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Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous
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Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous
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Nadine Ledetzky, Ayako Osawa, Kanoko Iki, Hendrik Pollmann, Sören Gassel, Jürgen Breitenbach, Kazutoshi Shindo, Gerhard Sandmann ⇑ J.W. Goethe University Frankfurt, Institute of Molecular Bioscience, Max von Laue Str. 9, D-60438 Frankfurt, Germany Department of Food and Nutrition, Japan Women’s University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
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Article history: Received 18 December 2013 Available online xxxx
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Keywords: Astaxanthin biosynthesis crtYB gene 3-HO-4-keto-70 ,80 -dihydro-b-carotene Ketocarotenoids Xanthophyllomces dendrorhous (=Phaffia rhodozyma)
a b s t r a c t Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4keto-70 ,80 -dihydro-b-carotene additionally by NMR spectroscopy. The others were 4-keto-b-zeacarotene and 4-keto-70 ,80 -dihydro-b-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed. Ó 2014 Elsevier Inc. All rights reserved.
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Introduction
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The basidiomycetous yeast Xanthophyllomces dendrorhous (sexual state, named Phaffia rhodozyma as the asexuel state) is the only known fungus which is capable of synthesizing the commercially high-value carotenoid astaxanthin (3,30 -dihydroxy-b,b-carotene4,40 -dione) [1,2]. It is used in large quantities as a feed additive in aquaculture. Together with b-carotene, astaxanthin is the carotenoid with the highest market value [3]. In the fungus, astaxanthin protects against oxidative stress [4]. This carotenoid is one of the best singlet oxygen quenchers [5] and free radical scavengers [6]. The carotenoid biosynthesis pathway is well-established in X. dendrorhous. After the provision of geranylgeranyl pyrophosphate via the mevalonate pathway, only three enzymes catalyze the entire reaction sequence from phytoene synthesis to astaxanthin. They include the gene product of crtYB, a fusion gene like in other fungi encoding a phytoene synthase together with a lycopene
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⇑ Corresponding author at: J.W. Goethe University Frankfurt, Institute of Molec
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ular Bioscience, Max von Laue Str. 9, D-60438 Frankfurt, Germany. Fax: +49 69 79829600. E-mail address: [email protected] (G. Sandmann).
cyclase [7], a phytoene desaturase CrtI [8] and an astaxanthin synthase Asy which utilizes b-carotene as a substrate [9]. The latter enzyme is a unique P450-type hydroxylase of the 3A monooxygenase sub-family found exclusively in X. dendrorhous. It carries out a multistep conversion of b-carotene to astaxanthin by insertion of a 3-hydroxy as well as a 4-keto group (via two hydroxylation steps) in both b-ionone rings [2]. The enzyme needs a specific P450 reductase CrtR for functionality [10]. Due to the catalytic mechanism of Asy which is different to conventional b-carotene ketolases, the product in X. dendrorhous is in the (3R,30 R)-configuration compared to the 3S,30 S-enantiomer formed in bacteria and fungi [11]. In addition to astaxanthin, 3-HO-4-ketotorulene is another minor end product of the carotenoid pathway in X. dendrorhous. It results from the insertion of one extra double bond into lycopene by CrtI leaving this end of the molecule acyclic whereas the other side is cyclised and processed by Asy exactly resembling one half of the astaxanthin molecule [2]. For genetic pathway engineering in X. dendrorhous, all necessary tools such as genome integration plasmids and transformation protocols are available [12]. This enables the genetic manipulation of the carotenoid biosynthesis pathway aiming at the increase of astaxanthin biosynthesis. It has already been shown by
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over-expression of the crtYB gene that phytoene synthesis is the limiting step in carotenogenesis of X. dendrorhous [13]. In the present investigation, we transformed X. dendrorhous with multiple copies of crtYB and analysed the resulting carotenoid composition which was quantitatively and qualitatively changed. One of the newly formed carotenoids was isolated and identified by NMR as a novel compound not described before.
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Experimental procedures
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Strains, cultivation and transformation
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X. dendrorhous strain CBS6938 (=ATCC96594) was grown in shaking cultures (50 ml in 500 ml baffled Erlenmeyer flasks, 180 rpm, over 8 days) at 20 °C in YM medium. Details including transformation by electroporation were described in Visser et al. [12]. Selection of transformants was with geneticin (G-418 sulfate, 100 lg/ml), hygromycin (60 lg/ml) or nourseothricin (30 lg/ml) on agar plates. Escherichia coli strains DH5a and JM110 used for genetic manipulations, plasmid amplification, and heterologous synthesis of standard carotenoids for HPLC by transformation with individual gene combinations were grown in LB medium containing appropriate antibiotic concentrations, ampicillin (100 lg/ml), chloramphenicol (34 lg/ml), kanamycin (25 lg/ml), and tetracyclin (25 lg/ml), according to Sambrook et al. [14].
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Plasmids for transformation of X. dendrorhous and E. coli and determination of copy number The crtYB containing plasmids for transformation of X. dendrorhous were derived from pPR2TN with geneticin resistance as selection marker [12] including pPR13F (Fig. 1) which resembles pPR2TN but carries one copy of crtYB [7]. In case of pPR2TNH-YB (Fig. 1) with one gene copy of crtYB, the geneticin resistance gene
nptII in pPR13F was replaced by the hph gene from E. coli encoding hygromycin phosphotransferase [15]. The geneticin resistance gene together with the the glyceraldehyde phosphate dehydrogenase promoter was cut out with NruI and partial digestion with SacI and the hph gene fused to the glyceraldehyde phosphate dehydrogenase promoter by PCR ligated into the same sites. Plasmid pPR2TN-YB-YB (Fig. 1) carrying two copies of crtYB with geneticin as selction marker was constructed by partial digestion of pPR13F with BamHI and insertion of the whole crtYB gene cassette with the promoter and terminator of glyceraldehyde phosphate dehydrogenase. Another modification of plasmid pPR2TN yielded pPR2TNNasy (Fig. 1) with an astaxanthin synthase gene which mediates the conversion of b-carotene and several other intermediates to astaxanthin and the nourseothricin resistance gene nat from Streptomyces noursei encoding nourseothricin acyl transferase [16]. First, geneticin resistance gene nptII was replaced by the nat gene, then the whole promoter terminator cassette with the asy cDNA from pPR2TN2BPAT [9] was inserted into the BamHI site. The different selection markers allows up to three transformations with combinations of all three plasmids. This yielded X. dendrorhous CBS6938-(YB)1 with one integration event for crtYB, CBS6938(YB)2 with the integration of a double cassette of crtYB or CBS6938-(YB)3 with a combination of both. Additionally, the latter strain was transformed a third time with pPR2TNN-asy resulting in CBS6938-(YB3asy). All plasmids used for E. coli transformation to generate the different reference carotenoids in E. coli are listed in Table 1. They include three plasmids pACCRT-EBIRc pACCAR16DcrtX and pACCAR25DcrtX for the synthesis of neurosporene, b-carotene and zeaxanthin, respectively. They carry chloramphenicol resistance as selection marker and are compatible with the other plasmids. The other plasmids in Table 1 mediate only a single modification reaction as indicated and carry different selection markers. Plasmid pBBR1-MCS2crtZ was constructed by PCR amplification of crtZ using pT7-7crtZ [17] as a template with the primers Z-forw:
Fig. 1. Map of the transformation plasmids for X. dendrorhous. They basically differ in the selection markers geneticin, hygromycin or nourseothricin and carry one or two crtYB gene inserts or the asy gene instead. nptII, geneticin resistance gene; hph, hygromycin resistance gene; nat, nourseothricin resistance gene; Amp, ampicillin resistance; rDNA, ribosomal DNA.
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N. Ledetzky et al. / Archives of Biochemistry and Biophysics xxx (2014) xxx–xxx Table 1 Plasmids used for combinatorial biosynthesis in E. coli to generate reference carotenoids for HPLC. Plasmid
Expressed enzyme(s)
Marker
Carotenoid product or modification reaction
Ref.
pACCRT-EBIRc pACCAR16DcrtX
GGPP-S, P-S, P-3D GGPP-S, P-S, P-4D L-C GGPP-S, P-S, P-4D L-C, bC-H L-C bC-K bC-K bC-H bC-H
chl chl
Neurosporene b-Carotene
[27] [28]
chl
Zeaxanthin
[28]
tet tet amp amp kan
Cyclization 4,40 -Ketolation 4,40 -Ketolation 3,30 -Hydroxylation 3,30 -Hydroxylation
[29] [30] [31] [32] This work
pACCAR25DcrtX pRKcrtY pRKbkt pCRBKT pUCbch pBBR1-MCS2crtZ
Abbreviations: bC-H, b-carotene 3-hydroxylase; bC-K, b-carotene 4-ketolase. GGPP-S, geranylgeranyl pyrophosphate synthase; L-C, lycopene cyclase; P-3D, 3-step phytoene desaturase; P-4D, 4-step phytoene desaturase; P-S, phytoene synthase.
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50 -CTC AAC GGG CCC ATT ATG CTG TGG-30 and Z-rev: 50 -CGT GCT GCA AGC TTC TCA GG-30 . The PCR product was then ligated into the Apa//HindIII sites of pBBR1-MCS1 with kanamycin as selection marker [18].
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Determination of crtYB copy numbers
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Genomic DNA was isolated from CBS6938 and the crtYB transformants after breaking the cells with glass beads in a Retsch homogeniser using classical phenol–chloroform extraction and isopropanol precipitation as described by Lecellier and Silar [19]. The purity was checked on 1% agarose gels and the amounts quantified after ethidium bromide staining by densitometry.qRT-PCR was carried out in the Rotor Gene PCR Cycler 3000 (Corbett Life Science) with the Kapa Sybr Fast Universal Mix (Peqlab, Erlangen, Germany) for the crtYB gene with the primers YB-forw: 50 -AGG GCT GAT CCC TCG ATA CC-30 and YB-rev: 50 -GTC TCG ATA GGC GTC TTC CG-30 . The PCR cycles were: 1 cycle of denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95 °C for 3 s, and extension at 60 °C for 20 s and a final melt curve with range from 50 to 59 °C in 0.5 °C steps. For relative quantification according to Pfaffl [20] DCT values and efficiency of amplification were used. The copy number of crtYB in non-transformed X. dendrorhous was set as one due to the expected haploid nature of our CBS6938 strain [10].
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Isolation of carotenoids and NMR analysis
Results
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The red pigments in the freeze-dried cells of transformant CBS 6938-(YB)3 (20.08 g, from 10 l culture) were extracted 6 times with 30 ml acetone by sonication for 5 min. To the combined supernatants (180 ml), n-hexane (180 ml) and H2O (180 ml) were added and the pigments partitioned between n-hexane–acetone/H2O. The n-hexane–acetone layer (upper layer) was analyzed by HPLC. Eight different carotenoids were separated including two unidentified carotenoids with a characteristic absorbance maximum at 451 nm (=type III). To isolate the unidentified carotenoids, the nhexane–acetone layer was concentrated to dryness (787.3 mg) and applied to silica gel column chromatography (Silica Gel 60, Kanto Chemicals) i.d. 30 130 mm, solvent: n-hexane). The silica gel column was developed with n-hexane (500 ml), n-hexane–acetone 20:1 (500 ml), 8:1 (500 ml), and 4:1 (200 ml), stepwisely. The n-hexane–acetone (20:1) eluate which contains the two type III carotenoids and 3-hydroxy echinenone was collected and concentrated to give a red oil (42.1 mg). The red oil was subjected to silica gel column chromatography (i.d. 20 mm 150 mm, solvent: CH2Cl2) and developed with CH2Cl2. The red fractions were collected and concentrated to dryness (7.3 mg), and subjected to preparative ODS HPLC on a l-Bondapack C18 column (Waters Associates) i.d. 7.8 300 mm with CH3CN–THF–MeOH (58:7:35) at a flow rate
Plasmids and transformants of X. dendrorhous
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Two pPR2TN based plasmids [12] with one or two copies of the phytoene synthase/lycopene cyclase gene crtYB from X. dendrorhous for genome integration were constructed. Details on their structures are given in Fig. 1. Both plasmids also differ by the selection marker which is geneticin in pPR2TN-YB-YB or hygromycin in pPR2TNH-YB. This allows the integration of both plasmids into the same transformant not only individually but also in combination. After transformation, three lines differing in crtYB integration were obtained: CBS6938-(YB)1 by integration of pPR2TNH-YB, CBS6938(YB)2 by integration pPR2TN-YB-YB containing two crtYB copies and finally a double transformant CBS6938-(YB)3 with both plasmids (Table 2). They all differ in their total carotenoid content which is lowest in the wild type and highest in the double transformant ranging from 258 to 798 lg/g dw. Although CBS6938(YB)3 exhibits the highest carotenoid synthesis, the concentration of the end product astaxanthin is not increased compared to CBS6938-(YB)2 and the percentage of astaxanthin within total carotenoids is even decreased. HPLC analysis of wild type and
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of 3 ml/min and PDA detection (250–600 nm). In this HPLC, 3-hydroxy echinenone was eluted at Rt 6.3 min as pure compound (0.9 mg) and the mixtures of type III carotenoids (2.0 mg) were eluted at Rt 7.3–7.7 min. This fraction was finally subjected to preparative ODS HPLC on Develosil C30-UG-5 (Nomura Chemical) i.d. 10 250 mm with solvent CH3CN–CH2Cl2–EtOH (60:40:2) and a flow rate of 3 ml/min resulting in pure compounds with Rt 11.6 min. 0.8 mg (compound 7, Fig. 2B) and Rt 12.4 min. 0.3 mg (compound 5, Fig. 2B). 1H- and 13C NMR spectra were measured at 400 MHz with a Bruker AVANCE 400. HRESI-MS was recorded with a JEOL JMS-T100LP mass spectrometer.
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Analytical high-performance liquid chromatography (HPLC)
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Carotenoid extracts from X. dendrorhous and E. coli were analyzed by analytical HPLC1 either on silica gel Cosmosil 5SL-II 25 cm column with solvent: n-hexane–acetone (85:15) or on a reversed phase 15 cm Nucleosil 100 C18, 5l column with acetonitrile /methanol/2-propanol (85:10:5, by volume) as the mobile phase with a flow rate of 1 ml/min. Spectra were recorded online with a photodiode array detector 440 (Kontron, Straubenhard, Germany). Carotenoid identification and quantification was carried out with authentic standards.
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Abbreviation used: HPLC, high-performance liquid chromatography.
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Fig. 2. HPLC separation of wild type (WT) X. dendrorhous and transformant CBS 6938-(YB)3 carotenoids together with extracts of E. coli engineered for the generation of reference carotenoids. Traces (A) WT, (B) CBS6938-(YB)3, (C) pACCAR16DcrtX + pRKbkt + pUCbch, (D) pACCRT-EBIRc + pCRBKT + pRKcrtY and (E) pACCRT-EBIRc + pCRBKT + pRKcrtY + pBBR1-MCS2crtZ. Peaks marked with ⁄ indicate a similar spectrum resembling that in Fig. 3 type III. For NMR identification of compound 7, see Table 3. Ast, astaxanthin; Adr, adonirubin; HOe, HO-echinenone; Ech, echinenone; Bcar, all-trans b-carotene; cBcar, cis b-carotene; 7,8Db, 7,8dihydro-b-carotene; diHO78Db, 3,30 -dihydroxy-70 ,80 -dihydro-b-carotene; HOK78Db, 3-HO-4-keto-70 ,80 -dihydro-b-carotene; HOBz, 3-HO-b-zeacarotene; KBz, 4-keto-b-zeacarotene; KBz, 3-HO-4-keto-b-zeacarotene.
CBS6938-(YB)3 indicate an increased accumulation of carotenoid intermediates (Fig. 2A and B). In order to channel these intermediates into the end product, a third plasmid pPR2TNN-asy with the astaxanthin synthase gene and nourseothricin as selection marker was constructed (Fig. 1C) and used for additional transformation of CBS6938-(YB)3 yielding CBS6938-(YB3-asy). In this final line the astaxanthin content reached 69% of total carotenoids. In addition, it synthesizes not only 3-fold more total carotenoids than the wild type but also shows a 2.8-fold higher astaxanthin content (Table 2).
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Reversed-phase HPLC analysis of wild type and CBS6938-(YB)3
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HPLC analysis of the pigments demonstrated that in the wild type CBS6938 (Fig. 2A) astaxanthin (1) is the dominating carotenoid together with small amounts of adonirubin (3-HO-4,40 -diketo-bcarotene) (2) and with 3-HO-4-ketotorulene (16) with its typical spectrum with a maximum at 490 nm (Fig. 3). In transformant CBS6938-(YB)3 several additional intermediates accumulated (Fig. 2B). Some of them, peak No. 2 and 4 exhibited the same type I spectrum (Fig. 3) as astaxanthin whereas peaks 6 and 8 exhibited the type II spectrum of echinenone (4-keto-b-carotene). Four of the intermediates 5, 7, 9, 11 labeled with an asterix showed the untypical type III bell-shaped spectrum with a maximum at 451 nm and a shoulder at 447 nm. In order to identify them, reference carotenoids were generated by gene combinations in E. coli. Due to the defined functions of the genes and their previous use [21–23], different ketolated and hydroxylated b-carotene and neurosporene derivates were obtained. Extracts in Fig. 2C from E. coli/ pACCAR16DcrtX + pRKbkt + pUCbch allowed the identification of astaxanthin (1), adonirubin (2), canthaxanthin (4,40 -diketo-b-carotene) (4), 3-HO-echinenone (6), echinenone (8) and b-carotene all-trans and cis isomer (12 and 13). E. coli/pACCRT-EBIRc + pCRBKT + pRKcrtY analyzed in Fig. 2D synthesized neurosporene related ketocarotenoids 4-keto-b-zeacarotene (9) and 4-keto-70 , 80 -dihydro-b-carotene (11) with the type III spectrum (Fig. 3) and also 7,8-dihydro-b-carotene (15). With an additional plasmid carrying a hydroxylase gene, E. coli/pACCRT-EBIRc + pCRBKT + pRKcrtY + pBBR1-MCS2crtZ produced two hydroxy products, 3,30 -diHO70 ,80 -dihydro-b-carotene and 3-HO-b-zeacarotene, and two hydroxy-keto products, 3-HO-4-keto-b-zeacarotene (5) and 3-HO-4keto-70 ,80 -dihydro-b-carotene (7) (Fig. 2E). Some of the carotenoids which accumulated in CBS6938-(YB)3 can be attributed to the pathway from b-carotene to astaxanthin. In addition, 7,8-dihydro-b-carotene was found together with several keto derivatives of this carotene and of b-zeacarotene which were tentatively identified by co-chromatography together with their optical spectra. These carotenoids are all related to cyclization products of neurosporene. In order to confirm their biosynthesis outlined in the dotted box of Fig. 4, we isolated and purified the most abundant carotenoid 7 which co-chromatographed with 3-HO-4-keto-70 ,80 -dihydro-b-carotene for NMR analysis.
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Table 2 Transformants of X. dendrorhous with their integrated plasmids, additional copy numbers and their carotenoid biosynthesis potential. Strains CBS6938 CBS6938-(YB)1 CBS6938-(YB)2 CBS6938-(YB)3 CBS6938-(YB3asy)
Transformation plasmids
GC#
Total carot. (lg/g dw)
Astaxanthin (%)
pR2TNH-YB (Hygromycin) pPR2TN-YB-YB (Geneticin) pPR2TNH-YB (Hygromycin) + pPR2TN-YB-YB (G418) pPR2TNH-YB (Hygromycin) + pPR2TN-YB-YB (Geneticin) + pPR2TNN-asy (Nourseothricin)
1 2 4 5 5
258 ± 26 617 ± 31 745 ± 37 798 ± 30 772 ± 13
191 ± 19 268 ± 22 323 ± 18 301 ± 21 533 ± 66
(74) (44) (43) (38) (69)
Total carotenoid values are means of three independent cultures ± SD, values are significantly different within at least a 1% confidence interval. GC#, genome copy number of crtYB; total Car, total carotenoid content.
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Fig. 3. Visible spectra of carotenoids in X. dendrorhous including putative ketolated and hydroxylated cyclization products of neurosporene. Type I resembles diketo-bcarotene derivatives like astaxanthin, type II mono keto derivatives like echinenone, type III monoketo-7,8-dihydro-b-carotene derivatives. Bcar, b-carotene; 78Db, 7,8dihydro-b-carotene; KHOT, 3-HO-4-ketotorulene.
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Structure elucidation of compound 7
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Purified compound 7 (Fig. 2B) was dissolved in MeOH and analyzed by positive ion HRESI-MS. The (M+Na)+ peak was observed at m/z 591.43972 [calcd. for 591.43652 (C40H56O2Na)], and the molecular formula of 7 was determined to be C40H56O2 [3-hydroxy echinenone (C40H54O2) + 2H]. The 1H NMR spectrum of 7 in CDCl3 was quite similar to that of 3-hydroxy echinenone [24], while new 2 sp3 methylene signals [d 2.12 (both) (H-70 and H-80 )] and up-field shifts of 2 singlet methyl [d 1.62 (H-180 ) and d 1.87 (H-190 )] and 1 sp3 methine [d 5.98 (H-100 )] were observed in 7 [3-hydroxy echinenone: d 1.72 (H-180 ), d 1.98 (H-190 ), and d 6.16 (H-100 )]. Considering these observations, 7 was proposed to be a dihydro-derivative of 3-hydroxy echinenone. The analyses of the 1H–1H DQF COSY, HMQC and HMBC spectra (the key long range couplings in the HMBC spectrum were shown in Table 3A) for 7 proved one end structure in 7 was 3-hydroxy 4-keto b-ionone ring (C-1–C-6) and the other was b-ionone ring (C-10 –C-60 ) like 3-hydroxy echinenone, while the 1H chemical shifts (Table 3B) at H-180 (d1.62) and H-190 (d 1.87) and the 13C chemical shifts at C-60 (from d 137.8 to 136.9) and C-180 (from d 21.7 to 19.5) in b-ionone ring were different from those of 3-hydroxy echinenone [3-hydroxy echinenone: d 137.8 (C-60 ) and d 21.7 (C-180 )]. All data mentioned above strongly suggested that compound 7 is 3-hydroxy-70 ,80 -dihydro-echinenone (see structure in Table 3A). The up-field shifts of H-100 (d 5.98) and H-190 (d 1.87) in the olefine structure, and the 1H–13C long-range couplings from H-190 (d 1.62) to C-80 (d 40.6) and C-100 (d 124.5) in the HMBC spectrum also
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Fig. 4. Carotenoid biosynthesis in X. dendrorhous transformant with over-expressed phytoene synthase and lycopene cyclase (arrows indicating both reactions are boxed) with an additional neurosporene derived extention (in dotted box). The NMR-identified derivative 3-HO-4-keto-70 ,80 -dihydro-b-carotene is circled.
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B. Mass and NMR parameters HRESI-MS (+) m/z 591.43972 (M+Na)+, C40H56NaO2(calcd. for 591.43652). 1H NMR (CDCl3) d: 1.01 (s, H-160 , 170 ), 1.21 (s, H-17), 1.32 (s, H-16), 1.44 (m, H-20 ), 1.57 (m, H-30 ), 1.62 (s, H-180 ), 1.81 (dd, J = 13.6, 14.4 Hz, H-2 a), 1.87 (s, H-190 ), 1.92 (t, J = 7.3 Hz, H-40 ), 1.95 (s, H-18), 1.96 (s, H-200 ), 1.98 (s, H-20), 2.00 (s, H-20), 2.12 (m, H-70 , 80 ), 2.15 (dd, J = 5.4, 14.4 Hz, H-2 b), 4.33 (dd, J = 5.4, 13.6 Hz, H-3), 5.98 (d, J = 10.7 Hz, H-100 ), 6.20 (d, J = 15.0 Hz, H-7), 6.21 (d, J = 11.2 Hz, H-140 ), 6.26 (d, J = 16.0, H-12), 6.30 (d, J = 11.5, H-10), 6.30 (d, J = 11.5,H-14), 6.43 (d, J = 15.0 Hz, H-8), 6.44 (d, J = 14.7 Hz, H-12), 6.53 (dd, J = 10.7, 16.0 Hz, H-110 ), 6.62 (dd, J = 11.5, 14.7 Hz, H-11), 6.63 (dd, J = 11.5, 14.9 Hz, H-15), 6.67 (dd, J = 15.3, 11.2 Hz, H-150 ). 13C NMR (CDCl3) d:12.7 (C-19, 20, 200 ), 13.8 (C-18), 17.0 (C-190 ), 19.5 (C-180 ), 19.7 (C-30 ), 26.1 (C-16), 27.5 (C-70 ), 28.4 (C-160 , 170 ), 30.5 (C-17), 35.0 (C-10 ), 32.6 (C-40 ), 36.8 (C-1), 39.8 (C-20 ), 40.6 (C-80 ), 45.7 (C-2), 69.1 (C-3), 123.2 (C-7), 124.5 (C-100 ), 124.6 (C-11), 125.0 (C-110 ), 126.6 (C-5), 127.2 (C-50 ), 130.2 (C-15), 130.5 (C-150 ), 131.1 (C-140 ), 133.6 (C-14), 134.2 (C-9), 135.0 (C-120 ), 135.1 (C-10), 136.2 (C-13), 136.7 (C-60 ), 136.9 (C-130 ), 140.1 (C-12), 140.9 (C-90 ), 142.2 (C-8), 162.3 (C-6), 200.2 (C-4)
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A. The structure of 3 hydroxy-70 ,80 -dihydro-echinenone with 1H-13C long range couplings
Table 3 NMR characterization of compound 7 as 3-HO-4-keto-70 ,80 -dihydro-b-carotene.
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supported the structure of 1 as 3-hydroxy-70 ,80 -dihydro-echinenone, a new carotenoid not described before. The IUPAC-IUB semi-systematic name of 7 is 3-hydroxy-70 ,80 -dihydro-b,b-carotene-4-one. The amounts of other compounds with a type III spectrum (e.g. compound 5 in Fig. 3) were too small for NMR structural analyses. Nevertheless, their optical spectra indicate that they possess either a 70 ,80 -dihydro-b-carotene or a b-zeacarotene related structure.
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Discussion
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X. dendrorhous is a promising microorganism for the synthesis of astaxanthin provided its yield can be increased by pathway engineering or other means [1,2]. It is well-known that the flux into the specific carotenoid pathway is limited by phytoene synthesis [13]. In our engineering approach, we attempted to saturate the level of phytoene synthase by transformation with multiple copies of its gene crtYB (Table 2). With this approach, it was possible to increase the total number of crtYB copies from one in the wild type via two in CBS6938-(YB), four in CBS6938-(YB)2 to five in CBS6938-(YB)3. Saturation levels indicated by formation of total carotenoids were almost reached by three crtYB integration steps and the resulting transformant CBS6938-(YB)3 with four crtYB copies additional to the one in the genome. Under these conditions, next limitations of the entire carotenoid pathway may be precursor supply and/or conversion of carotenoid intermediates to the end product. The latter was evident by accumulation of substrates and intermediates which can be metabolized to astaxanthin synthase (Fig. 2B). This limitation could be partially alleviated by additional transformation with a copy of the astaxanthin synthase gene asy which increased the percentage of astaxanthin in CBS6938-(YB3asy) almost back to the wild type proportion (Table 2). In addition to b-carotene, echinenone, HO-echineneone, canthaxanthin and adonirubin (Fig. 1) accumulating in CBS6938(YB)3 as intermediate of the pathway towards astaxanthin, three minor carotenoids with an unknown absorbance spectra with a maximum at 451 nm, a small shoulder at 447 nm and with an asymmetrical bell shape (type III of Fig. 3) were found exclusively in this crtYB transformant. These features may be indicative of ketocarotenoids. Two carotenes detected in the fungus Podospora anserina after over-expression of its al-2 gene equivalent to crtYB which from their spectra could be the non-ketolated precursors are b-zeacarotene and 7,8-dihydro-b-carotene [25]. Their main absorbance maxima are at 428 nm and together with one keto group at position 4 both products could exhibit the type III spectrum. Both carotenes are derived from neurosporene either by a one-step cyclization to b-zeacarotene or a second one at the other end of the carbon chain to 7,8-dihydro-b-carotene. Keto derivatives of both carotenes with or without additional 3-HO groups were synthesized as reference compounds in E. coli as reported previously [21,22] and used to identify the compounds with type III spectra in CBS6938-(YB)3. They co-chromatographed with compound 7, 3-HO-4-keto-70 ,80 -dihydro-b-carotene, 9, 4-keto-b-zeacarotene, and 11, 4-keto-70 ,80 -dihydro-b-carotene. Their synthesis is outlined in Fig. 4 (boxed part). For the most abundant compound 7, we were able to proof its structure more definitely by MS and NMR spectroscopy (Table 3). This fully supports the extra branch of carotenogenesis in transformant CBS6938-(YB)3 which diverts at the stage of neurosporene. This is due to a competition for neurosporene (which is not fully desaturated but can already be cyclised at least at one end of the molecule) by phytoene desaturase and lycopene cyclase with a much higher preference for desaturation which may be due to a poorer specificity of lycopene cyclase for the substrate neurosporene. In X.
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dendrorhous wild type, this leads to an exclusive formation of lycopene. However, unfavorable reactions can be forced by high enzyme concentrations as exemplified for phytoene desaturase in purple bacteria [26]. Since gene crtYB encodes a phytoene synthase/lycopene cyclase fusion protein, its over-expression in CBS6938-(YB)3 not only resulted in enhanced carotenogenesis due to higher amounts of phytoene synthase but also caused the formation of neurosporene derivatives by elevated lycopene cyclase levels. This lycopene cyclase should cyclise the end of the neurosporene molecule which resembles the end also found in lycopene in the same way as lycopene is cyclised at both identical ends. In addition, it is also able to cyclise the 7,8-dihydro side of neurosporene yielding 7,8-dihydro-b-carotene. This broad substrate specificity has already been reported before for the lycopene cyclase from the bacterium Pantoea ananas (formerly Erwinia uredovora) and from the plant Capsicum annuum [21]. Downstream in the pathway, it can be assumed that astaxanthin synthase of X. dendrorhous modifies the b-ionone ring with 3-HO and 4-keto groups regardless of the structure of the other end of the molecule carrying either a 7,8-dihydro-b-ionone ring or being acyclic (Fig. 4).
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Acknowledgment
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This work was supported by the LOEWE Project ‘‘Integrative Pilzforschung’’.
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Contents lists available at ScienceDirect
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Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous
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Nadine Ledetzky, Ayako Osawa, Kanoko Iki, Hendrik Pollmann, Sören Gassel, Jürgen Breitenbach, Kazutoshi Shindo, Gerhard Sandmann ⇑ J.W. Goethe University Frankfurt, Institute of Molecular Bioscience, Max von Laue Str. 9, D-60438 Frankfurt, Germany Department of Food and Nutrition, Japan Women’s University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
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a r t i c l e
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Article history: Received 18 December 2013 Available online xxxx
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Keywords: Astaxanthin biosynthesis crtYB gene 3-HO-4-keto-70 ,80 -dihydro-b-carotene Ketocarotenoids Xanthophyllomces dendrorhous (=Phaffia rhodozyma)
a b s t r a c t Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4keto-70 ,80 -dihydro-b-carotene additionally by NMR spectroscopy. The others were 4-keto-b-zeacarotene and 4-keto-70 ,80 -dihydro-b-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed. Ó 2014 Elsevier Inc. All rights reserved.
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Introduction
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The basidiomycetous yeast Xanthophyllomces dendrorhous (sexual state, named Phaffia rhodozyma as the asexuel state) is the only known fungus which is capable of synthesizing the commercially high-value carotenoid astaxanthin (3,30 -dihydroxy-b,b-carotene4,40 -dione) [1,2]. It is used in large quantities as a feed additive in aquaculture. Together with b-carotene, astaxanthin is the carotenoid with the highest market value [3]. In the fungus, astaxanthin protects against oxidative stress [4]. This carotenoid is one of the best singlet oxygen quenchers [5] and free radical scavengers [6]. The carotenoid biosynthesis pathway is well-established in X. dendrorhous. After the provision of geranylgeranyl pyrophosphate via the mevalonate pathway, only three enzymes catalyze the entire reaction sequence from phytoene synthesis to astaxanthin. They include the gene product of crtYB, a fusion gene like in other fungi encoding a phytoene synthase together with a lycopene
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⇑ Corresponding author at: J.W. Goethe University Frankfurt, Institute of Molec
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ular Bioscience, Max von Laue Str. 9, D-60438 Frankfurt, Germany. Fax: +49 69 79829600. E-mail address: [email protected] (G. Sandmann).
cyclase [7], a phytoene desaturase CrtI [8] and an astaxanthin synthase Asy which utilizes b-carotene as a substrate [9]. The latter enzyme is a unique P450-type hydroxylase of the 3A monooxygenase sub-family found exclusively in X. dendrorhous. It carries out a multistep conversion of b-carotene to astaxanthin by insertion of a 3-hydroxy as well as a 4-keto group (via two hydroxylation steps) in both b-ionone rings [2]. The enzyme needs a specific P450 reductase CrtR for functionality [10]. Due to the catalytic mechanism of Asy which is different to conventional b-carotene ketolases, the product in X. dendrorhous is in the (3R,30 R)-configuration compared to the 3S,30 S-enantiomer formed in bacteria and fungi [11]. In addition to astaxanthin, 3-HO-4-ketotorulene is another minor end product of the carotenoid pathway in X. dendrorhous. It results from the insertion of one extra double bond into lycopene by CrtI leaving this end of the molecule acyclic whereas the other side is cyclised and processed by Asy exactly resembling one half of the astaxanthin molecule [2]. For genetic pathway engineering in X. dendrorhous, all necessary tools such as genome integration plasmids and transformation protocols are available [12]. This enables the genetic manipulation of the carotenoid biosynthesis pathway aiming at the increase of astaxanthin biosynthesis. It has already been shown by
0003-9861/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2014.01.014
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over-expression of the crtYB gene that phytoene synthesis is the limiting step in carotenogenesis of X. dendrorhous [13]. In the present investigation, we transformed X. dendrorhous with multiple copies of crtYB and analysed the resulting carotenoid composition which was quantitatively and qualitatively changed. One of the newly formed carotenoids was isolated and identified by NMR as a novel compound not described before.
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Experimental procedures
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Strains, cultivation and transformation
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X. dendrorhous strain CBS6938 (=ATCC96594) was grown in shaking cultures (50 ml in 500 ml baffled Erlenmeyer flasks, 180 rpm, over 8 days) at 20 °C in YM medium. Details including transformation by electroporation were described in Visser et al. [12]. Selection of transformants was with geneticin (G-418 sulfate, 100 lg/ml), hygromycin (60 lg/ml) or nourseothricin (30 lg/ml) on agar plates. Escherichia coli strains DH5a and JM110 used for genetic manipulations, plasmid amplification, and heterologous synthesis of standard carotenoids for HPLC by transformation with individual gene combinations were grown in LB medium containing appropriate antibiotic concentrations, ampicillin (100 lg/ml), chloramphenicol (34 lg/ml), kanamycin (25 lg/ml), and tetracyclin (25 lg/ml), according to Sambrook et al. [14].
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Plasmids for transformation of X. dendrorhous and E. coli and determination of copy number The crtYB containing plasmids for transformation of X. dendrorhous were derived from pPR2TN with geneticin resistance as selection marker [12] including pPR13F (Fig. 1) which resembles pPR2TN but carries one copy of crtYB [7]. In case of pPR2TNH-YB (Fig. 1) with one gene copy of crtYB, the geneticin resistance gene
nptII in pPR13F was replaced by the hph gene from E. coli encoding hygromycin phosphotransferase [15]. The geneticin resistance gene together with the the glyceraldehyde phosphate dehydrogenase promoter was cut out with NruI and partial digestion with SacI and the hph gene fused to the glyceraldehyde phosphate dehydrogenase promoter by PCR ligated into the same sites. Plasmid pPR2TN-YB-YB (Fig. 1) carrying two copies of crtYB with geneticin as selction marker was constructed by partial digestion of pPR13F with BamHI and insertion of the whole crtYB gene cassette with the promoter and terminator of glyceraldehyde phosphate dehydrogenase. Another modification of plasmid pPR2TN yielded pPR2TNNasy (Fig. 1) with an astaxanthin synthase gene which mediates the conversion of b-carotene and several other intermediates to astaxanthin and the nourseothricin resistance gene nat from Streptomyces noursei encoding nourseothricin acyl transferase [16]. First, geneticin resistance gene nptII was replaced by the nat gene, then the whole promoter terminator cassette with the asy cDNA from pPR2TN2BPAT [9] was inserted into the BamHI site. The different selection markers allows up to three transformations with combinations of all three plasmids. This yielded X. dendrorhous CBS6938-(YB)1 with one integration event for crtYB, CBS6938(YB)2 with the integration of a double cassette of crtYB or CBS6938-(YB)3 with a combination of both. Additionally, the latter strain was transformed a third time with pPR2TNN-asy resulting in CBS6938-(YB3asy). All plasmids used for E. coli transformation to generate the different reference carotenoids in E. coli are listed in Table 1. They include three plasmids pACCRT-EBIRc pACCAR16DcrtX and pACCAR25DcrtX for the synthesis of neurosporene, b-carotene and zeaxanthin, respectively. They carry chloramphenicol resistance as selection marker and are compatible with the other plasmids. The other plasmids in Table 1 mediate only a single modification reaction as indicated and carry different selection markers. Plasmid pBBR1-MCS2crtZ was constructed by PCR amplification of crtZ using pT7-7crtZ [17] as a template with the primers Z-forw:
Fig. 1. Map of the transformation plasmids for X. dendrorhous. They basically differ in the selection markers geneticin, hygromycin or nourseothricin and carry one or two crtYB gene inserts or the asy gene instead. nptII, geneticin resistance gene; hph, hygromycin resistance gene; nat, nourseothricin resistance gene; Amp, ampicillin resistance; rDNA, ribosomal DNA.
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N. Ledetzky et al. / Archives of Biochemistry and Biophysics xxx (2014) xxx–xxx Table 1 Plasmids used for combinatorial biosynthesis in E. coli to generate reference carotenoids for HPLC. Plasmid
Expressed enzyme(s)
Marker
Carotenoid product or modification reaction
Ref.
pACCRT-EBIRc pACCAR16DcrtX
GGPP-S, P-S, P-3D GGPP-S, P-S, P-4D L-C GGPP-S, P-S, P-4D L-C, bC-H L-C bC-K bC-K bC-H bC-H
chl chl
Neurosporene b-Carotene
[27] [28]
chl
Zeaxanthin
[28]
tet tet amp amp kan
Cyclization 4,40 -Ketolation 4,40 -Ketolation 3,30 -Hydroxylation 3,30 -Hydroxylation
[29] [30] [31] [32] This work
pACCAR25DcrtX pRKcrtY pRKbkt pCRBKT pUCbch pBBR1-MCS2crtZ
Abbreviations: bC-H, b-carotene 3-hydroxylase; bC-K, b-carotene 4-ketolase. GGPP-S, geranylgeranyl pyrophosphate synthase; L-C, lycopene cyclase; P-3D, 3-step phytoene desaturase; P-4D, 4-step phytoene desaturase; P-S, phytoene synthase.
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50 -CTC AAC GGG CCC ATT ATG CTG TGG-30 and Z-rev: 50 -CGT GCT GCA AGC TTC TCA GG-30 . The PCR product was then ligated into the Apa//HindIII sites of pBBR1-MCS1 with kanamycin as selection marker [18].
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Determination of crtYB copy numbers
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Genomic DNA was isolated from CBS6938 and the crtYB transformants after breaking the cells with glass beads in a Retsch homogeniser using classical phenol–chloroform extraction and isopropanol precipitation as described by Lecellier and Silar [19]. The purity was checked on 1% agarose gels and the amounts quantified after ethidium bromide staining by densitometry.qRT-PCR was carried out in the Rotor Gene PCR Cycler 3000 (Corbett Life Science) with the Kapa Sybr Fast Universal Mix (Peqlab, Erlangen, Germany) for the crtYB gene with the primers YB-forw: 50 -AGG GCT GAT CCC TCG ATA CC-30 and YB-rev: 50 -GTC TCG ATA GGC GTC TTC CG-30 . The PCR cycles were: 1 cycle of denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95 °C for 3 s, and extension at 60 °C for 20 s and a final melt curve with range from 50 to 59 °C in 0.5 °C steps. For relative quantification according to Pfaffl [20] DCT values and efficiency of amplification were used. The copy number of crtYB in non-transformed X. dendrorhous was set as one due to the expected haploid nature of our CBS6938 strain [10].
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Isolation of carotenoids and NMR analysis
Results
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The red pigments in the freeze-dried cells of transformant CBS 6938-(YB)3 (20.08 g, from 10 l culture) were extracted 6 times with 30 ml acetone by sonication for 5 min. To the combined supernatants (180 ml), n-hexane (180 ml) and H2O (180 ml) were added and the pigments partitioned between n-hexane–acetone/H2O. The n-hexane–acetone layer (upper layer) was analyzed by HPLC. Eight different carotenoids were separated including two unidentified carotenoids with a characteristic absorbance maximum at 451 nm (=type III). To isolate the unidentified carotenoids, the nhexane–acetone layer was concentrated to dryness (787.3 mg) and applied to silica gel column chromatography (Silica Gel 60, Kanto Chemicals) i.d. 30 130 mm, solvent: n-hexane). The silica gel column was developed with n-hexane (500 ml), n-hexane–acetone 20:1 (500 ml), 8:1 (500 ml), and 4:1 (200 ml), stepwisely. The n-hexane–acetone (20:1) eluate which contains the two type III carotenoids and 3-hydroxy echinenone was collected and concentrated to give a red oil (42.1 mg). The red oil was subjected to silica gel column chromatography (i.d. 20 mm 150 mm, solvent: CH2Cl2) and developed with CH2Cl2. The red fractions were collected and concentrated to dryness (7.3 mg), and subjected to preparative ODS HPLC on a l-Bondapack C18 column (Waters Associates) i.d. 7.8 300 mm with CH3CN–THF–MeOH (58:7:35) at a flow rate
Plasmids and transformants of X. dendrorhous
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Two pPR2TN based plasmids [12] with one or two copies of the phytoene synthase/lycopene cyclase gene crtYB from X. dendrorhous for genome integration were constructed. Details on their structures are given in Fig. 1. Both plasmids also differ by the selection marker which is geneticin in pPR2TN-YB-YB or hygromycin in pPR2TNH-YB. This allows the integration of both plasmids into the same transformant not only individually but also in combination. After transformation, three lines differing in crtYB integration were obtained: CBS6938-(YB)1 by integration of pPR2TNH-YB, CBS6938(YB)2 by integration pPR2TN-YB-YB containing two crtYB copies and finally a double transformant CBS6938-(YB)3 with both plasmids (Table 2). They all differ in their total carotenoid content which is lowest in the wild type and highest in the double transformant ranging from 258 to 798 lg/g dw. Although CBS6938(YB)3 exhibits the highest carotenoid synthesis, the concentration of the end product astaxanthin is not increased compared to CBS6938-(YB)2 and the percentage of astaxanthin within total carotenoids is even decreased. HPLC analysis of wild type and
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148 149 150
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of 3 ml/min and PDA detection (250–600 nm). In this HPLC, 3-hydroxy echinenone was eluted at Rt 6.3 min as pure compound (0.9 mg) and the mixtures of type III carotenoids (2.0 mg) were eluted at Rt 7.3–7.7 min. This fraction was finally subjected to preparative ODS HPLC on Develosil C30-UG-5 (Nomura Chemical) i.d. 10 250 mm with solvent CH3CN–CH2Cl2–EtOH (60:40:2) and a flow rate of 3 ml/min resulting in pure compounds with Rt 11.6 min. 0.8 mg (compound 7, Fig. 2B) and Rt 12.4 min. 0.3 mg (compound 5, Fig. 2B). 1H- and 13C NMR spectra were measured at 400 MHz with a Bruker AVANCE 400. HRESI-MS was recorded with a JEOL JMS-T100LP mass spectrometer.
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Analytical high-performance liquid chromatography (HPLC)
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Carotenoid extracts from X. dendrorhous and E. coli were analyzed by analytical HPLC1 either on silica gel Cosmosil 5SL-II 25 cm column with solvent: n-hexane–acetone (85:15) or on a reversed phase 15 cm Nucleosil 100 C18, 5l column with acetonitrile /methanol/2-propanol (85:10:5, by volume) as the mobile phase with a flow rate of 1 ml/min. Spectra were recorded online with a photodiode array detector 440 (Kontron, Straubenhard, Germany). Carotenoid identification and quantification was carried out with authentic standards.
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1
Abbreviation used: HPLC, high-performance liquid chromatography.
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Fig. 2. HPLC separation of wild type (WT) X. dendrorhous and transformant CBS 6938-(YB)3 carotenoids together with extracts of E. coli engineered for the generation of reference carotenoids. Traces (A) WT, (B) CBS6938-(YB)3, (C) pACCAR16DcrtX + pRKbkt + pUCbch, (D) pACCRT-EBIRc + pCRBKT + pRKcrtY and (E) pACCRT-EBIRc + pCRBKT + pRKcrtY + pBBR1-MCS2crtZ. Peaks marked with ⁄ indicate a similar spectrum resembling that in Fig. 3 type III. For NMR identification of compound 7, see Table 3. Ast, astaxanthin; Adr, adonirubin; HOe, HO-echinenone; Ech, echinenone; Bcar, all-trans b-carotene; cBcar, cis b-carotene; 7,8Db, 7,8dihydro-b-carotene; diHO78Db, 3,30 -dihydroxy-70 ,80 -dihydro-b-carotene; HOK78Db, 3-HO-4-keto-70 ,80 -dihydro-b-carotene; HOBz, 3-HO-b-zeacarotene; KBz, 4-keto-b-zeacarotene; KBz, 3-HO-4-keto-b-zeacarotene.
CBS6938-(YB)3 indicate an increased accumulation of carotenoid intermediates (Fig. 2A and B). In order to channel these intermediates into the end product, a third plasmid pPR2TNN-asy with the astaxanthin synthase gene and nourseothricin as selection marker was constructed (Fig. 1C) and used for additional transformation of CBS6938-(YB)3 yielding CBS6938-(YB3-asy). In this final line the astaxanthin content reached 69% of total carotenoids. In addition, it synthesizes not only 3-fold more total carotenoids than the wild type but also shows a 2.8-fold higher astaxanthin content (Table 2).
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Reversed-phase HPLC analysis of wild type and CBS6938-(YB)3
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HPLC analysis of the pigments demonstrated that in the wild type CBS6938 (Fig. 2A) astaxanthin (1) is the dominating carotenoid together with small amounts of adonirubin (3-HO-4,40 -diketo-bcarotene) (2) and with 3-HO-4-ketotorulene (16) with its typical spectrum with a maximum at 490 nm (Fig. 3). In transformant CBS6938-(YB)3 several additional intermediates accumulated (Fig. 2B). Some of them, peak No. 2 and 4 exhibited the same type I spectrum (Fig. 3) as astaxanthin whereas peaks 6 and 8 exhibited the type II spectrum of echinenone (4-keto-b-carotene). Four of the intermediates 5, 7, 9, 11 labeled with an asterix showed the untypical type III bell-shaped spectrum with a maximum at 451 nm and a shoulder at 447 nm. In order to identify them, reference carotenoids were generated by gene combinations in E. coli. Due to the defined functions of the genes and their previous use [21–23], different ketolated and hydroxylated b-carotene and neurosporene derivates were obtained. Extracts in Fig. 2C from E. coli/ pACCAR16DcrtX + pRKbkt + pUCbch allowed the identification of astaxanthin (1), adonirubin (2), canthaxanthin (4,40 -diketo-b-carotene) (4), 3-HO-echinenone (6), echinenone (8) and b-carotene all-trans and cis isomer (12 and 13). E. coli/pACCRT-EBIRc + pCRBKT + pRKcrtY analyzed in Fig. 2D synthesized neurosporene related ketocarotenoids 4-keto-b-zeacarotene (9) and 4-keto-70 , 80 -dihydro-b-carotene (11) with the type III spectrum (Fig. 3) and also 7,8-dihydro-b-carotene (15). With an additional plasmid carrying a hydroxylase gene, E. coli/pACCRT-EBIRc + pCRBKT + pRKcrtY + pBBR1-MCS2crtZ produced two hydroxy products, 3,30 -diHO70 ,80 -dihydro-b-carotene and 3-HO-b-zeacarotene, and two hydroxy-keto products, 3-HO-4-keto-b-zeacarotene (5) and 3-HO-4keto-70 ,80 -dihydro-b-carotene (7) (Fig. 2E). Some of the carotenoids which accumulated in CBS6938-(YB)3 can be attributed to the pathway from b-carotene to astaxanthin. In addition, 7,8-dihydro-b-carotene was found together with several keto derivatives of this carotene and of b-zeacarotene which were tentatively identified by co-chromatography together with their optical spectra. These carotenoids are all related to cyclization products of neurosporene. In order to confirm their biosynthesis outlined in the dotted box of Fig. 4, we isolated and purified the most abundant carotenoid 7 which co-chromatographed with 3-HO-4-keto-70 ,80 -dihydro-b-carotene for NMR analysis.
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Table 2 Transformants of X. dendrorhous with their integrated plasmids, additional copy numbers and their carotenoid biosynthesis potential. Strains CBS6938 CBS6938-(YB)1 CBS6938-(YB)2 CBS6938-(YB)3 CBS6938-(YB3asy)
Transformation plasmids
GC#
Total carot. (lg/g dw)
Astaxanthin (%)
pR2TNH-YB (Hygromycin) pPR2TN-YB-YB (Geneticin) pPR2TNH-YB (Hygromycin) + pPR2TN-YB-YB (G418) pPR2TNH-YB (Hygromycin) + pPR2TN-YB-YB (Geneticin) + pPR2TNN-asy (Nourseothricin)
1 2 4 5 5
258 ± 26 617 ± 31 745 ± 37 798 ± 30 772 ± 13
191 ± 19 268 ± 22 323 ± 18 301 ± 21 533 ± 66
(74) (44) (43) (38) (69)
Total carotenoid values are means of three independent cultures ± SD, values are significantly different within at least a 1% confidence interval. GC#, genome copy number of crtYB; total Car, total carotenoid content.
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Fig. 3. Visible spectra of carotenoids in X. dendrorhous including putative ketolated and hydroxylated cyclization products of neurosporene. Type I resembles diketo-bcarotene derivatives like astaxanthin, type II mono keto derivatives like echinenone, type III monoketo-7,8-dihydro-b-carotene derivatives. Bcar, b-carotene; 78Db, 7,8dihydro-b-carotene; KHOT, 3-HO-4-ketotorulene.
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Structure elucidation of compound 7
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Purified compound 7 (Fig. 2B) was dissolved in MeOH and analyzed by positive ion HRESI-MS. The (M+Na)+ peak was observed at m/z 591.43972 [calcd. for 591.43652 (C40H56O2Na)], and the molecular formula of 7 was determined to be C40H56O2 [3-hydroxy echinenone (C40H54O2) + 2H]. The 1H NMR spectrum of 7 in CDCl3 was quite similar to that of 3-hydroxy echinenone [24], while new 2 sp3 methylene signals [d 2.12 (both) (H-70 and H-80 )] and up-field shifts of 2 singlet methyl [d 1.62 (H-180 ) and d 1.87 (H-190 )] and 1 sp3 methine [d 5.98 (H-100 )] were observed in 7 [3-hydroxy echinenone: d 1.72 (H-180 ), d 1.98 (H-190 ), and d 6.16 (H-100 )]. Considering these observations, 7 was proposed to be a dihydro-derivative of 3-hydroxy echinenone. The analyses of the 1H–1H DQF COSY, HMQC and HMBC spectra (the key long range couplings in the HMBC spectrum were shown in Table 3A) for 7 proved one end structure in 7 was 3-hydroxy 4-keto b-ionone ring (C-1–C-6) and the other was b-ionone ring (C-10 –C-60 ) like 3-hydroxy echinenone, while the 1H chemical shifts (Table 3B) at H-180 (d1.62) and H-190 (d 1.87) and the 13C chemical shifts at C-60 (from d 137.8 to 136.9) and C-180 (from d 21.7 to 19.5) in b-ionone ring were different from those of 3-hydroxy echinenone [3-hydroxy echinenone: d 137.8 (C-60 ) and d 21.7 (C-180 )]. All data mentioned above strongly suggested that compound 7 is 3-hydroxy-70 ,80 -dihydro-echinenone (see structure in Table 3A). The up-field shifts of H-100 (d 5.98) and H-190 (d 1.87) in the olefine structure, and the 1H–13C long-range couplings from H-190 (d 1.62) to C-80 (d 40.6) and C-100 (d 124.5) in the HMBC spectrum also
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Fig. 4. Carotenoid biosynthesis in X. dendrorhous transformant with over-expressed phytoene synthase and lycopene cyclase (arrows indicating both reactions are boxed) with an additional neurosporene derived extention (in dotted box). The NMR-identified derivative 3-HO-4-keto-70 ,80 -dihydro-b-carotene is circled.
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B. Mass and NMR parameters HRESI-MS (+) m/z 591.43972 (M+Na)+, C40H56NaO2(calcd. for 591.43652). 1H NMR (CDCl3) d: 1.01 (s, H-160 , 170 ), 1.21 (s, H-17), 1.32 (s, H-16), 1.44 (m, H-20 ), 1.57 (m, H-30 ), 1.62 (s, H-180 ), 1.81 (dd, J = 13.6, 14.4 Hz, H-2 a), 1.87 (s, H-190 ), 1.92 (t, J = 7.3 Hz, H-40 ), 1.95 (s, H-18), 1.96 (s, H-200 ), 1.98 (s, H-20), 2.00 (s, H-20), 2.12 (m, H-70 , 80 ), 2.15 (dd, J = 5.4, 14.4 Hz, H-2 b), 4.33 (dd, J = 5.4, 13.6 Hz, H-3), 5.98 (d, J = 10.7 Hz, H-100 ), 6.20 (d, J = 15.0 Hz, H-7), 6.21 (d, J = 11.2 Hz, H-140 ), 6.26 (d, J = 16.0, H-12), 6.30 (d, J = 11.5, H-10), 6.30 (d, J = 11.5,H-14), 6.43 (d, J = 15.0 Hz, H-8), 6.44 (d, J = 14.7 Hz, H-12), 6.53 (dd, J = 10.7, 16.0 Hz, H-110 ), 6.62 (dd, J = 11.5, 14.7 Hz, H-11), 6.63 (dd, J = 11.5, 14.9 Hz, H-15), 6.67 (dd, J = 15.3, 11.2 Hz, H-150 ). 13C NMR (CDCl3) d:12.7 (C-19, 20, 200 ), 13.8 (C-18), 17.0 (C-190 ), 19.5 (C-180 ), 19.7 (C-30 ), 26.1 (C-16), 27.5 (C-70 ), 28.4 (C-160 , 170 ), 30.5 (C-17), 35.0 (C-10 ), 32.6 (C-40 ), 36.8 (C-1), 39.8 (C-20 ), 40.6 (C-80 ), 45.7 (C-2), 69.1 (C-3), 123.2 (C-7), 124.5 (C-100 ), 124.6 (C-11), 125.0 (C-110 ), 126.6 (C-5), 127.2 (C-50 ), 130.2 (C-15), 130.5 (C-150 ), 131.1 (C-140 ), 133.6 (C-14), 134.2 (C-9), 135.0 (C-120 ), 135.1 (C-10), 136.2 (C-13), 136.7 (C-60 ), 136.9 (C-130 ), 140.1 (C-12), 140.9 (C-90 ), 142.2 (C-8), 162.3 (C-6), 200.2 (C-4)
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A. The structure of 3 hydroxy-70 ,80 -dihydro-echinenone with 1H-13C long range couplings
Table 3 NMR characterization of compound 7 as 3-HO-4-keto-70 ,80 -dihydro-b-carotene.
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supported the structure of 1 as 3-hydroxy-70 ,80 -dihydro-echinenone, a new carotenoid not described before. The IUPAC-IUB semi-systematic name of 7 is 3-hydroxy-70 ,80 -dihydro-b,b-carotene-4-one. The amounts of other compounds with a type III spectrum (e.g. compound 5 in Fig. 3) were too small for NMR structural analyses. Nevertheless, their optical spectra indicate that they possess either a 70 ,80 -dihydro-b-carotene or a b-zeacarotene related structure.
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Discussion
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X. dendrorhous is a promising microorganism for the synthesis of astaxanthin provided its yield can be increased by pathway engineering or other means [1,2]. It is well-known that the flux into the specific carotenoid pathway is limited by phytoene synthesis [13]. In our engineering approach, we attempted to saturate the level of phytoene synthase by transformation with multiple copies of its gene crtYB (Table 2). With this approach, it was possible to increase the total number of crtYB copies from one in the wild type via two in CBS6938-(YB), four in CBS6938-(YB)2 to five in CBS6938-(YB)3. Saturation levels indicated by formation of total carotenoids were almost reached by three crtYB integration steps and the resulting transformant CBS6938-(YB)3 with four crtYB copies additional to the one in the genome. Under these conditions, next limitations of the entire carotenoid pathway may be precursor supply and/or conversion of carotenoid intermediates to the end product. The latter was evident by accumulation of substrates and intermediates which can be metabolized to astaxanthin synthase (Fig. 2B). This limitation could be partially alleviated by additional transformation with a copy of the astaxanthin synthase gene asy which increased the percentage of astaxanthin in CBS6938-(YB3asy) almost back to the wild type proportion (Table 2). In addition to b-carotene, echinenone, HO-echineneone, canthaxanthin and adonirubin (Fig. 1) accumulating in CBS6938(YB)3 as intermediate of the pathway towards astaxanthin, three minor carotenoids with an unknown absorbance spectra with a maximum at 451 nm, a small shoulder at 447 nm and with an asymmetrical bell shape (type III of Fig. 3) were found exclusively in this crtYB transformant. These features may be indicative of ketocarotenoids. Two carotenes detected in the fungus Podospora anserina after over-expression of its al-2 gene equivalent to crtYB which from their spectra could be the non-ketolated precursors are b-zeacarotene and 7,8-dihydro-b-carotene [25]. Their main absorbance maxima are at 428 nm and together with one keto group at position 4 both products could exhibit the type III spectrum. Both carotenes are derived from neurosporene either by a one-step cyclization to b-zeacarotene or a second one at the other end of the carbon chain to 7,8-dihydro-b-carotene. Keto derivatives of both carotenes with or without additional 3-HO groups were synthesized as reference compounds in E. coli as reported previously [21,22] and used to identify the compounds with type III spectra in CBS6938-(YB)3. They co-chromatographed with compound 7, 3-HO-4-keto-70 ,80 -dihydro-b-carotene, 9, 4-keto-b-zeacarotene, and 11, 4-keto-70 ,80 -dihydro-b-carotene. Their synthesis is outlined in Fig. 4 (boxed part). For the most abundant compound 7, we were able to proof its structure more definitely by MS and NMR spectroscopy (Table 3). This fully supports the extra branch of carotenogenesis in transformant CBS6938-(YB)3 which diverts at the stage of neurosporene. This is due to a competition for neurosporene (which is not fully desaturated but can already be cyclised at least at one end of the molecule) by phytoene desaturase and lycopene cyclase with a much higher preference for desaturation which may be due to a poorer specificity of lycopene cyclase for the substrate neurosporene. In X.
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dendrorhous wild type, this leads to an exclusive formation of lycopene. However, unfavorable reactions can be forced by high enzyme concentrations as exemplified for phytoene desaturase in purple bacteria [26]. Since gene crtYB encodes a phytoene synthase/lycopene cyclase fusion protein, its over-expression in CBS6938-(YB)3 not only resulted in enhanced carotenogenesis due to higher amounts of phytoene synthase but also caused the formation of neurosporene derivatives by elevated lycopene cyclase levels. This lycopene cyclase should cyclise the end of the neurosporene molecule which resembles the end also found in lycopene in the same way as lycopene is cyclised at both identical ends. In addition, it is also able to cyclise the 7,8-dihydro side of neurosporene yielding 7,8-dihydro-b-carotene. This broad substrate specificity has already been reported before for the lycopene cyclase from the bacterium Pantoea ananas (formerly Erwinia uredovora) and from the plant Capsicum annuum [21]. Downstream in the pathway, it can be assumed that astaxanthin synthase of X. dendrorhous modifies the b-ionone ring with 3-HO and 4-keto groups regardless of the structure of the other end of the molecule carrying either a 7,8-dihydro-b-ionone ring or being acyclic (Fig. 4).
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Acknowledgment
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This work was supported by the LOEWE Project ‘‘Integrative Pilzforschung’’.
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