Metabolic engineering of resveratrol and other longevity boosting compounds

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1 Metabolic engineering of resveratrol and other longevity boosting compounds Yechun Wang, Hui Chen, and Oliver Yu* Donald Danforth Plant Science Center, St. Louis, MO, USA Abstract. Resveratrol, a compound commonly found in red wine, has attracted many attentions recently. It is a diphenolic natural product accumulated in grapes and a few other species under stress conditions. It possesses a special ability to increase the life span of eukaryotic organisms, ranging from yeast, to fruit fly, to obese mouse. The demand for resveratrol as a food and nutrition supplement has increased significantly in recent years. Extensive work has VC 2010 International Union of Biochemistry and Molecular Biology, Inc. Volume 36, Number 5, September/October 2010, Pages oyu@danforthcenter.org been carried out to increase the production of resveratrol in plants and microbes. In this review, we will discuss the biosynthetic pathway of resveratrol and engineering methods to heterologously express the pathway in various organisms. We will outline the shortcuts and limitations of common engineering efforts. We will also discuss briefly the features and engineering challenges of other longevity boosting compounds. Keywords: resveratrol, metabolic engineering, longevity 1. Introduction Resveratrol (3,5,4 0 -trihydroxy-trans-stilbene) is a low-molecular mass phenolic compound produced by a limited number of higher plants. It was first discovered from white hellebore (Veratrum album) in 1940s [1], and later, from the roots of Japanese knotweed (Polygonum cuspidatum) in 1963 [2]. Resveratrol can also be found in grapes, mulberries, peanuts, jackfruit, eucalyptus, rheum, and a few other species [3]. One of the most common dietary sources of resveratrol is red wine made from grapes. Resveratrol is a byproduct of plant response to many outside stresses, including fungal infection, wounding, or UV radiation [4]. With strong anti-microbial activities, resveratrol plays important roles in disease resistance in plants [5]. It is a major antimicrobial compound, called phytoalexin, of grapevine. Therefore, the accumulation of resveratrol is tightly linked to environmental and growth conditions of grapes. Fungal infection during fruit development drastically increases resveratrol levels in grapes. Resveratrol was found in all tissues of the grapevine, but the highest concentrations are found in the grape skins. As only trace amounts are present in the fruit flesh, resveratrol is hardly detectable in white wines, but is a key ingredient in red wines [6]. Resveratrol content of red wine is mostly *Address for correspondence: Oliver Yu, Ph.D., Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA. Tel.: ; Fax: ; oyu@danforthcenter.org. Received 14 July 2010; accepted 2 August 2010 DOI: /biof.126 Published online 16 September 2010 in Wiley Online Library (wileyonlinelibrary.com) between 0.2 and 5.8 mg/l depending on grape variety, location, season, and growth temperature [7]. In recent years, resveratrol was reported to have many human health benefits such as anti-oxidant, anti-oncogenic, antiviral, and anti-aging activities [8,9]. Research showed that resveratrol helps prevent damage to blood vessels, reduces cholesterol, and prevents blood clots due to its ability to act as an antioxidant and as an inhibitor of platelet aggregation [10]. Research in mice demonstrated that the antioxidant activity of resveratrol might also help protect mice from obesity and diabetes. Importantly, the specific effects of resveratrol on longevity attracted extensive attentions in aging research. Earlier reports suggested that resveratrol could mimic reduced caloric intake and extend lifespan in yeast and metazoans such as worms and fruit flies [10 12]. Because resveratrol has strong anti-oxidant activity and specific effects on longevity, the increasing demand for resveratrol, especially as a food supplement, skyrocketed in recent years. Metabolic engineering to increase resveratrol production thus has significant commercial value. All the genes encoding enzymes responsible for resveratrol biosynthesis have been cloned and characterized in detail, making biological engineering of this compound relatively straightforward. In this review, we will discuss recent progresses in metabolic engineering of resveratrol in various organisms. We will also discuss metabolic engineering of a few other compounds that have been shown to increase longevity in animal models. Engineering of resveratrol and related compounds is a fine example of what is the requirement for a successful engineering project, and the 394

2 pitfalls and future prospects of engineering metabolic pathways. 2. Resveratrol on longevity As a major scientific breakthrough, resveratrol was discovered in 2003 as one of the most potent compounds to increase the number of cell cycles in yeast Saccharomyces cerevisiae [10]. Since then, resveratrol has been shown to increase longevity in round worm (Caenorhabditis elegans), fruit fly (Drosophila melanogaster), a vertebrate fish (Nothobranchius furzeri), and obese mice [10,13,14]. In yeast, addition of resveratrol to the culture media nearly doubled the yeast lifespan as measured by the number of cell division cycles. Treatment with resveratrol increased longevity of round worm and fruit fly by 14% and 30%, respectively. In more complex animals, resveratrol feeding led to improved health and extended lifespan of fish by 59% [14]. More interestingly, high dosage of resveratrol feeding extended the life span of obese mice by several months, which also negated the dangerous effects of high-fat/high-cholesterol diets. The mechanism of resveratrol s effect on longevity is still a subject of debate [15]. The exact target of resveratrol remains controversial. The initial report demonstrated that the effects of resveratrol on cell cycles depending on a sirtuin gene. When mutated, sirtuin-null yeast did not respond to resveratrol treatment. Sirtuin is known as NAD þ -dependent deacetylase, a highly conserved metabolic regulator throughout eukaryotic organisms. Sirtuin has been implicated in promoting longevity by caloric restriction (CR). CR was one of the most efficient ways to increase life span. In mouse and other model systems, a simple reduction in caloric intake in the absence of malnutrition can increase longevity significantly [15]. Mutations in a couple of genes can block the effects of CR, suggesting a signal transduction pathway may exist to monitor the calorie intake and regulate cellular metabolism accordingly. One member of this signal transduction pathway was proven to be sirtuin, whose overexpression can extend lifespan in yeasts, round worms, and files [16 18]. On the other hand, AMP-activated protein kinase-deficient mice are resistant to the metabolic effects of resveratrol, suggesting that resveratrol may activate Sirt1 indirectly [19]. In support of sirtuin as the signal carrier of resveratrol, human sirtuin homolog, SIRT1, could be activated by resveratrol and mediate the beneficial effects on human health [20,21]. Resveratrol enhances binding and deacetylation of peptide substrate containing the Fluor de Lys group in vitro [21]. Subsequent studies showed that resveratrol could activate not only SIRT1 but also SIRT3 and SIRT4, as well as FOXO transcription factor and PBEF, which supplies NAD þ to the SIRT1 and has been linked to longevity [22]. Genetically, it was reported that resveratrol induced transcriptional co-activator PGC-1a, which is positively regulated by SIRT1-mediated deactylation. As in yeast, resveratrol did not extend lifespan of worm and fly if the sirtuin homologues were mutated. Similarly, behavior associated with caloric restriction did not occur when Sirt1 knockout mice were put on a calorie-restricted diet [23]. To date, resveratrol is the most potent natural compound able to activate SIRT1, mimicking the positive effect of calorie restriction to turn on longevity genes [24]. However, recent reports also expanded previous works that showed resveratrol was not the direct activator of SIRT1 [25,26]. Beher et al. reported that resveratrol could not activate SIRT1 activity using native p53 peptide as a substrate in vivo. Pacholec et al. further demonstrated that resveratrol did not lead to activation of SIRT1 with native peptide or fulllength protein substrates; instead, SIRT1 directly interacted with one or more fluorophore-containing peptide substrates [25]. Recently, other longevity genes, such as PHA-4, a forkhead transcription factor, WWP-1, and UBC-18 were discovered to mediate CR-induced longevity of C. elegans [27 29], which might serve as the alternative targets of resveratrol. At the same time, it is important to note that resveratrol failed to mimic other sirtuin-regulated physiological processes, such as reducing heart rate, increasing mitochondria content of muscle cells, and decreasing core body temperature [29]. 3. Resveratrol biosynthetic pathway Despite the controversial mechanisms, resveratrol attracted great attention as a proven compound to increase longevity in many animals. In plants, resveratrol exists in free (cis and trans isomers) and glycosylated forms (called piceid). All these forms of resveratrol are synthesized from the phenylpropanoid pathway. This pathway exists in all higher plants and is responsible for the synthesis of a wide range of phenolic natural compounds, such as flavonoids, lignins, and proanthocyanindins. Resveratrol biosynthesis forms a branch of phenylpropanoid pathway in related species (Fig. 1) [30]. Deamination of the amino acid phenylalanine by phenylalanine ammonia lyase (PAL) is the first step of resveratrol biosynthesis. The product of PAL, cinnamic acid, is used as a substrate of cinnamate-4-hydroxylase (C4H), which catalyzes it into p-coumaric acid. The third enzyme, p-coumarate:coa ligase (4CL), attaches the p-coumaric acid to the pantetheine group of coenzyme A to produce p-coumaroyl-coa. p-coumaroyl-coa is an important intermediate of all higher plants. It can be converted into monolignols for lignin biosynthesis, or flavonoids for anthocyanin and proanthocyanidin biosynthesis. In grapes and other resveratrol-accumulating plants, the last step of resveratrol synthesis is the stepwise condensation of one molecule of p-coumaroyl-coa with three molecules of malonyl-coa, catalyzed by stilbene synthase (STS). Malonyl-CoA is a precursor of flavonoids, and also an intermediate of fatty acid biosynthesis. Most bacteria and fungi do not make resveratrol, but p-coumaric acid synthesis has been reported. In some bacterial, the characterization of tyrosine ammonia lyase (TAL) simplified the pathway of p-coumaric acid synthesis by replacing the first 2 enzymes from the plant pathway with a single bacterial enzyme TAL (Fig. 1) [31]. 4. Resveratrol metabolic engineering The illustration of resveratrol synthetic pathway paved the way for metabolic engineering in plants and other Engineering of resveratrol and related compounds 395

3 Fig. 1. The schematic diagram of the resveratrol biosynthetic pathways. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] organisms. As resveratrol is a potent phytoalexin against plant pathogens and can enhance plant resistance to microbial disease, earlier applications of resveratrol engineering focused primarily on this anti-microbial potential [32,33]. The first report of heterologous expression of resveratrol was published in 1990, in which STS gene was introduced into tobacco via Agrobacterium-mediated transformation. Overexpression of STS led to resveratrol accumulation in tobacco leaves and flowers, making transgenic plants more resistant to infection of native fungal pathogen Botrytis cinerea [34,35]. In the transgenic plants, up to 400 lg resveratrol per gram fresh weight were detected. Since then, STS gene has been expressed in many other species such as rice [36], apple [37], kiwifruit [38], alfalfa [39], and lettuce [40]. The resveratrol levels in these plants are relatively low, ranging from 15 to 182 lg/g of fresh weight. However, in most of these transgenic plants, resveratrol engineering led to enhanced disease resistance against microbial pathogens. There was much interest in engineering resveratrol biosynthesis in bacteria and yeast even before the report of its longevity effects. Resveratrol can reduce serum lipids and cause other physiological effects associated with the health benefits called French Paradox, which suggests that French people have a relatively low incidence of coronary heart problems despite having a diet rich in saturated fats [41]. The consumption of red wine has long been suspected as the answer to the French Paradox. Therefore, resveratrol is the target of many clinical trials for its health benefits in the 1990s. However, one major variable is the concentration of resveratrol in red wines. Only wines made from grapes exposed to plant diseases contain high levels of resveratrol. Drier and cooler weather or application of chemical fungicides during plant growth seasons often reduced resveratrol accumulation significantly [42]. Thus, fermentation engineering to produce resveratrol will have major commercial potentials in the wine industry. Theoretically, the engineered yeast not only will ensure a stable high level of resveratrol in the red wine regardless of the growing conditions but can also introduce the health-promoting compound to many other fermentation products. Additionally, consumers would like to explore other sources of dietary resveratrol than alcoholic drinks. Not surprisingly, current efforts to increase the availability of resveratrol for commercial applications focus primarily on heterologous expression of the phenylpropanoid pathway genes in baker yeast S. cerevisiae. S. cerevisiae has been used as biological factories to synthesize different valuable compounds [43 45]. In heterologous microbial systems, tyrosine ammonia lyase (TAL) can replace PAL and C4H by producing p-coumaric acid directly from tyrosine. In vitro enzyme activity assay showed that the TAL gene, from bacterium Rhodobacter sphaeroides, was able to catalyze the formation of p-coumaric acid from L-tyrosine [31,45]. However, overexpression of TAL in yeast from in vivo yeast assays failed to produce any p-coumaric acid [45], even though the transcripts of TAL was detected at high levels in yeast cells containing TAL transgene [45]. These results seem to suggest that culture condition or differences in codon usage between Rhodobacter and yeast might limit protein expression in yeast. Transgenic S. cerevisiae coexpressing 4CL and STS produces resveratrol when fed p- coumaric acid; however, only low levels of resveratrol (<1.5 lg/l) were obtained. More recently, Trantas et al. constructed the complete resveratrol biosynthetic pathway in yeast to produce resveratrol from phenylalanine. When the medium was supplemented with 10 mm of phenylalanine, the strain produced 0.29 mg/l trans-resveratrol after about 120 h of cultivation [46]. If de novo biosynthesis is not required, feeding p-coumaric acid can reduce the cost of fermentation and increase the yield. Beekwilder et al. showed the yield of resveratrol can reach 6 mg/l in yeast [47]. Because protein protein interactions may increase metabolic efficiency by channeling intermediates between enzymes, expression of the 4CL::STS fusion protein increased resveratrol production significantly 396 BioFactors

4 in the culture [45]. Further improvement of the fermentation platform is still possible. An engineered Escherichia coli JM109 strain carrying 4CL and STS accumulated over mg/l resveratrol with feeding substrate of 1 mm p-coumaric acid [44,48]. More strikingly, industrial S. cerevisiae strain expressing 4CL and STS was reported to produce resveratrol up to 391 mg/l in rich medium [49]. The yield of resveratrol production has increased more than 250,000-fold because the initial report of resveratrol engineering in yeast [3]. 5. Potential of resveratrol metabolic engineering In short, metabolic engineering of resveratrol in plants, even in common crops, has been successful. To meet current industry demand for this compound, high-yield microbial engineering becomes necessary. Both S. cerevisiae and E. coli have been engineered to express genes involved in resveratrol biosynthesis. To date, the highest yield of resveratrol from engineered E. coli was over 171 mg/l in about 60 h of growth with feeding p-coumaric acid. Even higher resveratrol accumulation was achieved by using an industrial Brazilian sugar cane fermenting yeast (391 mg/l). The results revealed that under the same culture conditions, higher resveratrol production was achieved with industrial strain than with laboratory strain CEN.OK2-1 (261 mg/l), and by fermentation in YEPD-instead of SD-medium. Strains, culture media, and incubation time all affect resveratrol production [49]. These studies showed that both yeast and bacteria are well suited host for industrial resveratrol production. Although the amount of resveratrol produced is much higher today, the conversion efficiency is only between 34.3% and 43.5% from the added substrate p-coumaric acid. In addition, compared with the microbial production of other high value small molecules, resveratrol biosynthesis still has a huge room for improvement. For example, citric acid and erythritol produced by osmosis-tolerant fungi can reach several hundreds of grams per liter, which is almost a 1,000-fold higher than the high end of reported resveratrol production. Very few industrial fungal species have been tested for resveratrol production as of today. The plant enzymes of the resveratrol pathway are well characterized. The factors affecting enzyme activities have been documented and can be monitored and regulated during fermentation. The last step of resveratrol synthesis requires STS to catalyze the condensation of one molecule of p-coumaroyl-coa with three molecules of malonyl-coa. Bacteria and yeast produce only small amounts of cellular malonyl-coa, whichisalsoanintermediateoffattyacidbiosynthesis[48].it has been widely accepted that acetyl-coa carboxylase is the only source for malonyl-coa biosynthesis and the formation of malonyl-coa by acetyl-coa carboxylase is a well-established rate-limiting step in lipid biosynthesis. In transgenic cells engineered for resveratrol production, a portion of endogenous malonyl-coa will be diverted into resveratrol and malonyl-coa is potentially limiting the formation of resveratrol on a large scale. Therefore, if the cellular malonyl-coa is enhanced, resveratrol accumulation might be increased many folds. Fig. 2. Selected compounds that have been shown to increase longevity in animal models. 6. Other longevity boosting compounds Calorie restriction mimetic (CRM) compounds are chemical substances, which can mimic the physiological changes of CR in the absence of food restriction. The stress pathways activated by CR can be activated by CRMs [50]. As discussed above, resveratrol is considered as one example of the signal molecules mediating the lifespan benefits of CR. Extensive research are on-going to find additional CRMs to increase longevity. SRT1720 is a compound discovered based on screening of sirtuin-interacting compounds. Interestingly, resveratrol suppresses mitogen-induced phosphorylation of S6K and activates the kinase AMPK, suggesting it might also function as an indirect inhibitor of the target of rapamycin (TOR) signaling pathway [51]. The TOR signaling pathway is one of the sirtuin-independent pathways that showed major effects of longevity. Several compounds have been reported to interact with TOR pathway and have effects on extending life span. We have selected a few compounds that have longevity effects for comparisons and further discussions (Fig. 2). Engineering of resveratrol and related compounds 397

5 6.1. SRT1720 SRT1720 is a small molecule identified by Sirtris Pharmaceuticals as an activator of the sirtuin subtype SIRT1 [52]. It is structurally unrelated to resveratrol but was reported to activate SIRT1 with a potency 1,000-fold greater than resveratrol [52]. In vivo, SRT1720 produces a signaling profile that mirrors CR, improves glucose and insulin homeostasis [53]. As a man-made compound, SRT1720 can be obtained through chemical synthesis [52]. However, recent studies suggest that similar to resveratrol, SRT1720 may not be a direct activator of SIRT1 [25] Rapamycin Rapamycin, also known as sirolimus, is a macrocyclic polyketide antibiotic which was first isolated from the filamentous soil bacterium Streptomyces hygroscopicus [54]. It possesses antifungal, immunosuppressive, and antiproliferative activities. Its mechanism of action has been extensively studied and it is well established that rapamycin is an inhibitor of TOR signaling pathway. TOR is a phosphatidylinositol kinaserelated Ser/Thr kinase that regulates cell growth in response to nutrient availability. TOR-mediated nutrient-sensing pathway is also an evolutionarily conserved machinery in eukaryotes [50]. Recently, rapamycin was found to extend lifespan in mice [55] as well as abolish cognitive deficits and reduce amyloid-b levels in a mouse model of Alzheimer s disease [56]. In S. hygroscopicus, a type I polyketide synthase system consisting three clustered polyketide synthase genes rapa, rapb, and rapc among other genes is responsible for rapamycin biosynthesis. The three enzymes RapA, RapB, and RapC have a total of 14 modules, each catalyzing a specific round of chain elongation [57]. Rapamycin has been the subject of extensive biosynthetic investigations and a number of novel rapamycin analogues have been generated [58]. In addition to semisynthesis, novel techniques such as precursordirected biosynthesis and mutasynthesis are used to synthesize rapamycin analogues. Both approaches are based on the cellular uptake of modified biosynthetic intermediates and their incorporation into complex secondary metabolites [59,60] Nordihydroguaiaretic acid Nordihydroguaiaretic acid, also called as masoprocol, is a naturally occurring tetrahydroxy lignin, which makes up 5 10% of the dry weight of the leaves of creosote bush (Larrea tridentata) [61]. NDGA is a strong antioxidant with antineoplastic, antiviral, and anti-inflammatory activities [62]. It was also reported that NDGA inhibits tumor necrosis factor a- activation of microglia and extends survival of G93A-SOD1 transgenic mice [63]. More recently, NDGA was found to increase lifespan of genetically heterogeneous male mice [64]. Considering the fact that both resveratrol and NDGA are polyphenols derived from the phenopropanoid pathway, it is possible that NDGA exerts its life-extension activity through activating sirtuin-mediated pathway of CR [11]. The biosynthetic pathway leading to the formation of NDGA in creosote bush has been proposed and one of the genes in the pathway which encodes (þ)-larreatricin hydroxylase has been cloned [65,66] Metformin Metformin, a biguanide alkaloid originated from French lilac (Galega officinalis), is clinically used for the treatment of type 2 diabetes [51,67]. Metformin increases insulin sensitivity and lowers blood glucose levels through activating AMPactivated protein kinase (AMPK) [68] in an LKB1 (a proteinthreonine kinase) dependent manner [69]. Metformin also induces a dietary restriction-like state and the oxidative stress response to extend the lifespan of C. elegans [70]. It has been suggested that the anti-aging effects of metformin result from the inhibition of TOR [51]. The biosynthetic pathway of metformin is largely unknown Aspirin Salicylic acid is a plant hormone mediating the defense responses against microbial pathogens. It was first isolated from the willow bark in 1838 and the first synthetically produced conversion of SA to aspirin was achieved in 1859 [71]. Aspirin, a derivative of salicylic acid (SA) also known as acetylsalicylic acid, is clinically used as a popular nonsteroidal anti-inflammatory agent that also has antithrombotic and antioxidant properties, and its principal mechanism of action is to inhibit the activity of COX (cyclooxygenase)-1 and 2 [64]. Aspirin was reported to exert anti-diabetic activities through protecting insulin receptor substrate (IRS) proteins from serine phosphorylation catalyzed by multiple kinases [72]. More recent studies revealed that aspirin can increase lifespan of genetically heterogeneous male mice [64]. Because chemical synthesis of aspirin is well established, metabolic engineering of this compound in plants and microbes has not been widely reported. 7. Conclusions As a plant disease resistant agent and nutrition supplement, resveratrol is the target of engineering for almost 20 years. Along with other components of the plant phenylpropanoid pathway, the enzymes involved in making resveratrol have been investigated for decades, and their protein structures and catalytic mechanisms are mostly resolved. With this information, engineering of resveratrol in heterologous systems, in theory, should be easy. However, as evidenced in this review, engineering of resveratrol and related longevity boosting compounds still needs deeper understanding of the pathways and many cellular processes. Unknown limiting factors that prohibit further accumulation of resveratrol in plants and microbes abound. Although chemical de novo synthesis of trans-resveratrol can be achieved, the commercial values of natural resveratrol are driving scientists to explore additional methods to improve the biosynthetic approaches in native and heterologous systems. The efforts in engineering in turn will increase our knowledge of resveratrol and other longevity boosting compounds. 398 BioFactors

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