      Figure 4Modulations of valine and leucine biosynthesis impact the composition of acylsugar-specialized metabolites in tomato plants. Isopropylmalate synthase (IPMS) catalyzes the committed step of l-leucine biosynthesis and is typically feedback-inhibited by leucine (red line). S. lycopersicum (cultivated tomato) has an IPMS3 enzyme (SlIPMS3) that is truncated at its C-terminal regulatory domain and thus insensitive to leucine, leading to active synthesis of 3-methylbutanoate (iC5)-acylsugar chains derived from the keto acid of leucine, 4-methyl-2-oxopentanoate (4MOP, green). In contrast, the IPMS3 enzyme of S. pennellii (wild tomato) is further truncated into the catalytic domain and lacks its activity, leading to active synthesis of 2-methylpropanoate (iC4)-acylsugar chains derived from the keto acid of valine, 3-methyl-2-oxobutanoate (3MOB, blue).

      Genetic and molecular basis of primary metabolic diversity

      With the advent of genome editing, the identification of alleles (mutations) underlying key metabolic innovations (e.g. primary metabolic diversification) is critical for introducing a specific genetic modification(s) for rational and precise metabolic engineering. Thus, we now have a strong rationale to go beyond gene discovery and conduct structure–function analyses of encoded enzymes to identify key amino acid residues and mutations. This is particularly crucial when we try to manipulate plant primary metabolism, which is highly sensitive to genetic modification due to its tight integration with complex metabolic networks and plant growth and physiology.

      Phylogeny-guided structure–function analysis to identify mutations underlying key evolutionary innovations of plant metabolism

      Comparative analyses of enzyme variants from different plant species and accessions have identified causal mutations responsible for unique biochemical properties (e.g. substrate and product specificities) that evolved in certain plant lineages (
      • Louie G.V.
      • Bowman M.E.
      • Moffitt M.C.
      • Baiga T.J.
      • Moore B.S.
      • Noel J.P.
      Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonia-lyases.
      • Watts K.T.
      • Mijts B.N.
      • Lee P.C.
      • Manning A.J.
      • Schmidt-Dannert C.
      Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family.
      ,
      • Xu M.
      • Wilderman P.R.
      • Peters R.J.
      Following evolution's lead to a single residue switch for diterpene synthase product outcome.
      • Kang J.-H.
      • Gonzales-Vigil E.
      • Matsuba Y.
      • Pichersky E.
      • Barry C.S.
      Determination of residues responsible for substrate and product specificity of Solanum habrochaites short-chain cis-prenyltransferases.
      ). A rapidly increasing number of genome and transcriptome sequences (
      • Cheng S.
      • Melkonian M.
      • Smith S.A.
      • Brockington S.
      • Archibald J.M.
      • Delaux P.-M.
      • Li F.-W.
      • Melkonian B.
      • Mavrodiev E.V.
      • Sun W.
      • Fu Y.
      • Yang H.
      • Soltis D.E.
      • Graham S.W.
      • Soltis P.S.
      • et al.
      10KP: a phylodiverse genome sequencing plan.
      ,
      • Kersey P.J.
      Plant genome sequences: past, present, future.
      • Matasci N.
      • Hung L.-H.
      • Yan Z.
      • Carpenter E.J.
      • Wickett N.J.
      • Mirarab S.
      • Nguyen N.
      • Warnow T.
      • Ayyampalayam S.
      • Barker M.
      • Burleigh J.G.
      • Gitzendanner M.A.
      • Wafula E.
      • Der J.P.
      • dePamphilis C.W.
      • et al.
      Data access for the 1,000 plants (1KP) project.
      ) is further enabling “phylogeny-guided” structure–function analyses, which determine and utilize evolutionary transitions (i.e. gain and loss) of a lineage-specific enzyme property (
      • Fan P.
      • Moghe G.D.
      • Last R.L.
      Comparative biochemistry and in vitro pathway reconstruction as powerful partners in studies of metabolic diversity.
      ). Two groups of closely-related protein sequences but with distinct biochemical characteristics can be compared to identify residues that are conserved only in one group. The key is to utilize a large number of genome/transcriptome sequences and determine precise phylogenetic boundaries for the presence and absence of a certain biochemical property to pinpoint responsible residues. Based on a protein crystal structure or a structure model, these candidate residues can be further prioritized for validation by site-directed mutagenesis followed by biochemical analyses. This approach not only reduces the number of sites for mutagenesis but also informs which particular amino acid to mutate to, out of 19 amino acids. This method was recently employed to uncover metabolic enzyme diversification underlying chemical diversity of acylsugar-specialized metabolites, among the closely-related species of the Solanum genus and the Solanaceae family (
      • Fan P.
      • Moghe G.D.
      • Last R.L.
      Comparative biochemistry and in vitro pathway reconstruction as powerful partners in studies of metabolic diversity.
      ,
      • Fan P.
      • Miller A.M.
      • Liu X.
      • Jones A.D.
      • Last R.L.
      Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity.
      • Fan P.
      • Miller A.M.
      • Schilmiller A.L.
      • Liu X.
      • Ofner I.
      • Jones A.D.
      • Zamir D.
      • Last R.L.
      In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network.
      ). A similar approach has been also utilized to uncover the genetic basis of primary metabolic diversity in plants, as described below.

      Molecular basis of the evolution of plant prephenate aminotransferases and the arogenate Phe and tyrosine pathways

      PPA-ATs catalyze the committed step of the arogenate pathway of Phe and tyrosine biosynthesis (Fig. 3) (
      • Siehl D.L.
      • Connelly J.A.
      • Conn E.E.
      Tyrosine biosynthesis in Sorghum bicolor: characteristics of prephenate aminotransferase.
      • Bonner C.A.
      • Jensen R.A.
      Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris.
      ,
      • Maeda H.
      • Yoo H.
      • Dudareva N.
      Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate.
      • Graindorge M.
      • Giustini C.
      • Jacomin A.C.
      • Kraut A.
      • Curien G.
      • Matringe M.
      Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis.
      ,
      • Dal Cin V.
      • Tieman D.M.
      • Tohge T.
      • McQuinn R.
      • de Vos R.C.
      • Osorio S.
      • Schmelz E.A.
      • Taylor M.G.
      • Smits-Kroon M.T.
      • Schuurink R.C.
      • Haring M.A.
      • Giovannoni J.
      • Fernie A.R.
      • Klee H.J.
      Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit.
      ) and are found in plants and some microbes (
      • Graindorge M.
      • Giustini C.
      • Kraut A.
      • Moyet L.
      • Curien G.
      • Matringe M.
      Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms.
      ,
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ,
      • Giustini C.
      • Graindorge M.
      • Cobessi D.
      • Crouzy S.
      • Robin A.
      • Curien G.
      • Matringe M.
      Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase.
      ). Biochemical characterization of PPA-AT homologs from various plants and microbes determined the phylogenetic distribution of their functional orthologs that are capable of transaminating prephenate (
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ,
      • Giustini C.
      • Graindorge M.
      • Cobessi D.
      • Crouzy S.
      • Robin A.
      • Curien G.
      • Matringe M.
      Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase.
      ). The peptide sequence comparison of closely-related aminotransferases with and without prephenate transamination activity identified two amino acid residues required for this activity. Mutating these two residues converted Arabidopsis PPA-AT to a general aromatic amino acid aminotransferase having broad substrate specificity (
      • Dornfeld C.
      • Weisberg A.J.
      • K C R.
      • Dudareva N.
      • Jelesko J.G.
      • Maeda H.A.
      Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway.
      ). X-ray crystal structure analyses of plant and bacterial PPA-ATs further revealed the molecular basis of prephenate substrate recognition and identified two additional residues that further enhance prephenate specificity (
      • Giustini C.
      • Graindorge M.
      • Cobessi D.
      • Crouzy S.
      • Robin A.
      • Curien G.
      • Matringe M.
      Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase.
      ,
      • Holland C.K.
      • Berkovich D.A.
      • Kohn M.L.
      • Maeda H.
      • Jez J.M.
      Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis.
      ). Thus, these residues likely played key roles in the evolution of PPA-ATs that allow plants to synthesize Phe and tyrosine via the arogenate pathway.

      Determinants of TyrA dehydrogenase substrate specificity and feedback regulation

      Phylogenetic sampling of TyrA orthologs across the eudicots also identified key residues underlying the evolutionary transition and emergence of prephenate dehydrogenase (TyrAp) from arogenate dehydrogenase (TyrAa) within the legume family (
      • Schenck C.A.
      • Holland C.K.
      • Schneider M.R.
      • Men Y.
      • Lee S.G.
      • Jez J.M.
      • Maeda H.A.
      Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.
      ). Sequence comparisons of hundreds of protein sequences before and after the evolutionary transition from TyrAa to TyrAp identified a highly-conserved acidic aspartate residue that is responsible for the arogenate specificity and tyrosine sensitivity of TyrAa enzymes. Further crystal structure analyses demonstrated that the aspartate residue directly interacts with the side-chain amine that is present in arogenate and tyrosine but is absent in prephenate (Fig. 3). Furthermore, introducing the aspartate residue in a feedback-inhibited canonical TyrAa enzyme from Arabidopsis reduced arogenate substrate specificity and introduced prephenate dehydrogenase activity while simultaneously relaxing the tyrosine feedback inhibition (
      • Schenck C.A.
      • Holland C.K.
      • Schneider M.R.
      • Men Y.
      • Lee S.G.
      • Jez J.M.
      • Maeda H.A.
      Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.
      ). Thus, the identified residue can now be utilized to relax negative regulation of tyrosine biosynthesis in nonlegume plants and to enhance tyrosine supply and production of its downstream specialized metabolites. Of course, the situation may be more complex in other cases due to potential epistatic interactions between different amino acid residues. For example, introduction of a functional mutation(s) may not be sufficient to provide a desired biochemical property, if a background enzyme to be engineered either lacks a permissive mutation(s) or carries a constraining mutation(s), which is required for or prevents the functionality of the introduced mutation(s), respectively (
      • Bloom J.D.
      • Gong L.I.
      • Baltimore D.
      Permissive secondary mutations enable the evolution of influenza oseltamivir resistance.
      • Harms M.J.
      • Thornton J.W.
      Evolutionary biochemistry: revealing the historical and physical causes of protein properties.
      ,
      • Breen M.S.
      • Kemena C.
      • Vlasov P.K.
      • Notredame C.
      • Kondrashov F.A.
      Epistasis as the primary factor in molecular evolution.
      ,
      • Starr T.N.
      • Thornton J.W.
      Epistasis in protein evolution.
      • Bershtein S.
      • Segal M.
      • Bekerman R.
      • Tokuriki N.
      • Tawfik D.S.
      Robustness–epistasis link shapes the fitness landscape of a randomly drifting protein.
      ). Nevertheless, the phylogeny-guided structure–function analyses provide powerful tools to identify key evolutionary innovations and natural mutations underlying both primary and specialized metabolic diversification. The identified mutations can then be used to conduct targeted metabolic engineering to redesign specific metabolic traits, such as optimization of primary metabolite precursor supply, as discussed in the following section.

      Harnessing primary metabolic diversity for building and optimizing plant chemical production platforms

      The fundamental knowledge about the evolutionary diversification of primary metabolism in plants can be utilized to build plant chassis, or chemical production platforms, and to further optimize their primary metabolism for efficient production of certain classes of natural products. Aforementioned studies suggest that the precursor supply needs to be optimized for efficient production of specialized metabolites, such as ones derived from tyrosine and anthranilate, which typically accumulate at low concentrations in most plants. Indeed, simultaneous expression of the beet TyrAa and the downstream betalain biosynthetic enzymes in Nicotiana benthamiana transient expression system demonstrated that enhanced supply of the tyrosine precursor increases the production of betalains derived from tyrosine (
      • Timoneda A.
      • Sheehan H.
      • Feng T.
      • Lopez-Nieves S.
      • Maeda H.A.
      • Brockington S.
      Redirecting primary metabolism to boost production of tyrosine-derived specialised metabolites in planta.
      ). Even for synthesis of terpenoid and phenylpropanoid compounds that are supported by the dual pathways of IPP and Phe biosynthesis, respectively, in plants, coordinated up-regulation of upstream primary metabolism (“push”) and downstream natural product pathways (“pull”) appears to be important (
      • Wu S.
      • Schalk M.
      • Clark A.
      • Miles R.B.
      • Coates R.
      • Chappell J.
      Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants.
      ,
      • Zhang Y.
      • Butelli E.
      • Alseekh S.
      • Tohge T.
      • Rallapalli G.
      • Luo J.
      • Kawar P.G.
      • Hill L.
      • Santino A.
      • Fernie A.R.
      • Martin C.
      Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato.
      ,
      • Reed J.
      • Stephenson M.J.
      • Miettinen K.
      • Brouwer B.
      • Leveau A.
      • Brett P.
      • Goss R.J.M.
      • Goossens A.
      • O'Connell M.A.
      • Osbourn A.
      A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules.
      • Lange B.M.
      • Mahmoud S.S.
      • Wildung M.R.
      • Turner G.W.
      • Davis E.M.
      • Lange I.
      • Baker R.C.
      • Boydston R.A.
      • Croteau R.B.
      Improving peppermint essential oil yield and composition by metabolic engineering.
      ). For example, the expression of AtMYB12, which activates the pentose phosphate, shikimate, and Phe pathways, in the tomato background expressing Delila and Rosea 1 transcription factors that activate anthocyanin biosynthesis led to a further increase in anthocyanin accumulation (
      • Zhang Y.
      • Butelli E.
      • Alseekh S.
      • Tohge T.
      • Rallapalli G.
      • Luo J.
      • Kawar P.G.
      • Hill L.
      • Santino A.
      • Fernie A.R.
      • Martin C.
      Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato.
      ).
      Some microbial enzymes, which are often not subjected to regulation in plants, were introduced into plants to enhance accumulation of some primary metabolites, such as amino acids (
      • Shaul O.
      • Galili G.
      Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase.
      ,
      • Falco S.C.
      • Guida T.
      • Locke M.
      • Mauvais J.
      • Sanders C.
      • Ward R.T.
      • Webber P.
      Transgenic canola and soybean seeds with increased lysine.
      ,
      • Tzin V.
      • Malitsky S.
      • Aharoni A.
      • Galili G.
      Expression of a bacterial bi-functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis.
      ). However, drastic alterations in primary metabolism often negatively impact plant growth and development, especially in vegetative tissues where many developmental processes are still taking place (
      • Shaul O.
      • Galili G.
      Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase.
      • Bartlem D.
      • Lambein I.
      • Okamoto T.
      • Itaya A.
      • Uda Y.
      • Kijima F.
      • Tamaki Y.
      • Nambara E.
      • Naito S.
      Mutation in the threonine synthase gene results in an over-accumulation of soluble methionine in Arabidopsis.
      ,
      • Hacham Y.
      • Avraham T.
      • Amir R.
      The N-terminal region of Arabidopsis cystathionine γ-synthase plays an important regulatory role in methionine metabolism.
      ,
      • Li X.
      • Bonawitz N.D.
      • Weng J.K.
      • Chapple C.
      The growth reduction associated with repressed lignin biosynthesis in Arabidopsis thaliana is independent of flavonoids.
      ,
      • Kim J.I.
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      Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase.
      ). For example, expression of completely tyrosine-insensitive bacterial TyrAa or TyrAp enzyme in Arabidopsis severely compromised plant growth (
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      Expression of a bacterial bi-functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis.
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      Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana.
      ). One way to overcome this issue is to use tissue-specific promoters, which led to many successful cases of metabolic engineering in seeds and fruits (
      • Zhang Y.
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      Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato.
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      Transgenic canola and soybean seeds with increased lysine.
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      Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed-specific expression of lysine-insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii.