Ann Microbiol (2015) 65:817–826 DOI 10.1007/s13213-014-0922-z ORIGINAL ARTICLE Pterostilbene production by microorganisms expressing resveratrol O-methyltransferase Yechun Wang & Mohammad Wadud Bhuiya & Rui Zhou & Oliver Yu Received: 28 February 2014 /Accepted: 26 May 2014 /Published online: 26 June 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Pterostilbene (3,5-dimethoxy-4′-hydroxyl-trans- Introduction stilbene)—a derivative of resveratrol—is a natural dietary compound and the primary antioxidant component in berries. Stilbenes comprise a small class of plant secondary metabo- Pterostilbene has significant advantages over resveratrol in lites derived from the phenylpropanoid pathway. The best- bioavailability, half-life in the body, cellular uptake, oral ab- known stilbene is resveratrol (3,5,4′-trihydroxy-trans- sorption and metabolic stability. Here, we expressed the res- stilebene). Resveratrol is a byproduct of plant response to veratrol O-methyltransferase (ROMT) gene (VvROMT)from outside stresses such as UV irradiation and fungal infection grape (Vitis vinifera)inEscherichia coli and Saccharomyces (Wang et al. 2010). Besides its important role as a phytoalexin cerevisiae and confirmed its specific ability to catalyze the in plant defense responses, resveratrol and its derivatives production of pterostilbene from resveratrol. By co- exhibit human health benefits, including anti-oxidant, anti- expressing an additional two genes from the resveratrol bio- tumor and anti-aging activities (Jang et al. 1997;Campagna synthetic pathway—4-coumarate CoA-ligase (4CL) and stil- and Rivas 2010;Wangetal.2010). One particularly promis- bene synthase (STS)—a large amount of pterostilbene was ing derivative of resveratrol, pterostilbene, is found in grape produced, with a trace amount of pinostilbene detected. To (Schmidlin et al. 2008), indian kino tree (Paul et al. 1999)and understand the molecular basis of the catalytic activity, four blueberries (Rimando et al. 2004). Hundreds of studies have key amino acid residues were identified in a 3D-model of shown that pterostilbene associates with anti-cancer, anti- VvROMT and mutagenized and assayed for augmented cata- diabetic, anti-carcinogenic, anti-inflammatory and anti- lytic activity. Our results demonstrate the potential utility of lipogenic activities, as indicated for resveratrol (Rimando the engineered microorganisms for pterostilbene production et al. 2002; Pari and Satheesh 2006;Remsbergetal.2008; and provide protein engineering targets that will hopefully Lee et al. 2013). However, pterostilbene has several key lead to increased activity of the ROMT enzyme. advantages over resveratrol (Rimando et al. 2002;Pauletal. 2009; Fulda 2010), many of which can be attributed to the facts that pterostilbene contains two methoxy groups and one Keywords Pterostilbene . Resveratrol . Resveratrol hydroxyl group while resveratrol has three hydroxyl groups. O-methyltransferase . Metabolic engineering . Plant The two methoxy groups increase the hydrophobicity of antioxidants pterostilbene, which increases oral absorption and enhances cellular uptake (Remsberg et al. 2008; Kapetanovic et al. Electronic supplementary material The online version of this article 2011). Pterostilbene also has a longer half-life in the blood (doi:10.1007/s13213-014-0922-z) contains supplementary material, than resveratrol and has shown higher bioactivity in assays which is available to authorized users. designed to test effectiveness in heart health and oxidative Y. Wa ng (*) : M. W. Bhuiya : R. Zhou : O. Yu stress ( Rimando et al. 2005;Josephetal.2008). Wilson et al. Conagen, 1005 North Warson Rd, Saint Louis, MO 63132, USA (2008) demonstrated that methoxylation products of resvera- e-mail: [email protected] trol, including desoxyrhapontigenin, 3-hydroxy-5, 4′- dimethoxystilbene, pterostilbene, resveratrol-trimethylether O. Yu Wuxi NewWay Biotech, 401 Xingyuan North Road, Wuxi, Jiangsu but not pinostilbene, generally had increased bioactivity in Province, China 214043 the whole organism. Under their experimental conditions, 818 Ann Microbiol (2015) 65:817–826 both hydroxylated and monomethoxylated stilbene, or pinostilbene, had negligible or modestly beneficial effects on adult Caenorhabditis elegans survival. Rimando et al. (2005) have shown that only pterostilbene is an agonist of peroxi- some proliferator-activated receptor α (PPARα), but not res- veratrol, piceatannol, or resveratrol trimethylether. Results from their in vitro study suggest that pterostilbene may be a more effective hypolipidemic agent than other forms of reservatrol. Because both resveratrol and its methoxylated derivatives have various health benefits, metabolic engineering to in- crease their production has become an active field of research. Resveratrol production has been achieved in Saccharomyces cerevisiae and Escherichia coli (Becker et al. 2003; Beekwilder et al. 2006; Watts et al. 2006;Chemlerand Koffas 2008; Trantas et al. 2009; Sydor et al. 2010; Wang et al. 2010, 2011; Lim et al. 2011; Jeandet et al. 2012; Wang and Yu 2012;Wuetal. 2013). However, reports on the production of methylated derivatives of resveratrol in microorganism and plants are few (Rimando et al. 2012; Jeong et al. 2014). Jeong et al. (2014)attempted Fig. 1 Schematic diagram of the pterostilbene biosynthetic pathway. to produce pterostilbene from resveratrol in E. coli by 4CL 4-Coumarate:coenzyme A ligase, STS stilbene synthase, ROMT the expression of two putative genes encoding resveratrol resveratrol O-methyltransferase O-methyltransferase (ROMT) from frost grape (Vitis riparia, VrROMT) and sorghum (Sorghum bicolor, Materials and methods SbOMT). Expression of SbOMT led to the production of pinostilbene from resveratrol, with only a trace Strains, plasmids and chemicals amount of pterostilbene. Expression of VrROMT resulted in next to no methylated resveratrol (Jeong et al. 2014). Escherichia coli DH5α was used for plasmid cloning. E. coli Rimando et al. (2012) co-expressed O-methyltransferase BW27784 (Khlebnikov et al. 2001), kindly provided by Jay of Sorghum bicolor (SbOMT3) and stilbene synthase of D. Keasling from the University of California, contains an peanut (AhSTS) in tobacco and Arabidopsis, resulting in integrated arabinose-H transporter gene, important for high- the accumulation of pterostilbene in both species. level resveratrol accumulation (Watts et al. 2006;Wangetal. We previously showed that yeast cells expressing a 2011). Plasmids pAC-4CL1 (Watts et al. 2006) and pTrc- 4-coumarate CoA-ligase (4CL) and stilbene synthase AtIPI (Cunningham et al. 2000) were kindly provided by (STS) fusion and gene produced 15-fold more resvera- Claudia Schmidt-Dannert from the University of Minnesota trol when fed 4-coumaric acid, the substrate of 4CL and Francis X. Cunningham Jr from the University of Maryland, (Zhang et al. 2006). Resveratrol can be converted into respectively. S. cerevisiae strain WAT11 (Urban et al. 1997) pterostilbene and its methylated derivatives by express- was used as a host for gene expression. p-Coumaric acid, ing ROMT (Fig. 1). Many ROMTs have been identified resveratrol and pterostilbene standards were purchased from and characterized from different species (Baerson et al. Sigma (St. Louis, MO). Acetonitrile (HPLC grade) was 2008; Schmidlin et al. 2008; Jeong et al. 2014). All the purchased from EMD Chemicals (Gibbstown, NJ). Formic genes encoding enzymes responsible for pterostilbene acid (HPLC grade) was purchased from Fisher Scientific biosynthesis have been cloned, making metabolic engi- (Pittsburgh, PA). neering of this compound relatively straightforward. In the present study, we report the production of DNA manipulation pterostilbene by co-expressing 4CL, STS and ROMT in E. coli and S. cerevisiae. We showed that ROMT from All DNA manipulations were performed according to stan- grapevine (Vitis vinifera) was capable of yielding dard procedures. Restriction enzymes and T4 DNA Ligase pterostilbene as the major product. We identified four were purchased from New England Biolabs (Ipswich, MA). key amino acid residues that contribute to substrate bind- PCR amplification and cloning reactions were performed ing and affinity based on a three-dimensional (3D)-model using Phusion® High-Fidelity DNA Polymerase (New En- structure of ROMT and site-directed mutagenesis. gland Biolabs). Taq DNA polymerase was used to add an A Ann Microbiol (2015) 65:817–826 819 to the blunt-end DNA fragments following the manufacturer’s Table 1 Primer pairs used in this study. F Forward, R reverse protocol (New England Biolabs). Primer Sequence (5′-3′) RMOT-F ATG GAT TTG GCA AAC GGT GTG ATA TC RNA extraction and cDNA synthesis RMOT-R TCA AGG ATA AAC CTC AAT GAG GGA CC SumoROMTF CGC GAA CAG ATT GGA GGT GAT TTG GCA ROMT (accession No: FM178870) was cloned from grape AAC GGT GTG ATA TCA GC (V.vinifera). Grape total RNAwas extracted with a Trizol Plus SumoROMTR GTG GCG GCC GCT CTATTATCA AGG ATA AAC RNA Purification Kit (Life Technologies, Carlsbad, CA). The CTC AAT GAG GGA CC synthesis of cDNA was carried out with M-MuLV reverse 4CL-F ATG GCG CCA CAA GAA CAA GCA GTT TC transcriptase (New England Biolabs) following the manufac- STS-R TTA ATT TGT AAC CATAGG AAT GCT ATG turer’s manual. The genes were amplified from the synthe- 4CL-XbaIF GCT CTA GAA GGA GGA TTA CAA AAT GGC sized cDNA with New England Biolabs’ Phusion PCR Kit GCC ACA AGA ACA AGC AGT TTC TC with the primers listed in Table 1. STS-NotIR TTG CGG CCG CTT AAT