Engineering Plant Secondary Metabolism in Microbial Systems

Secondary metabolites are broadly defined as natural products synthesized by an organism that are not essential to support growth and life. The plant kingdom manufactures over 200,000 distinct chemical compounds, most of which arise from specialized metabolism. While these compounds play important roles in interspecies competition and defense, many plant natural products have been exploited for use as medicines, fragrances, flavors, nutrients, repellants, and colorants. Despite this vast chemical diversity, many secondary metabolites are present at very low concentrations in planta, eliminating crop-based manufacturing as a means of attaining these important products. The structural and stereochemical complexity of specialized metabolites hinders most attempts to access these compounds using chemical synthesis. Although native plants can be engineered to accumulate target pathway metabolites (Zhou et al., 2009; Glenn et al., 2013; Lange and Ahkami, 2013; Wilson and Roberts, 2014; Tatsis and O’Connor, 2016), metabolic engineering is technically more challenging in plants than in microbes. Advancements in synthetic biology have stimulated the synthesis of valuable natural products in tractable laboratory microbes by interfacing plant secondary pathways with core host metabolism. Microbial synthesis overcomes many of the obstacles hindering traditional chemical synthesis and plant metabolic engineering, thus providing an alternative avenue for exploring plant specialized pathways. This Update provides a brief overview of engineering plant secondary metabolism in microbial systems. We briefly outline biosynthetic pathways mediating formation of the major classes of natural products with an emphasis on high-value terpenoids, alkaloids, phenylpropanoids, and polyketides. We also highlight common themes, strategies, and challenges underlying efforts to reconstruct and engineer these pathways in microbial hosts. We focus chiefly on de novo biosynthetic approaches in which plant specialized metabolites are synthesized directly from sugar feedstocks rather than supplemented precursors or intermediates. Readers are directed to a selection of pioneering supplementation studies within the context of microbially sourced plant natural products (Becker et al., 2003; Kaneko et al., 2003; Yan et al., 2005; Watts et al., 2006; Leonard et al., 2007, 2008; Hawkins and Smolke, 2008; Fossati et al., 2014, 2015). Terpenoids (also called isoprenoids) are the largest class of plant secondary metabolites, comprising more than 50,000 natural products (Connolly and Hill, 1991). Whereas the terpene classification refers strictly to hydrocarbons, terpenoids possess a range of chemical functionalities. The central precursors geranyl pyrophosphate (GPP; C10), farnesyl pyrophosphate (FPP; C15), and geranylgeranylpyrophosphate (GGPP; C20) form the structural basis of most higher order terpenoids (Fig. 1). Plant terpene synthases convert these pyrophosphate intermediates to terpenes, which are then functionalized in downstream reactions. Interfacing plant terpenoid secondary metabolic pathways with microbial metabolism. Plant secondary metabolic reactions and pathways (green) are shown linked to core microbial metabolism (beige shading; black font). S. cerevisiae is shown as the prospective host species. Key yeast enzymes discussed in the main text are shown. Abbreviations: Bts1, geranylgeranyl diphosphate synthase; DMAPP, dimethylallyl pyrophosphate; Erg9, squalene synthase; Erg20, farnesyl pyrophosphate synthetase; FPP, farnesyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl pyrophosphate; MEV, mevalonate pathway. GPP forms the backbone of most monoterpenoids (C10), including linear (geraniol and linalool) and cyclic (camphor and eucalyptol) terpenoids, as well as monoterpenes (limonene and pinene). Geraniol can be modified to the pest repellant citronellol, while limonene gives rise to menthol, a flavoring agent and decongestant. GPP also supplies the prenyl group in the biosynthesis of cannabinoids (Ahmed et al., 2015; Vickery et al., 2016), a class of natural products with promising pharmaceutical properties (Aizpurua-Olaizola et al., 2016). Sesquiterpenes (C15) are derived from FPP, itself generated through condensation of GPP and isopentenyl pyrophosphate (IPP) in yeast (Saccharomyces cerevisiae) or directly from IPP and dimethylallyl pyrophosphate units in plants. The sesquiterpene amorphadiene gives rise to the antimalarial drug precursor artemisinic acid. The addition of another IPP unit to FPP in yeast gives rise to GGPP, which forms the scaffold of the diterpenes and diterpenoids (C20), such as taxadiene, a precursor to the taxane family of chemotherapeutics. Condensation of two FPP units yields the linear triterpene squalene (C30), which serves as the universal building block of all sterols, including ergosterol in yeast. In plants, squalene gives rise to a number of pharmacologically active triterpenoids, such as β-amyrin and lupeol. Tetraterpenes (C40) are produced through condensation of two molecules of GGPP, yielding phytoene, the precursor to the carotenoids, such as lycopene and β-carotene. Polyterpenes such as natural rubber (cis-polyisoprene) comprise thousands of isoprene units. In the broadest sense, alkaloids are defined as low-molecular-weight metabolites containing heterocyclic (true alkaloids) or exocyclic (protoalkaloids, amines, and polyamines) nitrogen atoms. Approximately 20,000 natural alkaloids are known, many of which exhibit analgesic (morphine), stimulant (caffeine and ephedrine), psychotropic (mescaline and cocaine), antibacterial (sanguinarine), anticancer (vinblastine and vincristine), antitussive (codeine), anti-inflammatory (berberine), antispasmodic (papaverine), or antimalarial (quinine) activities. Phe, Tyr, and their derivatives (e.g. phenethylamine, tyramine, and dopamine) are the source of a tremendous number of alkaloids, including the benzylisoquinoline alkaloids (BIAs) and the phenethylisoquinoline alkaloids (Fig. 2). BIAs are a large class of roughly 2,500 metabolites that includes berberine, noscapine, sanguinarine, and morphine. These important medicines are derived from the condensation of dopamine and 4-hydroxyphenylacetaldehyde, both of which are synthesized from Tyr. Dopamine can also condense with derivatives of Phe (4-hydroxydihydrocinnamaldehyde), yielding the phenethylisoquinoline class of alkaloids. Phe and Tyr form the basis of some simpler protoalkaloids and catecholamines, including modified amphetamines (ephedrine and cathinone), dopamine, mescaline, and adrenaline. Owing to the ubiquity of the indole group in nature, Trp also forms the basis of many important alkaloids, such as simple indoles, β-carbolines (serotonin and harmine), and the monoterpenoid indole alkaloids (MIAs). Interfacing plant alkaloid secondary metabolic pathways with microbial metabolism. S. cerevisiae is shown as the prospective host species. Refer to Figure 1 for abbreviations and color coding. Additional abbreviations: AAA path, aromatic amino acid pathway; Aro1, pentafunctional arom protein; Aro2, chorismate synthase and Flavin reductase; Aro3+Aro4, 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase isoenzymes; Aro7, chorismate mutase; E4P, erythrose 4-phosphate; 4-HPAA, 4-hydroxyphenylacetaldehyde; PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; R5P, ribose 5-phosphate; TCA cycle, tricarboxylic acid cycle. MIAs, derived from the condensation of the Trp analog tryptamine and a monoterpenoid (secologanin), are one of the largest and most complex classes of alkaloids, giving rise to more than 3,000 structures. The Trp precursor anthranilate also serves as a precursor to several alkaloid subclasses, including the quinazolines. Beyond the aromatic amino acids, Arg and Orn form the basis of the tropane, pyrrolidine, pyrrolizidine, and pyridine alkaloids, whereas Lys is the precursor to the piperidines and quinolizidines. Nucleosides also form the basis of some alkaloids, such as caffeine and theobromine. The phenylpropanoids represent a class of more than 8,000 plant phenolics derived from Tyr and Phe via the phenylpropanoid pathway (Wu and Chappell, 2008). The name phenylpropanoid refers to the distinctive C6-C3 structure of metabolites within this pathway. The key phenylpropanoid branch point intermediate is p-coumaroyl-CoA (Fig. 3), which forms the basis of the flavonoids, stilbenoids, coumarins, lignans, catechins, and aurones. In addition to the C6-C3 skeleton, the phenylpropanoid pathway also diverts at the level of cinnamic or ferulic acid to yield an array of C6-C1 benzoates, such as vanillin, benzaldehyde, and gallic acid (Vogt, 2010; Kallscheuer et al., 2018). Although the stilbenoids and flavonoids originate from the phenylpropanoid pathway, they are elongated by type III plant polyketide synthases (PKSs), underscoring the mixed biosynthetic nature of these specialized metabolites (Box 1). PKSs accept CoA-bound substrates (Yu et al., 2012), most often p-coumaroyl-CoA, although cinnamoyl- and feruloyl-CoA form the basis of some phenylpropanoid polyketides, such as pinosylvin and curcumin (Preisig-Müller et al., 1999; Kita et al., 2008). Once loaded with a starter molecule, malonyl-CoA moieties are incorporated into the growing polyketide chain. Interfacing plant phenylpropanoid and polyketide secondary metabolic pathways with microbial metabolism. A selection of natural plant pathways in a prospective S. cerevisiae host is shown. More extensive networks of natural and synthetic phenylpropanoid routes have been shown previously in Wang et al. (2015) and Zhao et al. (2015). Refer to Figures 1 and 2 for abbreviations and color coding. Additional abbreviations: Acc1, acetyl-CoA carboxylase; PAL, Phe ammonia-lyase; PKS, polyketide synthase; TAL, Tyr ammonia-lyase. Several different polyketide backbones can be produced, though the most heavily targeted for metabolic engineering are the naringenin chalcone and resveratrol scaffolds, which give rise to the respective flavonoid and stilbenoid subclasses (Lussier et al., 2013). These compounds are functionalized in downstream reactions including aromatic hydroxylation, NADP (NADPH)-dependent reduction, O-methylation, and glycosylation. The flavonoids alone encompass more than 6,000 natural products, including chalcones, catechins, and (Yu et al., In to the flavonoids and stilbenoids, are not polyketides, as their structure is derived from hydroxylation, and of or ferulic or the et al., 2008; et al., The most of synthetic biology for the synthesis of valuable plant products is an antimalarial terpenoid produced by a of more than a artemisinic acid of in yeast et al., whereas been engineered to of the amorphadiene precursor et al., The yeast artemisinic acid been as a of more than et al., 2016). is another terpenoid and microbes have been engineered to to 1 of the scaffold et al., 2010; et al., 2014; et al., 2015). terpenoid pathway and 1). not for the downstream acid. for forms of the to monoterpenoids exploited to intermediates in such as et al., et al., and et al., 2015). These efforts have to be to the de novo synthesis of functionalized in a microbial although pathway have been et al., 2015). 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In a use of of amorphadiene by a of to et al., of for polyketide synthesis not in polyketide also in to the et al., 2018). to in amino acid biosynthesis S. cerevisiae et al., of higher in than in et al., 2015). for the synthesis of the polyketide acid et al., 2015). These studies highlight the often of to to engineering a The of microbial have or S. cerevisiae to their of and metabolic a of their and and their active central metabolic pathways. Several these many microbial synthesis (Box 2). the of microbial exploited for the synthesis of plant natural products aromatic amino at well of and S. a major of these for the of alkaloids and is an with a tremendous to and as well as aromatic amino et al., et al., is another with a to plant terpenoids and phenylpropanoids et al., 2013; et al., 2018). 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The yeast and aromatic amino acid pathways are to of and The point to the pathway condensation of and is by 3-deoxy-d-arabino-heptulosonate-7-phosphate for which two in yeast and by Tyr can be in both enzymes using and et al., 2008; et al., 2018). chorismate is by Tyr, of the for Tyr biosynthesis et al., 2008). of and in the host to a in of aromatic The terpenoid branch of secondary metabolism also been the point of engineering precursor The of the mevalonate pathway by can be by a Owing to the of this studies terpenoid or have incorporated to of et al., 2013; et al., 2015). The of monoterpenoids a engineering to the nature of the yeast This the synthesis of GPP from IPP and dimethylallyl as well as the condensation of GPP and an unit of IPP to give FPP (Fig. 1). Owing to the central of FPP in ergosterol is an essential that FPP synthase be with properties have been et al., and exploited for the of monoterpenoids and et al., 2015; et al., 2016). the essential squalene synthase squalene from two FPP thus on the of all terpenoids from FPP (Fig. 1). The most means of host is native precursor pathways with a This in efforts to amorphadiene in the native pathway by the yeast mevalonate pathway et al., the yeast pathway within a both and S. cerevisiae of of a in the biosynthesis of terpenoids, polyketides, phenylpropanoids, and cannabinoids 1 and 3), and also been a target for In acetyl-CoA synthesis pathways in different that acetyl-CoA directly and This provides for engineering on the pathway that is most for a et al., 2016). acetyl-CoA pathways are of et al., et al., 2015; and et al., et al., 2016). The of a derived from acetyl-CoA is the synthesis of of the sesquiterpene et al., 2016). 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The most means of metabolic is to of enzymes by or In addition to of the pathway, the yeast pathway from acetyl-CoA to FPP in the artemisinic acid et al., 2013). pathway can also arise from the of pathway intermediates to by host or plant phenylpropanoid biosynthesis from Phe or Tyr, the yeast pathway for aromatic amino acid is through of amino acid and et al., 2015). biosynthesis a more of Tyr as the dopamine and precursors are derived from biosynthesis and et al., The of in yeast to or which can be by more than enzymes et al., 2008; et al., 2015). These enzymes have also been in the of intermediates within the pathway et al., that pathway intermediates is to by host activities. In a a yeast shown to p-coumaroyl-CoA within the phenylpropanoid pathway to an et al., can also be to routes by pathway and within the core pathway have been shown to exhibit a range et al., et al., 2003; Fossati et al., which by of plant for a more et al., In addition to the of synthase within the pathway to exhibit on intermediates et al., 2016). In this higher on the substrates than the be in in which a pathway is such that enzymes are from pathway intermediates et al., of intermediates is also through as in the of synthase or synthase through to yeast farnesyl diphosphate synthase et al., et al., have the to more by of pathway thus competition from host In addition to terpenoid of resveratrol biosynthetic enzymes a in in engineered S. cerevisiae et al., have the to or as through of synthase with the yielding a in et al., with also of to et al., of synthetic biology to microbial for drug In this plant and are exploited to structural and not in A common chemical within the pharmaceutical is in which or moieties are to drug to the properties of the many natural products from amino acids, can be supplemented to engineered microbes to the formation of products. This to yeast or in the synthesis of pathway intermediates et al., 2018). products not a more of downstream this on the supplementation of amino acid and be by engineering de novo of been produced de novo by a into engineered for biosynthesis et al., 2018). of pathway enzymes also been exploited for the of as and by the of synthase to accept an range of substrates et al., this on the supplementation of in this that are not synthesized by these in to de novo biosynthetic a Microbial synthesis provides a avenue for the of natural plant fragrances, nutrients, and colorants. Although this is in and many challenges the of in microbial biosynthetic as a alternative to natural and chemical synthesis. The of complex plant secondary pathways to the of and more challenging routes in microbial systems. the of enzymes in several pathways a number of natural products for de novo biosynthesis in a microbial species. Several microbially derived natural products have and these to the for The of microbial for drug to important structural and chemical that have thus not been in to and for is for a is of support from a for is to for their support of as

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