Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed andintroduced into Saccharomyces cerevisiae. The recombinant yeaststrain was fed with phenylpropanoid acids and produced the flavanonesnaringenin and pinocembrin 62 and 22 times more efficientlycompared to previously reported recombinant prokaryotic strains.Microbial biosynthesis of the flavanone eriodictyol was alsoachieved.

INTRODUCTION

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Introduction

Flavonoids are a diverse family of plant polyphenols that demonstrategreat potential in the treatment of various human pathologicalconditions (1, 10). The health-promoting effects of flavonoidshave stimulated significant research toward the elucidationof their biosynthetic networks, as well as the development ofproduction platforms using well-characterized hosts (8, 19).Within this context, Hwang et al. recently described recombinantEscherichia coli strains that lead to the synthesis of flavanones,the common precursors of the vast majority of flavonoids (7).

In plants, flavanone biosynthesis begins with the hydroxylationof cinnamic acid to p-coumaric acid by a membrane-bound P450monooxygenase, cinnamate 4-hydroxylase (C4H) (Fig. 1). p-Coumaricacid is then activated to p-coumaroyl-coenzyme A (CoA) by 4-coumaroyl:CoAligase. In the next step, three molecules of malonyl-CoA aresequentially added to one molecule of p-coumaroyl-CoA, yieldingtetrahydroxychalcone, by the action of a polyketide synthase,chalcone synthase. Finally, chalcone isomerase converts theC₁₅ compound tetrahydroxychalcone into (2S)-flavanones (Fig.1) (5).

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FIG. 1. Flavanone biosynthesis in plants. Abbreviations: C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase.

We present here the construction of a gene cluster that containsfour plant-derived genes of the early flavonoid biosyntheticpathway that allows the conversion of phenylpropanoid acidsinto flavanones in Saccharomyces cerevisiae. This resulted ina substantial increase in the amount of flavanones producedcompared to recombinant E. coli production and opens the possibilityof producing several other flavonoid molecules whose biosynthesisrequires the action of plant P450 monooxygenases.

Cloning of flavanone pathway in yeast.

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Cloning of flavanone pathway...

Vector YEplac181 was utilized for cloning the flavanone biosyntheticgene cluster in S. cerevisiae. This cluster included four structuralgenes of heterologous plant origins: C4H cDNA from Arabidopsisthaliana (2), isolated from expressed sequence tag clone RAFL06-11-J16(RIKEN BioResource Center) (14, 15); Pc4cL-2 cDNA from Petroselinumcrispum (GenBank accession number AF233638) isolated from parsleyyoung leaves by reverse transcription-PCR (RT-PCR) (9); CHI-Aand chs cDNAs from Petunia x hybrida (GenBank accession numbersX14589 and X13225, respectively) isolated from petunia corollaalso by RT-PCR (16, 17). We chose parsley 4-coumaroyl:CoA ligase-2because this enzyme has been documented to accept caffeic acid,ferulic acid, and cinnamic acid with 28%, 66%, and 21% efficiency,respectively, in addition to its natural substrate, p-coumaricacid (9). We chose CHI-A because its previous cloning and expressionin tomato plants led to a significant increase in the accumulationof flavanones (11).

Cloning of the four-gene cluster in yeast was accomplished byfollowing a cloning strategy that has previously been utilizedsuccessfully for E. coli (19). In the first step, each geneis cloned separately under the control of a species-specificpromoter (in the present study, the strong S. cerevisiae GAL1promoter). In the second step, each gene is amplified togetherwith the promoter and is cloned using restriction digestionsinto the plasmid of choice (in the present study, S. cerevisiaeplasmid YEplac181) (Fig. 2 and Table 1).

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FIG. 2. Schematic representation of the cloning strategy used for assembling plasmid Ycc4c181, carrying the four flavanone biosynthetic genes. Abbreviations for restriction enzymes: H, HindIII; P, PstI; X, XbaI; K, KpnI; E, EcoRI. The PCR and RT-PCR primer sequences used are presented in Table 1.

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TABLE 1. Primers used in this study and referred to in Fig. 1

The successful application of this strategy in E. coli cannotbe considered a precedent for success in yeast. This is becauseS. cerevisiae tends to be more recombination prone, and as aresult, tandemly repeated copies of DNA (such as the 451-bpGAL1 promoter) have a higher probability of being looped outby excisional recombination (12). Therefore, the stability ofthe insertions must be carefully determined (3, 18). For thatpurpose, we grew the recombinant yeast strain for a maximumof 65 h at 30°C in leucine minimal selection medium (SC-Leuminimal medium) (4). Culture samples were taken every 24 h,and after plasmid isolation, the presence of each of the fourgenes together with the GAL1 promoter was tested by restrictiondigestions and PCRs. No recombination events that would leadto the loss of a gene(s) or promoters were observed during the65-h time frame.

Exploring flavanone biosynthesis in S. cerevisiae.

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Exploring flavanone biosynthesis...

We tested the ability of the recombinant yeast strain to synthesizeflavanone compounds by feeding it with phenylpropanoid acids,such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulicacid. In order to reduce cell growth inhibition, phenylpropanoidprecursors were added every 13 h to the cultures in five equaldoses, reaching a final concentration of 1 mM. In all cases,cultures were terminated after 65 h of incubation and flavonoidsubstances were extracted from the culture broth with an equalvolume of ethyl acetate. They were further analyzed by reverse-phasehigh-performance liquid chromatography (HPLC) using an acetonitrile-watergradient, at a flow rate of 1.0 ml/min. The HPLC conditionswere as follows: 10 to 40% for 10 min, 40 to 60% for 5 min,and 60 to 10% for 2 min. The retention times under these HPLCconditions for the standard authentic samples are presentedin Table 2.

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TABLE 2. Retention times of standard (authentic) compounds used in the present study

When cinnamic acid was used as a precursor metabolite and galactoseas the sole carbon source and inducer, a large amount (16.3mg/liter) of the corresponding unhydroxylated flavanone pinocembrinaccumulated in the medium. This is a 22-fold increase comparedto the amount of pinocembrin produced by the most efficientE. coli recombinant strain (7). However, only a relatively lowconcentration of naringenin (0.2 mg/liter) was detected, demonstratingthat although C4H was functionally expressed in yeast, it isstill a rate-limiting step enzyme in the four-enzyme hybridpathway. It is possible that increasing the activity of CPR1,the yeast P450 reductase that is required for P450 monooxygenasefunction through episomal overexpression, could lead to increasedC4H activity (6). When p-coumaric acid was used as a precursor,a large amount of naringenin (28.3 mg/liter) accumulated inthe culture, which is 62 times higher compared to the amountof naringenin produced by the most efficient recombinant E.coli strain. Similarly, when caffeic acid was used as a precursor,natural (2S)-eriodictyol was produced in significant amounts(6.5 mg/liter). This is the first time eriodictyol biosynthesishas been achieved through microbial fermentation. Finally, ferulicacid, which carries a methoxy group on the aromatic ring, failedto be metabolized by the recombinant yeast strain. This resultis in agreement with recent data obtained by Schroeder et al.demonstrating that O-methylations on the B ring of flavonoidsubstrates result in complete loss of enzymatic activity. Itis therefore possible that B-ring methylations occur later inthe complex flavonoid pathway to the end products (i.e., afterflavanones have been synthesized) (13). The biotransformationresults are summarized in Table 3.

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TABLE 3. Production of natural flavanones by recombinant yeast S. cerevisiae INVSCI harboring plasmid Ycc4c181^a

In conclusion, we describe the biosynthesis of milligram quantitiesof flavanone substances from an S. cerevisiae recombinant strainthat carries a plant-derived gene cluster. Since many flavonoidsubstances are formed through the action of P450 monooxygenasesthat cannot be readily expressed in E. coli, our success inproducing a variety of flavanone skeletons from recombinantyeast will allow us to proceed in the future with the biosynthesisof several other high-value flavonoid molecules, such as genisteinand quercetin.

ACKNOWLEDGMENTS

This work was supported by a research grant from the NationalScience Foundation (BES-0331404) to M. A. G. Koffas.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, 904 Furnas Hall, Buffalo, NY 14260. Phone: (716) 645-2911, ext. 2221. Fax: (716) 645-3822. E-mail: mkoffas{at}buffalo.edu.

REFERENCES

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