- Open Access
De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae
© Koopman et al.; licensee BioMed Central Ltd. 2012
- Received: 4 September 2012
- Accepted: 15 November 2012
- Published: 8 December 2012
Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules.
Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 μM). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 μM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 μM were reached.
The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.
- Saccharomyces cerevisiae
- de novo
- Metabolic engineering
In recent years, plant flavonoids, which comprise a family of over 9000 compounds, have attracted a tremendous increase in research interest[1–3]. This interest is mainly attributed to highly promising human health applications of specific flavonoids[4–8]. The biological activities of flavonoid compounds have been investigated in relation to a multitude of human pathological conditions, including cancer, diabetes, obesity and Parkinson’s disease[6, 9–14]. The identified mechanisms of action include scavenging of oxygen radicals, anti-inflammatory, antiviral and antitumor activities[15, 16].
Both for health related research and commercial nutritional applications, availability of sufficient amounts of defined flavonoid preparations is important. To date, flavonoid production mostly relies on isolation from plants. However, investigation and subsequential industrialization from plants is hampered by their low production efficiency. In addition to the low growth rate of some of the producing plants, extraction and separation of flavonoids with highly related structures complicate plant-based production, thereby impeding progress in the exploration of the biological activities of flavonoids[13, 14]. Although flavonoids can be produced chemically, efficient production of flavonoids by organic synthesis is severely hindered by the complexity of the molecules, as well as by the necessity of utilizing toxic chemicals and extreme reaction conditions[13, 14].
Expression of enzyme combinations, originating from a variety of host organisms has yielded microbial strains capable of producing the key flavonoid precursor naringenin (Figure1). Several reports describe successful biotransformation processes in which a phenylpropanoid precursor, such as coumaric acid, is converted into naringenin by metabolically engineered E. coli or S. cerevisiae[1, 14, 17, 19, 20, 27, 28]. The highest naringenin titers obtained through biotransformation were achieved in E. coli, with naringenin titers reaching 1.74 mM (474 mg·l-1) from 2.6 mM coumaric acid supplied to the medium. The reliance on ‘expensive’ phenylpropanoid precursors might represent a major hurdle for economically feasible flavonoid production. Hitherto, only one study reports de novo naringenin production from glucose. Using an engineered E. coli strain, naringenin titers of up to 106.5 μM (29 mg·l-1) were obtained.
S. cerevisiae has several attractive characteristics as a metabolic engineering platform for flavonoid production. In addition to its excellent accessibility to molecular and synthetic biology techniques[29, 30], its eukaryotic nature may facilitate functional expression of plant-derived flavonoid-biosynthetic genes. For example, S. cerevisiae can functionally express cytochrome P450-containing enzymes and its subcellular compartmentation is comparable to that of plant cells. Finally, its GRAS (generally recognized as safe) status facilitates subsequent application for the production of pharma- and nutraceuticals.
The goal of the present study was to define a metabolic engineering strategy for de novo production of naringenin by S. cerevisiae, using glucose as sole carbon source. For optimal synergistic activity, the flavonoid biosynthetic genes PAL1, C4H, CPR1, 4CL3, CHS3 and CHI1 used in this study were derived from a single plant species, A. thaliana and selected for in planta co-expression profiles. After expression of the plant pathway genes, optimization of naringenin production was explored by engineering of precursor supply to the naringenin pathway and by reducing the formation of byproducts derived from yeast metabolism.
Selection of naringenin biosynthetic genes from A. thaliana
A. thaliana genes included in the expression correlation studies
A. thaliana locus
phenylalanine ammonia lyase
phenylalanine ammonia lyase
phenylalanine ammonia lyase
phenylalanine ammonia lyase
4-coumaric acid-CoA ligase
4-coumaric acid-CoA ligase
4-coumaric acid-CoA ligase
4-coumaric acid-CoA ligase
p-coumaric acid 3-hydroxylase
Since these results are in good agreement with a previous study on expression correlation in plants, the following genes for the pathway towards naringenin were selected for heterologous expression in S. cerevisiae: the flavonoid biosynthetic genes 4CL3 (at1g65060), CHS1 (at4g00040) and CHI1 (at5g05270), as well as, C4H (at2g30490) and PAL1 (at2g37040) from the lignin biosynthetic genes. PAL1 and CHI1 were preferred over PAL2 and CHI3, as their expression profiles showed stronger correlations with C4H and 4CL3 respectively (Figure2).
Construction and evaluation of a naringenin producing strain
Alleviation of tyrosine feedback inhibition of yeast 3-deoxy-D-arabinose-heptulosonate-7-phosphate (DAHP) synthase
Since phenylalanine is a key intermediate in naringenin biosynthesis (Figure1), further metabolic engineering was directed at improving its intracellular availability. Aromatic amino acid biosynthesis in S. cerevisiae is subject to strong feedback inhibition by phenylalanine and tyrosine. Introduction of a tyrosine insensitive ARO4 allele (ARO4 G226S )[38, 39] in conjunction with the deletion of the other allele of the 3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase (ARO3) has previously been shown to cause a 4-fold increase in flux through the aromatic amino acid pathway. To test the potential impact of this genetic intervention on naringenin synthesis, S. cerevisiae strain IMX183 (ARO4 G226S aro3Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑) was constructed. The deregulation of DAHP synthase in this strain lead to a 2-fold increase of the naringenin titer in shake flask cultures (10.4 μM) (Figure3). However, this increase was accompanied by a strongly enhanced accumulation of phenylethanol, whose extracellular concentration was increased 100-fold to a level ca. 20 fold higher than the improved naringenin concentration (Figure3B). Therefore, to improve further naringenin production, we adopted a strategy to reduce the diversion of aromatic amino acid biosynthesis into this byproduct.
Elimination of competing phenylpyruvate decarboxylase activity
Alleviate the bottleneck downstream of coumaric acid
Expression of a tyrosine ammonia lyase leads to increased naringenin synthesis
The engineered naringenin production pathway directs carbon via the aromatic amino acid phenylalanine to coumaric acid through phenylalanine ammonia lyase, trans-cinnamate 4-monooxygenase and subsequently to naringenin through the 4-coumaric acid-CoA ligase and chalcone synthase reactions (Figure1). Deregulation of aromatic amino acid synthesis in S. cerevisiae by eliminating feed-back inhibition of DAHP synthase not only increases intracellular phenylalanine levels, but also the intracellular concentration of tyrosine. Since S. cerevisiae cannot interconvert tyrosine and phenylalanine, this increased intracellular availability of tyrosine could not be exploited for naringenin production. In other organisms, deamination of tyrosine by tyrosine ammonia lyase does provide an alternative route to coumaric acid. This has previously been shown to increase naringenin production by E coli when fed with either glucose or tyrosine[18, 20, 47]. To investigate the added benefit of the direct conversion of tyrosine into coumaric acid in S. cerevisiae IMX197, a cotal1 gene from Rhodobacter capsulatus was expressed. This R. capsulatus gene was chosen based on its known high catalytic efficiency for tyrosine. Integration of this yeast codon-optimized cotal1 gene resulted in increased naringenin production in the resulting strain IMX198 (aro3 Δ ARO4 G226S aro10 Δ, pdc5 Δ, pdc6 Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑) (Figure5B), to a titer of 200 μM in shake-flask cultures. Additionally, the phloretic acid concentrations in this strain again increased compared to the parental strain IMX197 (623,9 ± 11,4 μM and 396,1 ± 6,9 respectively), while the coumaric acid concentrations in cultures of the two strains remained the same.
Naringenin production in controlled aerobic batch cultures
In summary, the naringenin production of IMX198 in a well aerated and pH controlled batch bioreactor cultivation required only 38 hours and yielded more than twice the naringenin titer that was achieved in equivalent shake flasks cultivations (200 vs. 415 μM).
The present study, for the first time, demonstrates de novo production of the key flavonoid intermediate naringenin, from glucose, by an engineered S. cerevisiae strain. Combined expression of the product pathway, codon optimization, improvement of precursor supply and reduction of byproduct formation led to concentrations of over 400 μM naringenin in aerobic, glucose-grown batch cultures. These concentrations are over 4-fold higher than reported in a previous study on de novo biosynthesis of naringenin by an engineered E. coli strain.
In previous studies on microbial biotransformation of aromatic precursors to naringenin, the core biosynthetic pathway for naringenin synthesis (phenylalanine and/or tyrosine ammonia lyase, trans-cinnamate 4-monooxygenase, P450 cytochrome reductase, 4-coumaric acid-CoA ligase, chalcone synthase) was, in most cases, reconstituted by co-expressing genes from different plants and/or bacteria[17–19, 27, 28]. Only one of those studies used naringenin biosynthetic genes originating from a single donor organism (A. thaliana), which resulted in the first demonstration of de novo naringenin biosynthesis in E. coli. In line with this observation, co-expression of a set of biosynthetic genes from A. thaliana, that were selected based on correlation of in planta expression profiles, was sufficient for low-level de novo production of naringenin in S. cerevisiae (Figure3). The observations that heterologous co-expression of genes originating from single plant species is beneficial for naringenin production, support the hypothesis that maintaining donor specific protein-protein interactions can improve pathway kinetics and specificity. Besides using genes from a single plant species, also the recently developed synthetic protein scaffolds may be used to explore the impact of the physical association of pathway enzymes[53–55]. The potential of this approach is illustrated by the recent demonstration that protein scaffolding in yeast led to a five-fold increase of the titer of the stilbenoid resveratrol. The optimized supply of precursors and decreased production of aromatic byproducts make the yeast strains developed in this study an interesting models to explore the impact of pathway topology on flavonoid production in a heterologous host.
The improvement of naringenin production in this study followed a typical metabolic engineering approach consisting of repeated cycles of design, construction, and analysis. After introduction of the pathway-encoding genes and observation of low levels of naringenin, alleviation of feedback inhibition of aromatic amino acid biosynthesis by phenylalanine and tyrosine further improved naringenin production. As seen previously. removal of feedback inhibition also resulted in an overflow of phenylalanine into the Ehrlich pathway and, consequently, production of phenylethanol as a major byproduct. Restricting the activity of the Ehrlich pathway by deletion of three decarboxylase-encoding genes strongly reduced formation of phenylethanol and had a strong positive impact on naringenin production (Figure3). Precursor supply was further improved by expression of a tyrosine ammonia lyase, which introduced a second route towards coumarate besides phenylalanine ammonia lyase. The subsequent introduction of two additional copies of the A. thaliana CHS3 gene further increased the flux from coumaric acid to naringenin (Figure5B). This observation may be attributed to the catalytic properties of CHS3. This enzyme combines 4 substrates (coumaroyl CoA and three copies of malonyl CoA) to synthesise naringenin, and it is well known to exhibit a very low catalytic activity. Although originating from a different plant, the catalytic constant kcat of the chalcone synthase CHS3 from alfalfa is 2 min-1,, which is roughly 25-fold lower than the kcat of the protein preceding the chalcone synthase in the metabolic pathway, the coumaroyl-CoA ligase (4CL3) from A.thaliana which has a kcat of 50 min-1.
In the naringenin-producing S. cerevisiae strains, the by-product phloretic acid was produced in concentrations similar to those achieved for naringenin. Phloretic acid production was only produced in cultures that both accumulated high concentrations of coumaric acid and expressed A. thaliana 4CL3. Phloretic acid has previously been detected in engineered S. cerevisiae, but the mechanism of its formation remains unknown. Even though it was observed that increasing the flux to naringenin by increasing the copy number of the A. thaliana CHS3 gene, further analysis of the mechanism of phloretic acid production by these engineered S. cerevisiae, e.g. via systematic gene deletion studies, provides a good opportunity to further improve naringenin production. Also improvement of the provision of malonyl-CoA, a key precursor for both naringenin production as well as for lipid production, might further improve naringenin production. It has previously been shown that overexpression of acetyl-CoA carboxylase, which carboxylates acetyl-CoA to malonyl-CoA, leads to increased production of fatty acids in the yeast Hansenula polymorpha and of biodiesel or 6-methylsalicylic acid production in S. cerevisiae[61, 62]. Moreover, malonyl-CoA synthase in E. coli combined with malonate supplementation, improved biotransformation of coumaric acid into flavonoids. Increasing the availability of malonyl-CoA therefore provides an interesting target for further research.
Demonstration of de novo production of naringenin in S. cerevisiae represents an important step towards commercial production of plant-derived flavonoids from glucose. Even though commercial production of naringenin for direct consumer applications will require substantial further improvement of product yields and titers. Furthermore, this engineered naringenin-producing yeast strain provides an attractive platform for expression of plant-derived pathways that convert naringenin into other flavonoids, either by reconstruction of individual plant pathways or by combinatorial approaches based on the powerful yeast-based methods for in vivo recombination[29, 64]. Availability of a robust yeast platform for such studies paves the way for identification, characterization and eventual industrialization of many plant-derived flavonoids with pharma- and nutraceutical properties.
Here we provide the first report on the production of the flavonoid naringenin from glucose as sole carbon source using the yeast S. cerevisiae, and the optimization of the strain to reach a naringenin titer of over 400 μM. Several obstacles were overcome when expressing a foreign pathway in S. cerevisiae, which include the choice of enzymes, the expression systems the optimization of flux and the inhibition of competing pathways. This study successfully describes the reduction of the phenyl ethanol side product formation. All were done to increase the final titer of flavonoid production.
This strain may be used as an ideal platform for production of a wide variety of flavonoids originating from naringenin, thereby facilitating the production, characterization and possible implementation of such compounds as pharmaceutical and neutraceutical.
Strains and maintenance
Strains and plasmids used in this study
MATalpha ura3 his3 leu2 trp1 MAL2-8cSUC2
MATa aro3 Δ ARO4 G226S
MATalpha ura3 his3 leu2 trp1 MAL2-8cSUC2, pUDE172, PYK2::pUDI065
MATalpha ura3 his3 leu2 trp1 MAL2-8cSUC2 Δaro3(46,1065)::loxP ARO4 G226S
IMK328 pdc6Δ(-6,-2)::loxP pdc5Δ(-6,-2)::loxP
IMK328 pUDE172, PYK2::pUDI065
IMK389 pUDE172, PYK2::pUDI065
IMK393 pUDE172, PYK2::pUDI065
IMK393 pUDE172, PYK2::pUDI065, TRP1::pUDI069
IMK393 pUDE172, PYK2::pUDI065, pUDE188
IMK393 pUDE172, PYK2::pUDI065, TRP1::pUDI069, pUDE188
PCR template for loxp-KanMX4-loxp cassette
Centromeric plasmid, URA3, PGAL1-Cre- TCYC1
Centromeric plasmid, URA3, PTDH3-ccdb-TCYC1, Addgene plasmid 14148
Integration plasmid, URA3, PTDH3-ccdb-TCYC1, Addgene plasmid 14140
Integration plasmid, LEU2, PTDH3-ccdb-TCYC1, Addgene plasmid 14138
Integration plasmid, TRP1, PTDH3-ccdb-TCYC1, Addgene plasmid 14136
2 μm ori, HIS3, PTDH3-ccdb-TCYC1, Addgene plasmid 14150
2 μm ori, URA3, PTEF –TTEF
2 μm ori, URA3 PTPI –TADH
Cloning vector, Ka, atCHS3 cDNA#
Cloning vector, Ka, atCHI1 cDNA
Cloning vector, Bla, at4CL3 cDNA
pMA cloning vector, Bla, A-PTDH3-atPAL1-TCYC1-B*
pMA cloning vector, Bla, B-PPGI-coCPR1-TPGI-C
pMA cloning vector, Bla, C-PTPI-coC4H-TADH-D
pMA cloning vector, Bla, E-PPGK-coCHS3-TPGK-F
2 μm ori, HIS3, PTDH3-coCHS3-TCYC1
2 μm ori, URA3, PTEF-coCHS3-TTEF
2 μm ori, HIS3, PTDH3-coCHS3-TCYC1, PTEF-coCHS3-TTEF
2 μm ori, URA3, PTEF-at4CL3-TTEF
Centromeric plasmid, URA3, PTDH3-atPAL1-TCYC1, PTPI-coC4H-TADH, PPGI-coCPR1-TPGI
Integration plasmid, LEU2, PTDH3-atCHI1-TCYC1
Integration plasmid, LEU2, PTDH3-atCHI1-TCYC1, PTEF-at4CL3-TTEF
Integration plasmid, LEU2, PTDH3-atCHI1-TCYC1, PTPI-atCHS3-TADH, PTEF-at4CL3-TTEF
Integration plasmid, LEU2, PTDH3-atCHI1-TCYC1, PTPI-atCHS3-TADH, PTEF-at4CL3-TTEF, PYK2(1-710)
Integration plasmid, TRP1, PTDH3-coTAL1-TCYC1
Introduction of chromosomal gene alteration
Oligonucleotide primers used in this study
Primers for knockouts
ARO3 KO for
ARO3 KO rev
PDC5 KO for
PDC5 KO rev
PDC6 KO for
PDC6 KO rev
ARO10 KO for
ARO10 KO rev
Primers for cloning
Primers for verification
The introduction of a feed-back-insensitive DAHP synthase activity in S.cerevisiae was accomplished in two steps. Firstly, the ARO3 allele was deleted in strain CEN.PK 2.1C using primers ARO3 KO for and ARO3 KO rev as described above. In this resulting strain, the chromosomal ARO4 allele was replaced by the feed-back-insensitive ARO4 G226S allele[38, 39]. Genomic DNA of strain CEN.PK717.5A was prepared using the genomic DNA isolation kit (Zymo Research, Orange, CA), according to the manufacturer’s recommendations. The ARO4 G226S allele was then amplified using primers Aro4 for/Aro4 rev, and transformed. Selection was performed by screening for tyrosine tolerance by plating on selective agar plates containing synthetic medium, 2% glucose and 1 g·l-1 tyrosine. The resulting strain was named IMK328 (Table2).
Plasmid pUDE172 was obtained by transformation-associated recombination methods as described previously[29, 30, 77]. The backbone fragment harboring the yeast and bacterial origins of replication and markers was amplified from pAG416GPD-ccdB (Table2) using primers FK86/87. Genes were optimized for S. cerevisiae using the JCat algorithm), based on the A. thaliana gene sequence. The DNA fragments were synthesized (Life Technologies, Bleiswijk, The Netherlands) harboring the yeast codon-optimized genes (codon, promoter, terminator and 2 unique 80 bp flanks that included an Eco RV sites at each end). The codon-optimized plant genes were flanked by different promoter and terminator combinations to prevent plasmid instability due to homologous recombination. Prior to transformation-associated recombination, the synthesized fragments were digested from the cloning vector using Eco RV and gel purified. The pAG416GPD-ccdB backbone fragment and the 3 gene cassettes atPAL1, coC4H and coCPR were mixed at a ratio of 1:2:2:2 (50ng :100ng; 100ng: 100ng) and transformed to CEN.PK2-1C. Colonies were selected on glucose synthetic medium plates in the absence of uracil and checked by multiplex PCR using the primers FK105 – FK112. After plasmid isolation, retransformation to E. coli and subsequent plasmid purification, the structure of the resulting plasmid pUDE172 (Genbank accession number: JX268037) was verified using restriction analysis.
The A. thaliana CHI1 gene was amplified using primers FK7/8 from plasmid pUNI.U21762 (Table2), and then agarose gel purified and digested using Spe I and Sal I. The restricted fragment was ligated into an Spe I and Sal I linearized pAG305-GPD-ccdB plasmid. The A. thaliana 4CL3 gene was amplified using primers FK9/10 from pDZ.4CL3, purified and then ligated into p426TEF (Table2) using Spe I and Sal I. Subsequently, the PTEF-at4CL3-TTEF cassette was amplified using primer FK11/12, purified and ligated into a Nae I digested and FastAP (Thermo Scientific, Etten-Leur, The Netherlands) treated pUDI053, yielding pUDI060. The A. thaliana CHS3 gene was amplified using primer FK13/14, purified and ligated into p426TPI (Table2) using Spe I and Xho I yielding pUDE101. The PTPI-atCHS3-TADH cassette was amplified using primer FK15/16, purified and digested by Sac I. The resulting fragment was ligated into Sac I digested and FastAP treated pUDI060 plasmid, yielding pUDI061. A fragment of the PYK2 gene (1-710bp) was amplified from the CEN.PK2-1C genome using primer FK29/30, and then purified and digested using Pfo I. This fragment was ligated into pUDI061 plasmid that had been digested by Pfo I and treated with alkaline phosphatase (FastAP, Thermo Scientific) yielding pUDI065 (Genbank accession number: JX268039). The resulting plasmid was linearized using Nco I and integrated into the chromosomal PYK2 locus.
The tal1 gene from R. capsulatus[48, 79], was codon optimized for yeast using JCat, and synthesized (GeneScript, Piscataway, NJ). The synthetic cotal1 was amplified using primers FK52/53 and ligated into pAG304-ccdB using Spe I and Xho I resulting in pUDI069 (Genbank accession number: JX268036). Integration of this plasmid into the trp1 locus was preceded by linearization using Eco RV.
A DNA fragment containing the custom-synthesized, yeast codon-optimized CHS3 sequence (Life Technologies, Bleiswijk, The Netherlands) was amplified using primers FK169/170 and ligated into both a pAG325GPD-ccdB and p426TEF (Table2) using Spe I and Xho I yielding respectively, pUDE185 and 186. Subsequently, the PTEF-coCHS3-TTEF cassette was amplified using primers FK 171/172 and restricted using Sac I. This cassette was ligated into a Sac I digested and FastAP (Thermo Scientific) treated pUDE185. Plasmids were screened for opposing gene orientation by restriction analysis, yielding pUDE188 (Genbank accession number: JX268038).
Molecular biology procedures
PCR amplification was performed using Phusion® Hot Start II High Fidelity Polymerase (Thermo Scientific) according to the manufacturer's instructions in a Biometra TGradient Thermocycler (Biometra, Gottingen, Germany). Agarose gel separation was performed using 1% (w/v) agarose (Sigma-Aldrich, Zwijdrecht, The Netherlands) gel in 1×TAE (40 mM Tris-acetate pH 8.0 and 1 mM EDTA). Isolation of agarose trapped fragments was performed using Zymoclean Gel DNA Recovery kit (Zymo Research, Orange, CA). Restriction endonucleases, DNA ligases and FastAP (Thermo Scientific) were used according to the manufacturer's instructions. Transformation and amplification of plasmids were performed in E. coli DH5α electrocompetent cells (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Plasmids were isolated from E. coli with the Sigma GenElute Plasmid Miniprep Kit (Sigma-Aldrich). Alternatively, plasmid purification was performed using a Zymoprep™™ Yeast Plasmid Miniprep (Zymo Research). Sequencing of constructs was performed by Baseclear BV (Baseclear, Leiden, The Netherlands).
Cultivation and media
E. coli was grown at 37°C in Luria Broth medium containing the appropriate antibiotic, 100 μg·l-1 ampicillin or 50 μg·l-1 kanamycin (Sigma-Aldrich, Zwijndrecht, The Netherlands). S. cerevisiae was grown at 30°C in medium containing demineralized water, 20 g·l-1 glucose, 5 g·l-1 (NH4)2SO4, 3 g·l-1 KH2PO4, 0.5 g·l-1 MgSO4.7H2O, vitamins and trace elements. The pH of the medium was set to 6.0 using KOH. Appropriate growth factors to supplement the auxotrophic requirements of the strains were added at 150 mg·l-1 for uracil, 500 mg·l-1 for leucine, 75 mg·l-1 for tryptophan and 125 mg·l-1 for histidine. Unless otherwise stated, cultures were grown in 500 ml shake flasks with 100 ml medium by adding 1 ml frozen stock culture and incubating at 30°C in an Innova incubator shaker (New Brunswick Scientific, Edison, NJ) set at 200 rpm. Controlled aerobic batch cultures were grown at 30°C in 2l bioreactors (Applikon, Schiedam, The Netherlands), using a working volume of 1l. In the bioreactor experiments, the (NH4)2SO4 concentration in the synthetic medium was increased to 10 g·l-1 to avoid nitrogen depletion towards the end of the culture. Emulsion C antifoam at 0.05% (w/v) (Sigma-Aldrich, Zwijndrecht, The Netherlands) was added separately after autoclaving. The pH was maintained at pH 5.0 by automatic addition of either 2 M KOH or 2 M H2SO4, the stirrer speed was fixed at 800 rpm and the aeration rate was set at 500 ml·min-1.
Optical density was measured at 660 nm using a Libra S11 spectrophotometer (Biochrom, Cambridge, UK). Biomass dry weights were determined by filtration of 10 ml culture over dry, preweighed 0.45 μm nitrocellulose filters (Gelman Laboratory, Ann Arbor, USA). After removal of the medium, the filter was washed twice with demineralized water and dried for 20 min using a microwave set at 350 W. Glucose, ethanol, glycerol and acetate were analyzed using an Aminex HPX-87H ion exchange column (BioRad, Veenendaal, The Netherlands) operated at 60°C with 5 mM H2SO4 as mobile phase at a flow rate of 0.6 ml·min-1. For measurement of phenylpropanoic compounds, culture samples were diluted with an equal volume of 100% ethanol. After vigorous mixing, cells were spun down at 13000 rpm in a Sorval Heraeus #3328 rotor for 2 min. The supernatant was analysed using HPLC. For phenylpropanoic compounds, measurements were made using a Waters 2695 separation module and a Waters 996 photodiode array detector. Phloretic acid, coumaric acid, phenyl ethanol, cinnamic acid and naringenin were measured respectively at 275, 309, 214, 277, 289 nm using an Agilent Zorbax SB-C18 Column (4.6 × 5.0, 3.5 micron) operating at 30oC. A gradient of acetonitrile and 20 mM KH2PO4 (pH 2) with 1% acetonitrile was used as eluent, at a flow rate of 1 ml·min-1, increasing from 0 to 10% acetonitrile in 6 min followed by an increase to 40% acetonitrile until 23 min. From 23 min to 27 min, the flow was set to 100% KH2PO4. Naringenin, coumaric acid, cinnamic acid, phloretic acid and phenylethanol standards were obtained from Sigma Aldrich (Sigma-Aldrich, Zwijndrecht, The Netherlands).
For analysis of carbon dioxide production in bioreactor cultures, the off-gas was first cooled in a condenser (2°C) and dried with a Perma Pure Dryer (Permapure, Toms River, NJ). CO2 concentrations in the off-gas were then measured with a NGA 2000 Rosemount gas analyzer (Rosemount Analytical, Orrville,OH).
Identification of phenylpropanoid intermediates and naringenin was performed using liquid chromatography coupled to both photodiode-array detection and accurate mass quadrupole time-of-flight mass spectrometry (LC–PDA-QTOF MS) was performed using a Waters Alliance 2795 HPLC connected to a Waters 2996 PDA detector and subsequently a QTOF Ultima V4.00.00 mass spectrometer (Waters, MS technologies, UK) operating in negative ionization mode, an analytical column (Luna 3 μ C18/2 100A; 2.0 × 150 mm) attached to a C18 pre-column (2.0 × 4 mm; AJO-4286; both Phenomenex, USA). Eluents A (ultrapure water:formic acid (1000:1, v/v) ) and B (acetonitrile : formic acid (1000 : 1, v/v) ) were used at 0.19 ml.min. The gradient started at 5% B and increased linearly to 35% B in 45 min, after which the column was washed and equilibrated for 15 min before the next injection. The injection volume was 5 μl. Leucine enkephalin ([M-H]- = 554.2620) was used as a lock mass for on-line accurate mass correction. Data were recorded using MassLynx 4.0 software (Waters).
Co-expression correlation analysis of naringenin biosynthetic genes in A. thaliana
Expression correlation analysis was performed using the BAR Expression Angler. Each candidate gene listed in Table1 was used as “bait”, and expression was correlated over a set of 392 micro-array experiments generated using the ATH1 Affymetrix Whole Genome GeneChip, from the Nottingham Arabidopsis Stock Centre's microarray database. Correlations between expression levels of genes from Table1 were recorded when the Pearson correlation coefficient was above 0.6.
We thank Marinka Almering, Erik de Hulster, Bart Oud and Victor Guadalupe Medina for their technical assistance. The research group of JTP is part of the Kluyver Centre for Genomics of Industrial Fermentation (http://www.kluyvercentre.nl/pro1/general/home.asp/); the research group of RDH is part of the Centre for BioSYstems Genomics, both of which are supported by The Netherlands Genomics Initiative (http://www.genomics.nl/). FK, JTP and J-MD. were also supported by the “Platform Green Synthetic Biology” programme (http://www.pgsb.nl/) funded by NGI. FK, J-MD, AJAM were also financially supported by NGI through the Horizon breakthrough programme (project 93519011). JB, AH and RDH acknowledge support by the EU 7th Framework ATHENA project (FP7-KBBE-2009-3-245121-ATHENA). This research was also supported by BIOFLAVOUR, COST Action FA0907 (http://www.bioflavour.insa-toulouse.fr).
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