Biosynthetic Pathway and Metabolic Engineering of Plant

Review pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. 2018, 66, 2273−2280

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones Mwafaq Ibdah,*,† Stefan Martens,‡ and David R. Gang§ †

Newe Ya’ar Research Center, Agriculture Research Organization, Post Office Box 1021, Ramat Yishay 30095, Israel Department of Food Quality and Nutrition, Centro Ricerca e Innovazione, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all’Adige, Trentino, Italy § Institute of Biological Chemistry, Washington State University, Post Office Box 646340, Pullman, Washington 99164-6340, United States ‡

ABSTRACT: Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones. KEYWORDS: dihydrochalcones, biosynthesis, biological activity, metabolic engineering

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INTRODUCTION Flavonoid compounds represent a highly different class of specialized plant metabolites with more than 9000 structures, and dihydrochalcones (DHCs) define a major subclass of this group. Chemically, DHCs are open-chain flavonoids, in which the two aromatic rings are linked by a three-carbon α,βsaturated carbonyl system (Figure 1). Similar to other

isolated from many medicinal plants belonging to diverse plant families. DHC occurrence in general in the plant kingdom has been recently intensively reviewed by Rivière.8 Approximately 265 diverse DHCs are presently known to be formed in over 46 plant families.8,9 DHC distributions are excessively heterogeneous in the plant kingdom, with members of this compound class having been isolated and characterized from the angiosperms and from pteridophytes.8 DHCs, similar to many other classes of natural products, have been shown to play different important roles in human health. High intake of apple fruits, which are rich in DHCs, has been linked to a lower risk of many degenerative diseases, particularly diabetes, Alzheimer’s disease, and cardiovascular disease.8,10−13 Various investigations have described the potential benefits of DHCs in human health, especially because of their antioxidant properties. Indeed, specific DHC compounds may be effective in preventing different human physiological disorders, notably diabetes,14 bone resorption,15 and free-radical-involving disease, by inhibition of the formation of advanced glycation end products.1 Also, DHCs have proven chemopreventative and antitumor activities.16 DHCs have also been reported to act as flavor enhancers and bitterness blockers with various uses in the food, beverage, and pharmaceutical industries.17,18

Figure 1. Structure and numbering of phloretin as a representative backbone for all DHCs.

polyphenols, e.g. flavonoids, DHCs display a wide variation of hydroxyl and glucosyl substitution patterns. For example, 3hydroxyphloretin, phloridzin (phloretin-2′-O-glucoside), trilobatin (phloretin-4′-O-glucoside), and sieboldin (3-hydroxyphloretin-4′-O-glucoside) accumulate in diverse plant species, including Malus species (Figure 2).1−6 DHCs are perhaps best known as a class of compounds found in apple trees (Figure 3) that have been intensively reviewed by Gosch et al.,7 but they are also occasionally encountered in other edible plants. Importantly, they have been © 2017 American Chemical Society

Special Issue: 11th Wartburg Symposium on Flavor Chemistry and Biology Received: Revised: Accepted: Published: 2273

September 26, 2017 November 23, 2017 November 24, 2017 November 24, 2017 DOI: 10.1021/acs.jafc.7b04445 J. Agric. Food Chem. 2018, 66, 2273−2280

Review

Journal of Agricultural and Food Chemistry

interactions with other organisms and the environment. The physiological role for these compounds in planta appears to be largely in defense mechanisms.1,2 The synthesis and accumulation of DHCs and their derivatives can also occurs when plants are faced with serious diseases, such as scab19 and fire blight caused by the bacterium Erwinia amylovora.20 Evidence for bioactivity of DHCs as functional antioxidants was suggested by the lowering of oxidative stress of apple leaves.1 As is the case for several natural products, the isolation of DHCs from different plant species is limited by low production levels and the complexity of the mixtures recovered from plants. Moreover, the total chemical syntheses of these structurally complex metabolites are economically impractical in many cases. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts, such as Escherichia coli and Saccharomyces cerevisiae, and to engineer the metabolic pathway/host to improve the production of these metabolites.21,22 Microbial production offers many benefits over each field and plant cell cultivation because of the rapid growth of microbes compared to plants, the convenience of genetic manipulation, and also the well-established metabolic engineering tools developed to be used in microbes. In addition, microbial biosynthesis is more environmentally friendly than chemical synthesis and can produce much more pure products from the culture than is obtainable from plant tissues or plant cultures, thus allowing for simpler (and “greener”) purification strategies. However, the functional reconstruction of plant biosynthetic pathways in microbes and the application of microbial biosynthesis for the industrial production of important compounds is still challenging.21,23,24 It requires not only a full understanding of the biochemical pathway of interest, but information about the interactions between the pathway members (e.g., protein−protein interactions, steric hindrances, substrate channeling, and side reactions) as well as a strong foundational platform upon which to build the production system (e.g., reduced/eliminated feedback inhibition of early pathway steps, precursor availability within the production strains, high growth and production capacity of the production strains, etc.). As an important foundational step for production of DHCs in microbial culture systems, the biosynthesis of DHCs was recently investigated, with genes for pathway members identified and recombinant enzymes characterized.25−31 The objective of this review is to highlight the latest advances in DHC biosynthetic research and the recent efforts in the microbial production of representative compounds of the DHCs. The current challenges and the potential of these approaches are also briefly discussed.

Figure 2. Metabolic profiling of DHCs from four different Malus genotypes: (A) Malus sieversii, (B) Malus trilobata, (C) Malus hybrid ‘Evereste’, and (D) Malus sieboldii syn. toringo.

Figure 3. Phloretin and phloridzin distribution in leaves, seeds, flesh, and peel of apples. Data were re-elaborated from Lata et al.,82 DudaChodak et al.,83 and Yahyaa et al.44

DHCs are specialized metabolites (also called “secondary metabolites”) that plants produce to protect themselves in their

Table 1. Biochemically Characterized Malus Enzymes Involved in DHC Biosynthesis enzyme name MdHCDBR MdCHS3 MdPh-2′-OGT MdPh-2′-OGT MdPh-4′-OGT

substrate

Km (μM)

Vmax (pkat μg−1 of protein)

reference

p-coumaryl-CoA feruloyl-CoA p-dihydrocoumaryl-CoA p-coumaryl-CoA phloretin phloretin phloretin

96.6 92.9 5.07 5.09 0.62 82 26.1

47.7 102.9 1.05 0.94

26

2274

8 1.86

44 27 59 28 DOI: 10.1021/acs.jafc.7b04445 J. Agric. Food Chem. 2018, 66, 2273−2280

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Figure 4. Proposed biosynthetic routes to several hydroxylated, glycosylated, and methylated DHC derivatives. HCDBR, hydroxycinnamoyl-CoA double-bond reductase; CHS, chalcone synthase; Ph-2′-OGT, phloretin-2′-O-glycosyltransferase; Ph-4′-OGT, phloretin-4′-O-glycosyltransferase; 1,2 RhaT, 1,2-rhamnosyltransferase; CPR, cytochrome P450 reductase; CYP, cytochrome P450; and OMT, O-methyltransferase. The red-labeled genes/ enzymes have been biochemically characterized from Malus.

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BIOSYNTHESIS OF DHCS Overview. The DHC pathway is a part of the large phenylpropanoid network, which produces a range of other specialized metabolites, such as flavonoids, phenolic acids, lignins, stilbenes, and lignans. The substitution pattern and type of substituents present on DHCs lead to the diversity within the chemical class. From a biosynthetic point of view and in contrast to the extensive literature on flavonoid and chalcone biosynthesis, little information was available on the biosynthesis of DHCs in plants until very recently (Table 1). Flavonoid compounds are biosynthesized via the phenylpropanoid−acetate network.32 Phenylalanine ammonia lyase catalyzes the first step in this metabolic network: the conversion of L-phenylalanine to tcinnamic acid. Cinnamate 4-hydroxylase (C4H) then catalyzes the synthesis of p-hydroxycinnamate (p-coumaric acid) from tcinnamic acid. p-Coumaric acid is further converted by pcoumarate:CoA ligase (4CL) to its coenzyme A (CoA) ester. From these central intermediates, the pathway diverges into several side branches, each resulting in a different class of phenolic compounds (Figure 4).33 The DHC pathway was originally thought to be a subpathway of the larger flavonoid biosynthetic network, with the working hypothesis being that reduction of the double bond of the propenyl linker occurred after the action of the type-III polyketide synthase (PKS) known as chalcone synthase (CHS). However, work by us and others clearly demonstrated that

DHCs belong to their own special network, being separated from flavones, flavonols, flavanol, anthocyanins, proanthocyanidins, etc., by action of a double-bond reductase (DBR) prior to the action of a CHS-like PKS (Figure 4).26,29,34 Significant recent advances in our understanding of DHC biosynthesis include characterization of the formation of pdihydrocoumaroyl-CoA from p-coumaroyl-CoA, progress toward elucidating phloretin and other DHC derivative formation, molecular characterization of several genes encoding enzymes that modify the DHC core structures, and analysis of enzyme function. Data are also starting to emerge from knockout studies of DHC biosynthesis genes. However, there are still main areas where data are lacking. The range of genes encoding secondary modification enzymes that have been characterized is still limited in comparison to the great array of known DHC structures (Table 1). Moreover, cDNAs or genes have not been described yet for some of the enzymes carrying out the hydroxylation or methylation of the core DHC structure. Formation of p-Dihydrocoumaroyl-CoA. The first committed enzyme in DHC biosynthesis is catalyzed by a DBR, which belongs to the medium-chain dehydrogenase/ reductase (MDR) superfamily and that catalyzes the reduction of the α,β unsaturated double bond of the enoyl moiety in planta, such as p-coumaroyl-CoA. The resulting p-dihydrocoumaroyl-CoA serves as the substrate for a CHS-like enzyme (perhaps canonical CHS in some plant families) in subsequent 2275

DOI: 10.1021/acs.jafc.7b04445 J. Agric. Food Chem. 2018, 66, 2273−2280

Review


Formation of Phloretin from p-DihydrocoumaroylCoA. The chemical structure similarity of p-coumaroyl-CoA and p-dihydrocoumaroyl-CoA led to the assumption that CHS could use either molecule as a substrate, with condensation of three molecules of malonyl-CoA to form naringenin chalcone or phloretin, respectively. Gosch et al.34 suggested that the formation of phloretin is catalyzed by a common CHS and not by a specific CHS-like enzyme. However, that is yet to be determined across the plant kingdom. CHS catalyzes the first committed step in the flavonoid biosynthesis (via naringenin chalcone) and potentially DHCs (via phloretin). The reaction catalyzed by CHS includes one molecule of p-coumaryl-CoA for naringenin chalcone or one molecule of p-dihydrocoumaroyl-CoA for phloretin as the starter substrate and three C2 units from malonyl-CoA as the extender molecules (Figure 4).34,42−44 CHS belongs to the superfamily of enzymes called type-III PKSs that also comprises stilbene synthase, p-coumaroyltriacitic acid synthase, acridone synthase, pyrone synthase, and bibenzyl synthase, among others.42 CHS is structurally and mechanistically among the best studied of the PKSs.45,46 The isolation of a cDNA for CHS represented the first gene cloned for a flavonoid pathway enzyme.47,48 CHS sequences and a series of CHS-like sequences have been extensively studied in numerous plant species, e.g., M. domestica,44 Solanum lycopersicum,49 Medicaco sativa,50 Petunia × hybrida,51 Psilotum nudum,35 and strawberry (Fragaria × ananassa).52 Austin and Noel identified about 650 CHS and CHS-like sequences in the public sequence database.45 The number of sequences belonging to this class has ballooned since then, thanks to genome sequencing efforts. GenBank now lists over 4250 genes as being annotated as CHS, with another 3450+ CHS-like genes being from the plant kingdom. It is likely (probable) that most of these genes do not code for canonically functional CHSs and that some may play specific roles in biosynthesis of certain subfamilies of phenylpropanoids, such as DHCs, in particular, plant families.44,45 Recently, three genes of the CHS superfamily (MdCHS1, MdCHS2, and MdCHS3) from M. domestica were functionally characterized.44 All displayed kinetic parameters in a range to be expected for CHS relative to its standard substrates. However, the three recombinant Malus CHSs converted cinnamoyl-CoA, p-coumaroyl-CoA, and p-dihydrocoumaroylCoA substrates to their corresponding products with varying conversion rates.44 These three enzymes appeared to be fully functional CHS enzymes that had the ability to catalyze the formation of additional compounds beyond naringenin chalcone and, thus, could be viewed as perhaps being in the middle of new functionalization. DHC Modification Enzymes. DHCs are substrates for a range of modification reactions, including hydroxylation, glycosylation, prenylation, methylation, and polymerization. To date, genes encoding proteins have been isolated that catalyze some of these conversions. For example, two uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) from Oryza sativa (rice) 53 and Fagopyrum esculentum (buckwheat) were identified that are able to C-glycosylate the 3′ position of a 2-hydroxyflavanone to form nothofagin (2′,4,4′,6′-tetrahydroxy-3-C-β-D-glucopyranosyldihydrochalcone) (Figure 5).54 Recently, the isolation of a cDNA encoding an enzyme involved in the 3-hydroxylation of DHCs was accomplished.55

substitution/modification reactions that produce DHCs (Figure 4). Several different enzymes from apple were suggested to catalyze this reduction of the α,β unsaturated double bond of the enoyl moiety in planta. It had been assumed that pdihydrocoumaroyl-CoA is formed from p-coumaroyl-CoA by a NADPH-dependent dehydrogenase (NADPH: p-coumaroylCoA oxidoreductase) or a DBR. Gosch et al.34 showed the formation of phloretin when the CoA ester of p-coumaric acid, radiolabeled malonyl-CoA, and NADPH were incubated with protein extracts from apple leaves. Enzyme assays with recombinant proteins from Psilotum nudum showed that pdihydrocoumaroyl-CoA can act as a precursor for phloretin.35 To date, more than 1000 protein sequences have been identified as MDR superfamily members, with a broad range of enzymatic activities. They are found in all kingdoms of life and are involved in metabolism, regulatory processes, and protection against cell damage. Despite their low sequence similarity, they have a similar size of 350−400 residues and a conserved overall structure formed by two domains, a cofactor binding domain and a catalytic domain. While all MDRs use NAD(H) or NADP(H) as a cofactor, they can be divided into two classes with a different reaction mechanisms: zinccontaining and non-zinc-containing MDRs.36,37 Enone reductases are the best-characterized enzymes that can recognize and act on the α,β unsaturated double bond of the enoyl moiety of specialized metabolites, such as 3-methoxy-4hydroxybenzalacetone, 4-hydroxybenzalacetone,38 coniferyl aldehydes, dehydrodiconiferyl aldehydes and p-coumarylaldehyde,39 and (+)-pulegone.40 However, none of these enzymes have been shown to catalyze reduction of p-coumaroyl-CoA or feruloyl-CoA. In Arabidopsis, a DBR specific for alkenals catalyzed the 7,8 double-bond reduction of phenylpropanal substrates to their corresponding dihydro compounds.41 That enzyme was active with several of phenylpropanal substrates, although phenylpropenoyl-CoA esters, including p-coumaroylCoA, were not tested as substrates. An enoyl reductase-like (ENRL) enzyme that can generate pdihydrocoumaroyl-CoA from p-coumaroyl-CoA was cloned by Dare et al.,29 who showed in a RNAi-based study that reduction of the transcript levels of ENRL-3 in transgenic ‘Royal Gala’ led to a 66% decrease in the concentration of DHCs in the leaves in one silenced line. The isolation of a cDNA for a NADPH-dependent hydroxycinnamoyl-CoA DBR gene was first reported from Malus domestica by us.26 That gene shared significant amino acid sequence homology to the Arabidopsis alkenal DBR.41 Enzyme properties were also determined for the NADPHdependent hydroxycinnamoyl-CoA DBR protein from M. domestica.26 Similar Km values toward p-coumaroyl-CoA and feruloyl-CoA, at 96 and 101 μM, respectively, were determined for the plant-based and recombinant protein. p-Dihydrocoumaroyl-CoA and dihydroferuloyl-CoA were found to be the in vivo products of this enzyme in apple leaves, thus confirming its role as the first step in the DHC pathway and branch-point enzyme off of the general phenylpropanoid network (Table 1).26 Hence, it cannot be excluded that other reductases play a similar role, e.g., the ENRL-3/5 described by Dare et al.29 or other as yet unidentified enzymes. Eichenberger et al.21 demonstrate that the reduction can been performed by a apparently unrelated (yeast) enzyme, which is known to have homologues in plants. 2276


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sugar moieties is directly bonded to a carbon atom of aglycone. C-Glycosides are unusually stable, because their C−C bonds are resistant to acid hydrolysis or glycosidase. A diversity of plant species are known to accumulate C-glycosyl DHC and Cglycosylflavonoids.53,54,65,66 Gutmann and Nidetzky67 showed that OsCGT isolated from O. sativa formed 3′-C-glycoside nothofagin exclusively. Two cDNAs were isolated from the dicot plant F. esculentum, and the recombinant proteins [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)] were found to exhibit C-glucosylation activity toward phloretin.54 More recently, the isolation of two cDNAs for a CGT (CuCGT and FuCGT) genes were reported from Citrus unshiu and Frotunella crassifolia, respectively, that catalyzed the formation of di-C-glucosyl phloretin.68 DHC Hydroxylase. Introduction of the hydroxyl groups in the B ring of flavonoids and DHCs is catalyzed by the wellknown cytochrome P450-dependent monooxygenases flavonoid 3′-hydroxylase (F3′H) and chalcone 3-hydroxylase (CH3H).55,69−71 The F3′H recombinant proteins of Perilla frutescens, Petunia hybrid, and Arabidopsis thaliana accept flavones, flavanones, and dihydroflavonols as substrates, as do enzyme preparations from plant tissues.72−74 Indeed, recombinant P. frutescens F3′H showed a similar Km (about 20 μM) for naringenin, dihydrokaempferol, and apigenin.73 The A. thaliana F3′H amino acid sequence has been used to generate a model of the enzyme and examine the active site architecture and substrate recognition.74 Hydroxylation of position 3 of DHCs shows high similarity to the introduction of the second hydroxyl group in the B ring of flavonoids and chalcone. This was shown for the first time with microsomal enzyme preparations of Dahlia variabilis petals, where conversion of 6′-deoxychalcone, isoliquiritigenin, to the corresponding 3,4-hydroxylated product, butein, occurred.75 Transgenic apple plants overexpressing the Cosmos sulphureus CH3H gene show increased levels of 3-hydroxyphloridzin, but no 3-hydroxyphloretin accumulation was observed.55 This indicated that highly reactive 3-hydroxyphloretin is immediately converted to 3-hydroxyphloridzin to avoid undesired cell damage.

Figure 5. Structure and numbering of nothofagin (2′,4,4′,6′tetrahydroxy-3-C-β-D-glucopyranosyldihydrochalcone).

However, in vitro activities of recombinant proteins may not reflect their in vivo activities. Factors such as the abundance of protein in relation to the potential substrate and involvement in the metabolic channeling affect in vivo activity. In several transgenic experiments, endogenous flavonoid glucosyltransferases (GTs) have been shown to accept substrates that are not naturally present in the recipient species, such as 6′deoxychalcones and isoflavonoids, suggesting that broad substrate acceptance for some modification enzyme types may be common.56−58 DHCs O-Glycosyltransferase. cDNAs have been isolated for several UGTs with O-glycosylation activity on phloretin. The recombinant proteins showed a wide substrate acceptance.25,59 In general, UGTs characterized in flavonoid (and to date DHC) biosynthesis show high regiospecificity but broad substrate acceptance, although there are some exceptions.60−62 Several cDNAs encoding activities that can glycosylate the 2′hydroxyl of phloretin have been identified and characterized. MdPGT1 from M. domestica glycosylates phloretin in the presence of UDP-glucose into phloridzin and accepts only phloretin as a substrate (Figure 4).27 The recombinant proteins from the UGT71A15 and UGT71K1 genes of M. domestica show activities against several types of flavonoids and phenylpropanoids, including the DHC phloretin.25 However, their in vivo activities are likely to be UDP-glucose:phloretin 2′O-glycosyltransferase.25,27,59,63 The recombinant Pyrus communis UGT71A16 and UGT71K2 proteins also showed wide regiospecificity, adding glucose to the 2′-hydroxyl of phloretin and to position 5 of flavonoids, producing monoglucosides.25 The lack of phloretin in pear suggests that UGT71A16 and UGT71K2 accept other aglycones as substrates in vivo. Dianthus caryophyllus GT also showed activity toward phloretin, although phloretin is not present in this plant species.64 Recently, targeted downregulation of the apple phloretinspecific glycosyltransferase UGT88F1 leads to changes in the concentration of a wide range of polyphenolic compounds, including dihydrochalcone phloretin.31 In contrast to these activities, a cDNA from M. domestica (Golden Delicious) encoding a protein with UDP-glucose:phloretin 4′-O-glycosyltransferase (MdPh-4′-OGT) activity was recently isolated and functionally characterized (Table 1 and Figure 4).28 The recombinant apple MdPh-4′-OGT was found to be position-specific for its putative substrate phloretin. It would accept trilobatin, phloridzin, quercetin, and naringenin as substrates at lower efficiency. The Km value toward phloretin was 96 μM, and trilobatin was found in the in vivo product. Unlike most of the GTs discussed previously, MdPh-4′-OGT showed strong catalytic efficiency with phloretin.28 DHC C-Glycosyltransferase. C-Glycosides are characterized by their C−C bonds, in which anomeric carbon of the

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METABOLIC ENGINEERING OF DHCS IN A MICROBIAL CELL FACTORY The increasing prevalence of several diseases, such as Alzheimer’s disease, obesity, cancer, and diabetes, in humans in recent decades worldwide, accompanied by rising concern with regard to the safety of many synthetic chemistry-based pharmaceuticals, has raised public demand for phytochemicalbased medicines.6,76 This, in turn, has led to increasing interest in metabolic engineering as an approach to produce such natural products on an industrial scale, which has the potential to decrease production costs of, for example, desired DHCs. Eukaryotic and prokaryotic microbes, such as S. cerevisiae and E. coli, are widely used as a cell factories for the overexpression of targeted genes.21,77 These metabolically engineered microorganisms can be used to synthesize natural and non-natural desired compounds via a precursor-directed biosynthesis approach.78 Metabolic engineering involves enhancing or redirecting flux through metabolic pathways by making genetic modifications, e.g., deletion of genes and replacement of the gene expression signal, that alter the activity of specific enzymatic reactions. These strategies often include increasing activity at flux-controlling steps and introduction of irreversible 2277



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reactions that drive the flux in desired directions and often comprise elimination of unwanted activities.23,77 When it comes to industrial production and developing new platform strains, the host strain matters. In addition, there are some central aims that the strains must achieve to be ready for industrial production. For example, it should be considered whether the chosen host can survive under the desired process conditions, e.g., temperature, ionic strength, and pH, and if the host is genetically stable. In addition, the largest bottleneck for industrial implementation of novel bioprocesses is often in the scale-up step that usually appears late in the process, where the host strain has to be chosen absolutely, because it will generally be too expensive to change at a later stage.23,77 Several compounds with high value, such as pharmaceutical compounds (e.g., artemisinin), fragrance (e.g., nootkatone), biofuels (e.g., ethanol and isobutanol), and food chemicals (e.g., vanillin and resveratrol), were produced as end products in microbes.23 Such biotechnological processes compared to the extraction from plant material are environmentally friendly, less expensive, and allow for efficient production of specific desired compounds.79 Although many genes responsible for DHC biosynthesis have been characterized, there has been limited progress on the metabolic engineering of DHC production. To date, there have been only four reports of the engineering of DHC production in E. coli and S. cerevisiae.21,24,80,81 The co-expression of the 4CL gene from A. thaliana and CHS from Hypericum androsaemum was achieved, and the direct production of phloretin by feeding phloretic acid was detected.24 Recently, it was reported that overexpression of 4CL, C4H, CHS, DBR, and UGT from different sources greatly increased the production of several DHCs, such as phloretin, phloridzin, 3-hydroxyphloretin, nothofagin, and the sweet tasting molecule naringin dihydrochalcone.21 Thus, to increase the production of DHCs, several key steps must be followed: (i) a host strain must be chosen that can be used under industrial conditions (e.g., high osmotolerance and tolerance to low pH), and (ii) several specific enzymes must be chosen and overexpressed, e.g., DBR,26 CHS, and UGT.28,44 In conclusion, many DHCs with potential benefits for humans have been identified in a wide diversity of plant species. Recent advances in our understanding of the DHC biosynthetic pathway have revealed several key enzymes, but some of the enzymes responsible for functional modifications on the phloretin (or other DHC) backbone remain to be discovered. Furthermore, our ability to genetically engineer the DHC biosynthetic pathway is still limited, and there is much work yet to be performed to identify additional genes and enzymes involved in DHC formation and to develop a better heterologous system for an economically feasible industrial production process.

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ACKNOWLEDGMENTS

This research was supported by the Ministry of Agriculture & Rural Development (Grant 261-1043 to Mwafaq Ibdah) and partially funded by the European Region Tyrol−South Tyrol− Trentino (EGTC) through the Euregio Science Fund, Project ExpoApple2-IPN 55, 2nd call 2016, and the autonomous province of Trento (ADP 2010-2017, Italy, to Stefan Martens).

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ABBREVIATIONS USED 4CL, p-coumarate:CoA ligase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; CH3H, chalcone 3-hydroxylase; DBR, double-bond reductase; DHC, dihydrochalcone; ENRL, enoyl reductase-like; F3′H, flavonoid 3′-hydroxylase; GT, glucosyltransferase; Md, Malus domestica; MDR, medium-chain dehydrogenase/reductase; MdPh-4′-OGT, UDP-glucose:phloretin 4′-O-glycosyltransferase; PKS, polyketide synthase; UGT, UDP-dependent glycosyltransferase

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +972-4-953-9509. E-mail: mwafaq@volcani. agri.gov.il. ORCID

Mwafaq Ibdah: 0000-0002-7459-5356 Notes

The authors declare no competing financial interest. 2278


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