Using Recombinant Microorganisms for the Synthesis and Modification of Flavonoids and Stilbenes

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1 C H A P T E R 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 discovery 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 characteristic example, phytochemicals such as flavonoids and resveratrol have recently emerged as the underlying molecules behind the French paradox, 3,4 which is described as the observation that the French enjoy a relatively 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, cardiovascular disease, and aging. Over the decades, as a variety 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 economically and in high quantity and purity. Novel metabolic pathways have also been created by mixing and matching 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 functions in plant and chemical structures. 5 Flavonoids and related compounds are made through the phenylpropanoid pathway, as depicted in Figure The biosynthesis begins with the amino acid phenylalanine, which is deaminated to cinnamic acid by phenylalanine 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 catalyzed 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 Polyphenols in Human Health and Disease. DOI: Elsevier Inc. All rights reserved.

2 USING RECOMBINANT MICROORGANISMS FOR THE SYNTHESIS AND MODIFICATION OF FLAVONOIDS AND STILBENES FIGURE 36.1 Flavonoid biosynthesis. Adapted from Cress et al. 6 selectively to (2S)-flavanones, which are then hydroxylated by flavanone 3β-hydroxylase (FHT) at the 3-carbon position to give dihydroflavonols. These are reduced by dihydroflavonol 4-reductase (DFR) at the 4-carbon position, yielding the unstable leucoanthocyanidins. Leucoanthocyanidin reductase (LAR) catalyzes the subsequent reduction to flavan-3-ols (also called catechins). Both the leucoanthocyanidins and the flavan-3-ols are possible substrates for anthocyanidin synthase (ANS), which catalyzes the reaction to anthocyanidins. Finally, UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT) catalyzes the glycosylation at the 3-carbon, yielding anthocyanins. 8,9 Additional enzymes exist to catalyze addition of functional groups or manipulation of the skeleton to 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, catalyzing the formation of either resveratrol from p-coumaryl-coa or pinosylvin from cinnamoyl-coa, is a unique, distinct polyketide synthase that is closely related to CHS. While chalcone synthase is present in higher plants, stilbene synthase has a much more restricted distribution in the plant kingdom. Also, stilbene (or resveratrol) synthase exhibits wide substrate-specificity and can also accept other CoA esters aliphatic as well as aromatic ones as primers for polyketide synthesis. 11,12 Therefore, it is considered as one of the most important enzymes to participate in carbon backbone diversity in natural product pathways. 13

3 4. SIGNIFICANCE OF FLAVONOIDS AND STILBENES IN HUMAN HEALTH AND DISEASE 3. RECOMBINANT MICROBES Metabolic engineering is a powerful tool to generate desirable products at high productivity by manipulating the cellular and metabolic characteristics of a host organism. One of the big challenges of metabolic engineering is to identify optimal organisms and to determine targets for manipulations in individual genes, whole pathways, or even in transcriptional and translational control elements. In general, metabolic engineering of natural product biosynthesis in microbes consists of the following steps: bioprospecting and recombinant pathway design (recombineering); selection and cloning or synthesis of heterologous genes; production host choice, vector choice, and transformation of heterologous genes into host; troubleshooting expression, folding, and activity of plant proteins in microbial hosts (often via protein engineering); strain improvement via carbon flux redistribution, toxicity reduction, transporter engineering, removal of regulatory restrictions, and enzyme colocalization or compartmentalization; and fermentation optimization. Although the whole procedure for metabolic engineering is standardized and conceptualized, many regulatory control mechanisms in nature are not fully understood, and therefore, it is becoming typical to utilize systematic and informatics-based approaches combining genomic, proteomic, and metabolomic analyses. 14 In addition to the engineering techniques that allow modification of pathways for better production, other strategies like enzyme engineering and mutasynthesis can result in the creation of libraries of natural products and non-natural analogs that can be evaluated as drug candidates using high-throughput screening experiments. Metabolic engineering of natural product biosynthesis in microbes has the capability to generate immense amounts of target compounds to be used for discovery of novel nonnatural compounds for pharmaceutical or nutritional applications. 4. SIGNIFICANCE OF FLAVONOIDS AND STILBENES IN HUMAN HEALTH AND DISEASE Flavonoids are the largest group of phenolic groups among plant secondary metabolites. In general, this diverse class of compounds can be categorized into six major categories: isoflavones, flavanones, flavones, flavonols, catechins, and anthocyanins Table 36.1, all of which are common in fruits, vegetables, herbs, red wine, tea, and other foods that are part of a regular human diet. 6 Research on flavonoids was initiated by Hungarian scientist Albert Szent-Gyorgi, who showed 485 the synergistic effect between pure vitamin C and yet unidentified co-factors from the peels of lemons. 15 The potent antioxidant activity of flavonoids is of interest with respect to human health. Excess reactive oxygen species (ROS) impair the immune system and cause tissue injury followed by cardiovascular disease, inflammation, and cancer. 16 The antioxidant effect or free radical scavenging capacity of flavonoids has been studied extensively both in vitro and in vivo. 17,18 Scientists studied the antioxidant potency of anthocyanins (a subclass of the flavonoid family of molecules) in vivo using vitamin E-deficient rats. When the rats were fed with purified anthocyanins extracted from Abies koreana, decreased concentrations of hydroperoxides and 8-oxo-deoxyguanosine were measured in the livers, indicating anthocyanin-related prevention of some lipid peroxidation and DNA damage otherwise associated with vitamin E deficiency. 19 Flavonoids have been shown to exhibit many mechanisms of cancer interference, including antimutagenic activity, inhibition of oxidative DNA damage, induction of apoptosis, and anti-angiogenic effects. 15,20 Catechins from tea inhibit signaling cascades from epidermal growth factor receptors and induce apoptosis, or programed cell death, in various cancer models Furthermore, the soy isoflavone genistein shows anticancer activities through modulation of cell cycle and apoptosis by activating nuclear factor kappa-b (NF-κB) and Akt signaling pathways. Moreover, genistein antagonizes estrogen- and androgen-mediated signaling pathways in the processes of carcinogenesis in both in vivo and in vitro studies. 24 Flavonoids have also been studied for activity against type 2 diabetes. An in vitro study explained the effect of the flavan-3-ols (1)-catechin and (1)-afzelechin on glucose-induced insulin secretion of pancreatic β-cells. 25 Matsui et al. have focused on the antidiabetic activity of anthocyanins and investigated a two-phase study on the inhibition of rat intestinal α-glucosidase. The first report showed that plant extracts of anthocyanins inhibited α-glucosidase activity against maltose. 26 Inhibition improved when the α-glucosidase was immobilized to mimic the natural membrane-bound state of the enzyme. The second part of the study confirmed that the α-glucosidase inhibition was due to the anthocyanins and not to other compounds in the extracts, and the most active compounds were acylated anthocyanins. 27 The following year, the research group demonstrated in vivo effects of anthocyanins on blood glucose levels by verifying that a single dose of anthocyanin extract reduced the rate of increase of the blood glucose level in rats. 28 Stilbenes are produced by the aldol condensation of the tetraketide intermediate formed by the addition of three acetyl groups to p-coumaroyl-coa by STS.

4 USING RECOMBINANT MICROORGANISMS FOR THE SYNTHESIS AND MODIFICATION OF FLAVONOIDS AND STILBENES TABLE 36.1 Six Major Categories of Flavonoids Flavonoid Subclass Phenylalanine Precursor Tyrosine Precursor Caffeic Acid Precursor (R 1 5 H; R 2 5 H) (R 1 5 OH; R 2 5 H) (R 1 5 OH; R 2 5 H; R 2 5 OMe) 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 Among them, resveratrol is the most well-known and attractive compound. Resveratrol (3,5,4 0 -trihydroxystilbene) was first isolated from the roots of white hellebore (Veratrum grandiflorum O. Loes) in Since the cardioprotective effects of red wine were demonstrated, several reports have shown that resveratrol can prevent cancer, cardiovascular diseases, ischemic injuries, and Alzheimer s disease, and can also enhance stress resistance. 4 Its anticancer activity has been examined by investigating its antiproliferative and pro-apoptotic effects in vitro and in vivo. Resveratrol has been shown to decrease platelet aggregation, suppress atherosclerosis, reduce lipid peroxidation, and improve serum cholesterol and triglyceride concentrations. 5. CURRENT TECHNIQUES USING RECOMBINANT MICROBES FOR THE PRODUCTION OF FLAVONOIDS AND STILBENES Although the use of natural products for prevention and treatment of human diseases has many advantages, isolation of natural products can be limited due to their low bioavailability and environmental restrictions. Therefore, microbes and plants have been metabolically engineered to overcome these limitations by overproducing these compounds and making the resulting processes practical and productive. 5.1 Flavonoids E. coli is the most widely studied microbial platform for the production of flavonoids. A higher production level of anthocyanins from catechins was achieved by feeding with flavonoid intermediates in fermentation culture. 9 In one study, it was found that S. cerevisiae harbored a glucosidase that hydrolyzes flavonoid glucosides, something that may hinder heterologous production of glycosylated anthocyanins. This challenge may be overcome by inactivating the glucosidases by mutations and/or gene knockouts. 30 Carbon flux manipulation towards heterologous production of flavonoids is another target to be examined. Miyahisa and coworkers 31 overexpressed the enzyme acetyl-coa carboxylase (ACC), which converts acetyl-coa to malonyl-coa in the fatty acid biosynthesis pathway. A three-fold increase in the production of naringenin from tyrosine and a four-fold increase in pinocembrin production from phenylalanine were observed. Another group extended these efforts to further increase malonyl-coa bioavailability for flavonoid production. 32 They found that the overexpression of the four-subunit ACC from Photorhabdus luminescens resulted in a better enhancement of flavanone production than the two-subunit ACC from Corynbacterium glutamicum used in the study previously mentioned. The authors enhanced carbon flux toward malonyl-coa by overexpressing the acetate assimilation pathways by way of acka and pta overexpression or acs overexpression in addition to ACC. The acetate assimilation pathways improved availability of acetyl-coa for conversion to malonyl-coa by ACC. This led to flavanone production of up to 14 times higher than control strains lacking the overexpressions. Another report presented two alternate approaches to increase the pool of malonyl-coa in the engineered E. coli. 33 The first was to introduce the genes matb and matc from R. trifolii into the E. coli strain, which encode the malonate assimilation pathway, allowing conversion of malonate directly to malonyl-coa as opposed to the native conversion from glucose. This approach led to over 250% increase in flavanone production. Next, the authors attenuated the fatty acid biosynthesis pathway, which competes with the grafted flavonoid pathway for malonyl-coa. In order to achieve this, they added cerulenin to inhibit fatty acid biosynthesis. This led to more than 900% increase in flavanone levels. UDP-glucose has been identified as another important co-factor for

5 REFERENCES 487 production of anthocyanins and glycosylated flavonoids. The E. coli strain used (BL21) was already lacking the genes gale and galt that convert UDP-glucose to UDPgalactose, but the gene udg for UDP-glucose 6-dehydrogenase, which converts UDP-glucose to UDPgluconorate, was still active. When the authors deleted it, they observed additional improvement to anthocyanin production, with the overexpression of ndk and supplementation of orotic acid. In a separate study aimed at producing flavonoid glycosides in S. cerevisiae, researchers found that addition of orotic acid improved the yield of glycosides produced, likely due to increased production of UTP for UDP-glucose availability Stilbenes There have been many attempts to produce resveratrol in heterologous hosts, such as bacteria and yeast. Wang and co-workers 35 applied different methods to improve the biosynthesis of resveratrol in S. cerevisiae. Firstly, the enzyme tyrosine ammonia lyase (TAL) was mutated and re-synthesized replacing the bacteria codons with yeast codons, which increased the production of p-coumaric acid and resveratrol by up to 2.5-fold. Secondly, Becker and coworkers also tried to generate resveratrol with engineered yeast, introducing the phenylpropanoid pathway in S. cerevisiae to produce p-coumaroyl-coa. 36 To this end, the coenzyme-a ligase-encoding gene (4CL216) and the grapevine resveratrol synthase gene (vst1) were co-expressed in S. cerevisiae. Using this approach, Wang and co-workers observed a 2 6-fold improvement in resveratrol yields. Mathematical algorithms like OptForce have been used to guide genetic interventions for redirecting malonyl-coa flux towards the optimization of natural products. Finally, Bhan and co-workers 37 improved titers of resveratrol by B60% implementing one such strategy in E. coli. 6. PERSPECTIVES Apart from being potential drug candidates, flavonoids and stilbenes are widely used in the area of cosmetics, fragrances, nutraceuticals and food colorants. Increasing demand for these molecules makes their mass production at high yields and high purity to industrial scale indispensible. Such high-yield production would also allow the creation of well-defined mixtures for the more detailed investigation of synergistic health benefits of combinations of these compounds. In this chapter, we depict the importance of flavonoids and stilbenes in human health and disease and recent advances towards the development of recombinant microorganisms for their production. Several challenges, however, still remain. Firstly, the production of flavonoids has been achieved at high titers only when phenylpropanoic acids are fed as precursors to the recombinant organism, primarily due to low activity of PAL restricting aromatic amino acids (such as phenylalanine and tyrosine) conversion toward flavonoid metabolism. This is a problem that can potentially be addressed through either bioprospecting of more PAL enzymes derived from plant and fungal sources or through protein engineering. However, once more efficient PAL enzymes have been identified, a concerted effort should be made towards optimizing the carbon flux towards flavonoid precursor aromatic amino acids. Secondly, another important challenge is the functional expression of P450 monooxygenases in simple prokaryotes such as E. coli. A number of such enzymes are involved in the biosynthesis and functionalization of flavonoids; as proper function is dependent upon successful binding to the endoplasmic reticulum membrane, their efficient functional expression in E. coli remains an engineering conundrum. Furthermore, in order to achieve flavonoid production at the maximal theoretical yield, a substantial reduction in the carbon flux that enters the fatty acid metabolism is necessary, something that could potentially be achieved through antisense RNA and promoter and ribosome binding site engineering. Finally, and not least, there is little doubt that the creation of protein scaffolds will be yet another engineering task that can potentially enhance the production yields of flavonoids from recombinant micoorganisms. Such scaffolds would enable metabolite channeling through proteinprotein interactions and metabolons, similar to what has been speculated to exist in plants and in plant secondary metabolic pathways. 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