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CHAPTER 36 Using Recombinant Microorganisms for the Synthesis and Modification of Flavonoids and Stilbenes Eun Ji Joo*, Brady F. Cress and Mattheos A.G. Koffas ,*Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY, USA Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY, USA Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY, USA 1. INTRODUCTION Natural products have been the focus of drug discov- ery and development, with some of their advantages including their substantiated efficacy and abundant sources. The structures of at least 100,000 secondary metabolites from medicinal plants and 4000 flavonoids have been revealed. 1 With constant interest and effort, more than 50% of synthetic drugs have come from the mimics or precursors of natural products. 2 As a charac- teristic example, phytochemicals such as flavonoids and resveratrol have recently emerged as the underly- ing molecules behind the “French paradox,” 3,4 which is described as the observation that the French enjoy a rel- atively low risk of cardiovascular disease despite a diet that is high in saturated fat. In addition to the French paradox, flavonoids show several other health benefits and play multiple roles in cancer, inflammation, cardio- vascular disease, and aging. Over the decades, as a vari- ety of their biological and pharmacological effects have become more apparent, researchers in academia and the food and pharmaceutical industries have become interested in metabolically engineering their production in microbes to obtain those natural products economi- cally and in high quantity and purity. Novel metabolic pathways have also been created by mixing and match- ing biosynthetic enzymes from different sources or altering the biochemical properties of enzymes in order to generate novel molecules. 2. BIOSYNTHESIS OF FLAVONOIDS AND STILBENES Flavonoids are synthesized via the phenylpropanoid pathway from the common precursor phenylalanine or tyrosine. Stilbenes are not classified as flavonoids but share a high resemblance to flavonoids in both func- tions in plant and chemical structures. 5 Flavonoids and related compounds are made through the phenylpro- panoid pathway, as depicted in Figure 36.1. 6 The bio- synthesis begins with the amino acid phenylalanine, which is deaminated to cinnamic acid by phenylala- nine ammonia lyase (PAL). The P450 monooxygenase cinnamate-4-hydroxylase (C4H) oxidizes cinnamic acid to 4-coumaric acid. This carboxylic acid is activated by the addition of a coenzyme A (CoA) unit, which is cat- alyzed by 4-coumarate:CoA ligase (4CL), yielding 4- coumaroyl-CoA. A type III polyketide synthase then sequentially adds three acetate extender units, derived from malonyl-CoA, to a single activated 4-coumaroyl- CoA starter unit. Depending on the polyketide synthase activity, chalcone synthase (CHS) or stilbene synthase (STS), subsequent folding and cyclization of the generated tetraketide intermediate results either in the production of a chalcone or stilbene ring structure. 7 Among them, the sequential addition of three malonyl-CoA molecules by CHS commits the resulting chalcone to the flavonoid biosynthetic pathway. 8 Chalcone isomerase (CHI) isomerizes chalcones 483 Polyphenols in Human Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00036-0 © 2014 Elsevier Inc. All rights reserved.
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Page 1: Polyphenols in Human Health and Diseasehomepages.rpi.edu/~koffam/papers/2014_Joo_Cress_Koffas.pdf ·  · 2014-06-23Polyphenols in Human Health and Disease. DOI: ... which are common

C H A P T E R

36

Using Recombinant Microorganisms for theSynthesis and Modification of Flavonoids and

StilbenesEun Ji Joo*, Brady F. Cress† and Mattheos A.G. Koffas†,‡

*Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer

Polytechnic Institute, Troy NY, USA †Department of Chemical and Biological Engineering, Center for Biotechnology

and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY, USA ‡Department of Biology, Center for

Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY, USA

1. INTRODUCTION

Natural products have been the focus of drug discov-ery and development, with some of their advantagesincluding their substantiated efficacy and abundantsources. The structures of at least 100,000 secondarymetabolites from medicinal plants and 4000 flavonoidshave been revealed.1 With constant interest and effort,more than 50% of synthetic drugs have come from themimics or precursors of natural products.2 As a charac-teristic example, phytochemicals such as flavonoidsand resveratrol have recently emerged as the underly-ing molecules behind the “French paradox,”3,4 which isdescribed as the observation that the French enjoy a rel-atively low risk of cardiovascular disease despite a dietthat is high in saturated fat. In addition to the Frenchparadox, flavonoids show several other health benefitsand play multiple roles in cancer, inflammation, cardio-vascular disease, and aging. Over the decades, as a vari-ety of their biological and pharmacological effects havebecome more apparent, researchers in academia andthe food and pharmaceutical industries have becomeinterested in metabolically engineering their productionin microbes to obtain those natural products economi-cally and in high quantity and purity. Novel metabolicpathways have also been created by mixing and match-ing biosynthetic enzymes from different sources oraltering the biochemical properties of enzymes in orderto generate novel molecules.

2. BIOSYNTHESIS OF FLAVONOIDS ANDSTILBENES

Flavonoids are synthesized via the phenylpropanoidpathway from the common precursor phenylalanine ortyrosine. Stilbenes are not classified as flavonoids butshare a high resemblance to flavonoids in both func-tions in plant and chemical structures.5 Flavonoids andrelated compounds are made through the phenylpro-panoid pathway, as depicted in Figure 36.1.6 The bio-synthesis begins with the amino acid phenylalanine,which is deaminated to cinnamic acid by phenylala-nine ammonia lyase (PAL). The P450 monooxygenasecinnamate-4-hydroxylase (C4H) oxidizes cinnamic acidto 4-coumaric acid. This carboxylic acid is activated bythe addition of a coenzyme A (CoA) unit, which is cat-alyzed by 4-coumarate:CoA ligase (4CL), yielding 4-coumaroyl-CoA. A type III polyketide synthase thensequentially adds three acetate extender units, derivedfrom malonyl-CoA, to a single activated 4-coumaroyl-CoA starter unit. Depending on the polyketidesynthase activity, chalcone synthase (CHS) or stilbenesynthase (STS), subsequent folding and cyclization ofthe generated tetraketide intermediate results either inthe production of a chalcone or stilbene ring structure.7

Among them, the sequential addition of threemalonyl-CoA molecules by CHS commits the resultingchalcone to the flavonoid biosynthetic pathway.8

Chalcone isomerase (CHI) isomerizes chalcones

483Polyphenols in Human Health and Disease.

DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00036-0 © 2014 Elsevier Inc. All rights reserved.

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selectively to (2S)-flavanones, which are then hydrox-ylated by flavanone 3β-hydroxylase (FHT) at the3-carbon position to give dihydroflavonols. These arereduced by dihydroflavonol 4-reductase (DFR) at the4-carbon position, yielding the unstable leucoanthocya-nidins. Leucoanthocyanidin reductase (LAR) catalyzesthe subsequent reduction to flavan-3-ols (also calledcatechins). Both the leucoanthocyanidins and theflavan-3-ols are possible substrates for antho-cyanidin synthase (ANS), which catalyzes the reactionto anthocyanidins. Finally, UDP-glucose:flavonoid3-O-glucosyltransferase (3GT) catalyzes the glycosy-lation at the 3-carbon, yielding anthocyanins.8,9

Additional enzymes exist to catalyze addition offunctional groups or manipulation of the skeletonto lend structural diversity or related structures

including isoflavonoids, condensed tannins, aurones,and stilbenes.10

Stilbenes originate from condensation of p-coumaroyl-CoA with three malonyl-CoA residues. STS, catalyzingthe formation of either resveratrol from p-coumaryl-CoAor pinosylvin from cinnamoyl-CoA, is a unique, distinctpolyketide synthase that is closely related to CHS. Whilechalcone synthase is present in higher plants, stilbenesynthase has a much more restricted distribution in theplant kingdom. Also, stilbene (or resveratrol) synthaseexhibits wide substrate-specificity and can also acceptother CoA esters—aliphatic as well as aromatic ones—asprimers for polyketide synthesis.11,12 Therefore, it is con-sidered as one of the most important enzymes to partici-pate in carbon backbone diversity in natural productpathways.13

FIGURE 36.1 Flavonoid biosynthesis. Adapted from Cress et al.6

484 36. USING RECOMBINANT MICROORGANISMS FOR THE SYNTHESIS AND MODIFICATION OF FLAVONOIDS AND STILBENES

5. INFLAMMATION AND POLYPHENOLS

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3. RECOMBINANT MICROBES

Metabolic engineering is a powerful tool to generatedesirable products at high productivity by manipulat-ing the cellular and metabolic characteristics of a hostorganism. One of the big challenges of metabolic engi-neering is to identify optimal organisms and to deter-mine targets for manipulations in individual genes,whole pathways, or even in transcriptional and trans-lational control elements. In general, metabolic engi-neering of natural product biosynthesis in microbesconsists of the following steps: bioprospecting andrecombinant pathway design (recombineering); selec-tion and cloning or synthesis of heterologous genes;production host choice, vector choice, and transforma-tion of heterologous genes into host; troubleshootingexpression, folding, and activity of plant proteins inmicrobial hosts (often via protein engineering); strainimprovement via carbon flux redistribution, toxicityreduction, transporter engineering, removal of regula-tory restrictions, and enzyme colocalization or compart-mentalization; and fermentation optimization. Althoughthe whole procedure for metabolic engineering is stan-dardized and conceptualized, many regulatory controlmechanisms in nature are not fully understood, andtherefore, it is becoming typical to utilize systematic andinformatics-based approaches combining genomic,proteomic, and metabolomic analyses.14 In addition tothe engineering techniques that allow modification ofpathways for better production, other strategies likeenzyme engineering and mutasynthesis can result in thecreation of libraries of natural products and non-naturalanalogs that can be evaluated as drug candidates usinghigh-throughput screening experiments. Metabolic engi-neering of natural product biosynthesis in microbes hasthe capability to generate immense amounts of targetcompounds to be used for discovery of novel non-natural compounds for pharmaceutical or nutritionalapplications.

4. SIGNIFICANCE OF FLAVONOIDS ANDSTILBENES IN HUMAN HEALTH AND

DISEASE

Flavonoids are the largest group of phenolic groupsamong plant secondary metabolites. In general, thisdiverse class of compounds can be categorized into sixmajor categories: isoflavones, flavanones, flavones, fla-vonols, catechins, and anthocyanins Table 36.1, all ofwhich are common in fruits, vegetables, herbs, redwine, tea, and other foods that are part of a regularhuman diet.6 Research on flavonoids was initiated byHungarian scientist Albert Szent-Gyorgi, who showed

the synergistic effect between pure vitamin C and yetunidentified co-factors from the peels of lemons.15 Thepotent antioxidant activity of flavonoids is of interestwith respect to human health. Excess reactive oxygenspecies (ROS) impair the immune system and causetissue injury followed by cardiovascular disease,inflammation, and cancer.16 The antioxidant effect orfree radical scavenging capacity of flavonoids has beenstudied extensively both in vitro and in vivo.17,18

Scientists studied the antioxidant potency of anthocya-nins (a subclass of the flavonoid family of molecules)in vivo using vitamin E-deficient rats. When the ratswere fed with purified anthocyanins extracted fromAbies koreana, decreased concentrations of hydroperox-ides and 8-oxo-deoxyguanosine were measured in thelivers, indicating anthocyanin-related prevention ofsome lipid peroxidation and DNA damage otherwiseassociated with vitamin E deficiency.19 Flavonoidshave been shown to exhibit many mechanisms of can-cer interference, including antimutagenic activity, inhi-bition of oxidative DNA damage, induction ofapoptosis, and anti-angiogenic effects.15,20 Catechinsfrom tea inhibit signaling cascades from epidermalgrowth factor receptors and induce apoptosis, or pro-gramed cell death, in various cancer models.21�23

Furthermore, the soy isoflavone genistein showsanticancer activities through modulation of cell cycleand apoptosis by activating nuclear factor kappa-B(NF-κB) and Akt signaling pathways. Moreover, genis-tein antagonizes estrogen- and androgen-mediated sig-naling pathways in the processes of carcinogenesis inboth in vivo and in vitro studies.24 Flavonoids have alsobeen studied for activity against type 2 diabetes. Anin vitro study explained the effect of the flavan-3-ols(1)-catechin and (1)-afzelechin on glucose-inducedinsulin secretion of pancreatic β-cells.25 Matsui et al.have focused on the antidiabetic activity of anthocya-nins and investigated a two-phase study on the inhibi-tion of rat intestinal α-glucosidase. The first reportshowed that plant extracts of anthocyanins inhibitedα-glucosidase activity against maltose.26 Inhibitionimproved when the α-glucosidase was immobilized tomimic the natural membrane-bound state of theenzyme. The second part of the study confirmed thatthe α-glucosidase inhibition was due to the anthocya-nins and not to other compounds in the extracts, andthe most active compounds were acylated anthocya-nins.27 The following year, the research group demon-strated in vivo effects of anthocyanins on blood glucoselevels by verifying that a single dose of anthocyaninextract reduced the rate of increase of the blood glu-cose level in rats.28

Stilbenes are produced by the aldol condensation ofthe tetraketide intermediate formed by the addition ofthree acetyl groups to p-coumaroyl-CoA by STS.

4854. SIGNIFICANCE OF FLAVONOIDS AND STILBENES IN HUMAN HEALTH AND DISEASE

5. INFLAMMATION AND POLYPHENOLS

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Among them, resveratrol is the most well-known andattractive compound. Resveratrol (3,5,40-trihydroxystil-bene) was first isolated from the roots of white helle-bore (Veratrum grandiflorum O. Loes) in 1940.29 Sincethe cardioprotective effects of red wine were demon-strated, several reports have shown that resveratrolcan prevent cancer, cardiovascular diseases, ischemicinjuries, and Alzheimer’s disease, and can alsoenhance stress resistance.4 Its anticancer activity hasbeen examined by investigating its antiproliferativeand pro-apoptotic effects in vitro and in vivo.Resveratrol has been shown to decrease platelet aggre-gation, suppress atherosclerosis, reduce lipid peroxida-tion, and improve serum cholesterol and triglycerideconcentrations.

5. CURRENT TECHNIQUES USINGRECOMBINANT MICROBES FOR THEPRODUCTION OF FLAVONOIDS AND

STILBENES

Although the use of natural products for preventionand treatment of human diseases has many advan-tages, isolation of natural products can be limited dueto their low bioavailability and environmental restric-tions. Therefore, microbes and plants have been meta-bolically engineered to overcome these limitations byoverproducing these compounds and making theresulting processes practical and productive.

5.1 Flavonoids

E. coli is the most widely studied microbial platformfor the production of flavonoids. A higher productionlevel of anthocyanins from catechins was achieved byfeeding with flavonoid intermediates in fermentationculture.9 In one study, it was found that S. cerevisiae har-bored a glucosidase that hydrolyzes flavonoid

glucosides, something that may hinder heterologousproduction of glycosylated anthocyanins. This challengemay be overcome by inactivating the glucosidases bymutations and/or gene knockouts.30 Carbon flux manip-ulation towards heterologous production of flavonoidsis another target to be examined. Miyahisa and co-workers31 overexpressed the enzyme acetyl-CoA carbox-ylase (ACC), which converts acetyl-CoA to malonyl-CoAin the fatty acid biosynthesis pathway. A three-foldincrease in the production of naringenin from tyrosineand a four-fold increase in pinocembrin production fromphenylalanine were observed. Another group extendedthese efforts to further increase malonyl-CoA bioavail-ability for flavonoid production.32 They found that theoverexpression of the four-subunit ACC fromPhotorhabdus luminescens resulted in a better enhance-ment of flavanone production than the two-subunitACC from Corynbacterium glutamicum used in the studypreviously mentioned. The authors enhanced carbonflux toward malonyl-CoA by overexpressing the acetateassimilation pathways by way of ackA and pta overex-pression or acs overexpression in addition to ACC. Theacetate assimilation pathways improved availability ofacetyl-CoA for conversion to malonyl-CoA by ACC. Thisled to flavanone production of up to 14 times higherthan control strains lacking the overexpressions.Another report presented two alternate approaches toincrease the pool of malonyl-CoA in the engineered E.coli.33 The first was to introduce the genes matB andmatC from R. trifolii into the E. coli strain, which encodethe malonate assimilation pathway, allowing conversionof malonate directly to malonyl-CoA as opposed to thenative conversion from glucose. This approach led toover 250% increase in flavanone production. Next, theauthors attenuated the fatty acid biosynthesis pathway,which competes with the grafted flavonoid pathway formalonyl-CoA. In order to achieve this, they added ceru-lenin to inhibit fatty acid biosynthesis. This led to morethan 900% increase in flavanone levels. UDP-glucose hasbeen identified as another important co-factor for

TABLE 36.1 Six Major Categories of Flavonoids

Flavonoid Subclass Phenylalanine Precursor Tyrosine Precursor Caffeic Acid Precursor

(R15H; R25H) (R15OH; R25H) (R15OH; R25H; R25OMe)

Flavonones (2S)-pinocembrin (2S)-naringen (2S)-eriodictyol

Isoflavones 5,7-dihydroxyisoflavone Genistein Orobol

Flavones Apigenin Luteolin Chrysin

Flavonols Kaempferol Quercetin Myrecetin

Anthocyanin 3-O-glucosides Pelargonidin 3-O-glucoside Cyanidin 3-O-glucoside Delphinidin 3-O-glucoside

Stilbenoids Pinosylvin Resveratrol Piceatannol

Curcuminoids Dicinnamoylmethane Bisdemethoxycurcumin Curcumin

486 36. USING RECOMBINANT MICROORGANISMS FOR THE SYNTHESIS AND MODIFICATION OF FLAVONOIDS AND STILBENES

5. INFLAMMATION AND POLYPHENOLS

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production of anthocyanins and glycosylated flavonoids.The E. coli strain used (BL21) was already lacking thegenes galE and galT that convert UDP-glucose to UDP-galactose, but the gene udg for UDP-glucose 6-dehydro-genase, which converts UDP-glucose to UDP-gluconorate, was still active. When the authors deletedit, they observed additional improvement to anthocyaninproduction, with the overexpression of ndk and supple-mentation of orotic acid. In a separate study aimed atproducing flavonoid glycosides in S. cerevisiae, research-ers found that addition of orotic acid improved the yieldof glycosides produced, likely due to increased produc-tion of UTP for UDP-glucose availability.34

5.2 Stilbenes

There have been many attempts to produce res-veratrol in heterologous hosts, such as bacteria andyeast. Wang and co-workers35 applied differentmethods to improve the biosynthesis of resveratrolin S. cerevisiae. Firstly, the enzyme tyrosine ammonialyase (TAL) was mutated and re-synthesized repla-cing the bacteria codons with yeast codons, whichincreased the production of p-coumaric acid and res-veratrol by up to 2.5-fold. Secondly, Becker and co-workers also tried to generate resveratrol with engi-neered yeast, introducing the phenylpropanoid path-way in S. cerevisiae to produce p-coumaroyl-CoA.36

To this end, the coenzyme-A ligase-encoding gene(4CL216) and the grapevine resveratrol synthasegene (vst1) were co-expressed in S. cerevisiae. Usingthis approach, Wang and co-workers observed a2�6-fold improvement in resveratrol yields.Mathematical algorithms like OptForce have beenused to guide genetic interventions for redirectingmalonyl-CoA flux towards the optimization of natu-ral products. Finally, Bhan and co-workers37

improved titers of resveratrol by B60% implement-ing one such strategy in E. coli.

6. PERSPECTIVES

Apart from being potential drug candidates, flavo-noids and stilbenes are widely used in the area of cos-metics, fragrances, nutraceuticals and food colorants.Increasing demand for these molecules makes theirmass production at high yields and high purity toindustrial scale indispensible. Such high-yield produc-tion would also allow the creation of well-defined mix-tures for the more detailed investigation of synergistichealth benefits of combinations of these compounds.

In this chapter, we depict the importance of flavo-noids and stilbenes in human health and disease and

recent advances towards the development of recombi-nant microorganisms for their production. Severalchallenges, however, still remain. Firstly, the produc-tion of flavonoids has been achieved at high titers onlywhen phenylpropanoic acids are fed as precursors tothe recombinant organism, primarily due to low activ-ity of PAL restricting aromatic amino acids (such asphenylalanine and tyrosine) conversion toward flavo-noid metabolism. This is a problem that can potentiallybe addressed through either bioprospecting of morePAL enzymes derived from plant and fungal sourcesor through protein engineering. However, once moreefficient PAL enzymes have been identified, a con-certed effort should be made towards optimizing thecarbon flux towards flavonoid precursor aromaticamino acids. Secondly, another important challenge isthe functional expression of P450 monooxygenases insimple prokaryotes such as E. coli. A number of suchenzymes are involved in the biosynthesis and functio-nalization of flavonoids; as proper function is depen-dent upon successful binding to the endoplasmicreticulum membrane, their efficient functional expres-sion in E. coli remains an engineering conundrum.Furthermore, in order to achieve flavonoid productionat the maximal theoretical yield, a substantial reduc-tion in the carbon flux that enters the fatty acid metab-olism is necessary, something that could potentially beachieved through antisense RNA and promoter andribosome binding site engineering. Finally, and notleast, there is little doubt that the creation of proteinscaffolds will be yet another engineering task that canpotentially enhance the production yields of flavonoidsfrom recombinant micoorganisms. Such scaffoldswould enable metabolite channeling through protein-protein interactions and metabolons, similar to whathas been speculated to exist in plants and in plant sec-ondary metabolic pathways.

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488 36. USING RECOMBINANT MICROORGANISMS FOR THE SYNTHESIS AND MODIFICATION OF FLAVONOIDS AND STILBENES

5. INFLAMMATION AND POLYPHENOLS