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Home , Atropa, Hyoscyamus, Hyoscyamus niger, Tropane alkaloid

Engineering biosynthetic pathway in niger hairy root cultures

Lei Zhang*†, Ruxian Ding†, Yourong Chai‡§, Mercedes Bonfill¶, Elisabet Moyanoʈ, Kirsi-Marja Oksman-Caldentey**, Tiefeng Xu§, Yan Pi*, Zinan Wang*, Hanming Zhang†, Guoyin Kai§, Zhihua Liao*, Xiaofen Sun*, and Kexuan Tang*††

*State Key Laboratory of Genetic Engineering, Morgan-Tan International Center for Life Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China; †School of Pharmacy, Second Military Medical University, Shanghai 200433, China; ¶Unidad de Fisiologı´aVegetal, Facultad de Farmacia, Universidad de Barcelona, Diagonal 643, E-08028 Barcelona, Spain; ʈDepartament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avenida Dr. Aiguader 80, E-08003 Barcelona, Spain; **VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT (Espoo), Finland; ‡School of Agronomy and Life Sciences, Southwest Agricultural University, Chongqing 400716, China; and § Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, China

Communicated by Jiazhen Tan, Fudan University, Shanghai, China, March 2, 2004 (received for review July 10, 2003) is a pharmaceutically important tropane methylation of putrescine catalyzed by PMT is the first commit- extensively used as an agent. Here, we report the ted step in the biosynthesis of these (7). Scopolamine, simultaneous introduction and overexpression of genes encoding which is the 6,7-␤-epoxide of , is formed from the rate-limiting upstream enzyme putrescine N-methyltransferase hyoscyamine by means of 6␤-hydroxyhyoscyamine. Hyoscya- (PMT) and the downstream enzyme hyoscyamine 6 ␤-hydroxylase mine 6␤-hydroxylase (H6H, EC 1.14.11.11), a 2-oxo-glutarate- (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscy- dependent dioxygenase, catalyzes the hydroxylation of hyoscy- amus niger) hairy root cultures. Transgenic hairy root lines express- amine to 6␤-hydroxyhyoscyamine, as well as the epoxidation of ing both pmt and h6h produced significantly higher (P < 0.05) 6␤-hydroxyhyoscyamine to scopolamine (1, 8, 9) (Fig. 1). The levels of scopolamine compared with the wild-type and transgenic tropane alkaloids hyoscyamine (its racemic form being ) lines harboring a single gene (pmt or h6h). The best line (T3) and scopolamine are structurally related and are derived from a produced 411 mg͞liter scopolamine, which was over nine times common intermediate, the N-methylpyrrolinium cation. They more than that in the wild type (43 mg͞liter) and more than twice are used medicinally as anticholinergic agents that act on the the amount in the highest scopolamine–producing h6h single-gene parasympathetic nerve system. Because they differ in their transgenic line H11 (184 mg͞liter). To our knowledge, this is the actions on the central nervous system, there is currently a 10-fold highest scopolamine content achieved through genetic engineer- higher commercial demand for scopolamine, in the N- ing of a plant. We conclude that transgenic harboring both butylbromide form, than for hyoscyamine and atropine com- pmt and h6h possessed an increased flux in the bined (9). Hyoscyamine and scopolamine are mostly synthesized biosynthetic pathway that enhanced scopolamine yield, which was in young root cells and translocated to the aerial parts of the more efficient than plants harboring only one of the two genes. It plant (10). Hence, cultured roots are capable of accumulating seems that the pulling force of the downstream enzyme (the faucet high concentrations of these metabolites. Small-scale jar fer- enzyme) H6H plays a more important role in stimulating scopol- menters for the hairy roots of several Solanaceous species have amine accumulation in H. niger whereas the functioning of the been developed as prospective in vitro systems for commercial upstream enzyme PMT is increased proportionally. This study production of tropane alkaloids, but the scopolamine levels in provides an effective approach for large-scale commercial pro- these systems are often much lower than those of hyoscyamine duction of scopolamine by using hairy root culture systems as (11). Researchers have also carried out genetic engineering of bioreactors. pharmaceutically important tropane alkaloids (12), in which the conversion of hyoscyamine to the much more valuable scopol- Agrobacterium ͉ hyoscyamine 6␤-hydroxylase ͉ putrescine amine is the major goal. Releasing the expression of key N-methyltransferase ͉ scopolamine ͉ transformation enzymatic activities from the strict regulation to which they are normally subjected is expected to increase product formation. A econdary metabolites are low-molecular-weight compounds rough correlation has been found between H6H activity and the Sproduced widely throughout the plant kingdom. Plant alka- ratio of scopolamine to hyoscyamine in scopolamine-producing loids constitute the largest groups of natural products, providing cultured roots (9). H6H, therefore, is a promising target enzyme many pharmacologically active compounds. The in-depth un- that, if overexpressed in hyoscyamine-accumulating tissues, derstanding of biosynthetic pathways, along with the increasing would result in increased scopolamine levels in the transfor- number of cloned genes involved in biosynthesis, enable the mants. Several hyoscyamine-rich but scopolamine-poor plants exploration of metabolic engineering as a potential effective that had been considered unattractive for commercial exploita- approach to increase the yield of specific metabolites by enhanc- tion may now become promising candidates for large-scale ing rate-limiting steps or by blocking competitive pathways. A scopolamine production by means of cultured roots. The hy- few genera of the plant family , including Hyoscyamus, droxylase gene from has been introduced into Duboisia, , and Scopolia, are able to produce biologically , a typical hyoscyamine-rich tropane alkaloid- active and tropane alkaloids simultaneously (1–3). Both producing plant species (13). Several transgenic root clones tropane and pyridine alkaloid biosynthetic pathways share a showed 5-fold higher concentrations of scopolamine than the common polyamine metabolism in their early steps. Putrescine wild-type hairy roots. By overexpressing h6h in Hyoscyamus is a common precursor of both polyamines, such as spermidine muticus hairy root cultures, the best transgenic clone had a and spermine, and tropane͞pyridine alkaloids (4–6). Putrescine N-methyltransferase (PMT; EC 2.1.1.53) is the enzyme involved Abbreviations: H6H, hyoscyamine 6 ␤-hydroxylase; PMT, putrescine N-methyltransferase; P, in the removal of putrescine from the polyamine pool because it transgenic hairy root lines generated from pmt single gene transformation; H, transgenic catalyses the N-methylation of this diamine to form N- hairy root lines generated from h6h single gene-transformation; T, transgenic hairy root methylputrescine (mP). Because both the tropane ring moiety of lines generated from pmt͞h6h double gene-transformation. the tropane alkaloids and the pyrrolidine ring of nicotine are ††To whom correspondence should be addressed. E-mail: [email protected]. derived from putrescine by way of mP synthesis, the N- © 2004 by The National Academy of Sciences of the USA

6786–6791 ͉ PNAS ͉ April 27, 2004 ͉ vol. 101 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401391101 Downloaded by guest on October 1, 2021 genes that control the expression of multiple pathway enzyme genes, or both. A good example is the bioengineering of the ␤-carotene biosynthetic pathway in the major staple food crop rice (Oryza sativa). By overexpressing the combination of genes encoding for three key enzymes (phytoene synthase, phytoene desaturase, and lycopene ␤-cyclase), a final ␤-carotene (previ- tamin A) concentration of 2 mg͞kg was detected in dry rice endosperm (18). So far, explants of H. niger have not been used for scopolamine bioengineering. Commercial cultivars of H. niger contain the natural amounts of scopolamine, which are not very high. Hence, there is a very strong need to increase scopolamine production rates for commercial production. Here, we report the introduc- tion of gene constructs containing cDNA clones of pmt and h6h, driven by the constitutive cauliflower mosaic virus 35S promoter, into H. niger, either one gene at a time, or both genes together. The morphology, growth rate, activities of alkaloid pathway enzymes, and alkaloid production capacities of these engineered hairy root lines were investigated. Materials and Methods Construction of pmt and h6h Binary Expression Vector. The mono- valent pmt expression plasmid pBMI was previously constructed (16). Disarmed Agrobacterium tumefaciens strain C58C1 harbor- ing both pBMI and Agrobacterium rhizogenes Ri plasmid pRiA4, containing a single pmt gene, were used for plant transformation (16, 19). The monovalent h6h expression plasmid pLAL21, constructed by Jouhikainen et al. (14), was used in the study. Plasmid LAL21 was isolated from Escherichia coli strain DH5␣ and transformed into A. rhizogenes LBA9402 by electroporation (20). A positive clone, after confirmation by PCR and enzymatic digestion analysis for the presence of the h6h gene, was used for plant transformation. The pBMI and pLAL21 were used to construct a bivalent expression plasmid containing both pmt and h6h genes. The pBMI was linearized by HindIII digestion and blunted with Klenow fragment of E. coli DNA polymerase I. After double digestion of pLAL21 with EcoRI and EcoRV, the 1,250-bp fragment, containing the h6h coding sequence and 87-bp 3Ј-end sequence of the cauliflower mosaic virus (CaMV) 35S promoter, Fig. 1. Biosynthetic pathway of nicotine and tropane alkaloids (1). ArgDC, was recovered. It was then ligated with an EcoRI͞EcoRV arginine decarboxylase; OrnDC, ornithine decarboxylase; PMT, putrescine fragment from the double digestion of pGFP to form the N-methyltransferase; DAO, diamine oxidase; TR, tropinone reductase; H6H, hyoscyamine 6␤-hydroxylase. [Adapted with permission from ref. 1 (Copyright intermediate plasmid GFP-h6h. After digesting pGFP-h6h with 1994, Annual Reviews, Inc.).] SphI, the 2,100-bp fragment, containing the complete h6h expression cassette, was recovered, blunted with E. coli T4 DNA polymerase, and finally ligated with the blunted, linearized 100-fold increase of scopolamine. Furthermore, this clone pro- pBMI to form the recombinant expression vector pXI (Fig. 2A). duced hyoscyamine as the major alkaloid in similar amounts The pXI contained two separate expression cassettes for pmt and compared with controls (14). Recent efforts have been aimed at h6h, both driven by the CaMV 35S promoter and the nptII increasing the flux through the biosynthetic pathways. Overex- cassette for conferring kanamycin resistance. pression of PMT increased the nicotine content in Nicotiana The three plasmids, pBMI, pLAL21 and pXI, were separately sylvestris, implying increased flux through tropane and pyridine joined to an expression vector within the left and right T-DNA pathways whereas suppression of endogenous PMT activity [portion of the Ti (tumor-inducing) plasmid that is transferred to severely decreased the nicotine content and induced abnormal plant cells] borders. The pXI was isolated from E. coli strain DH5␣ and transformed into disarmed A. tumefaciens strain morphologies (15). When overexpressing the pmt gene in A. C58C1 containing A. rhizogenes Ri plasmid pRiA4 (20). A belladonna (15) and Duboisia hybrid (16), there was no signif- positive clone, after confirmation by PCR and enzymatic diges- icant increase in either tropane- or pyridine-type alkaloid

tion analysis for the presence of both pmt and h6h genes, was PLANT BIOLOGY concentrations. used to transform plant tissues for simultaneous expression of Although a substantial increase in productivity is feasible pmt and h6h. when a rate-limiting enzyme is targeted (17), in most biosyn- thetic pathways for secondary metabolites there often exists Plant Transformation and Root Cultivation. H. niger seeds were more than one rate-limiting step. Overexpression of one enzyme obtained from the Second Military Medical University (Shang- often renders subsequent reactions more rate-limiting, and thus hai, China) and germinated into plants. Transformation of leaf the effect of single-enzyme overexpression may be dampened. explants from these H. niger plants was carried out basically Strategies should include fortification of multiple steps by over- following the previously described method for leaf disk expressing multiple biosynthetic genes, manipulating regulatory transformation (21), with the simultaneous transformation with

Zhang et al. PNAS ͉ April 27, 2004 ͉ vol. 101 ͉ no. 17 ͉ 6787 Downloaded by guest on October 1, 2021 Fig. 2. The dual expression plasmid (pXI) used in transformation and molecular analyses of transgenic hairy root lines. (A) Schematic representation of the pXI. (B) Representative PCR analyses for the presence of rolB and rolC genes in transgenic hairy root lines. M, DL-2000 Marker (100–2,000 bp); P, pRiA4 (positive control); N, the wild-type H. niger root (negative control); T, transgenic hairy root lines containing both pmt and h6h genes induced by A. tumefaciens C58C1 strain (pRiA4, pXI). (C) Representative PCR analyses for the presence of pmt and h6h genes in transgenic hairy root lines. M, DL-2000 Marker (100–2,000bp); PC, pXI (positive control); NC, pRiA4 (negative control). (D) Northern blot analyses for the expression of pmt and h6h in transgenic hairy root lines. A4, transformed hairy root lines generated through blank transformation with A. rhizogenes strain A4; H, single h6h transgenic hairy root lines; P, single pmt transgenic hairy root lines; WT, wild type; T1 to T10, different transgenic lines.

strains A4 and LBA9402 of A. rhizogenes as controls. Wild-type dNTPs, 5 ␮lof10ϫ PCR buffer (Mg2ϩ plus) and 2.5 units of Taqr plants were grown in the same growth chamber. Roots developed DNA polymerase (TaKaRa) with 200 ng of genomic DNA as at cut edges 2–3 weeks after cocultivation were excised and template. For detection of the pmt gene, the template was cultured on solid, hormone-free, half-strength B5 medium (22), denatured at 94°C for 5 min followed by 35 cycles of amplifica- supplemented with 30 g͞liter sucrose as the carbon source. All tion (1 min at 94°C, 1 min at 60°C,45sat72°C) and by 5 min of the culture media contained 100 ␮g͞ml kanamycin and 500 at 72°C. For the detection of the h6h gene, the template was ␮g͞ml cefotaxime. Root culture clones were maintained at 26°C denatured at 94°C for 5 min followed by 35 cycles of amplifica- tion (1 min at 94°C, 1 min at 54°C, 1.5 min at 72°C) and by 5 min in the dark and routinely subcultured every 25–30 days. The at 72°C. The mixture of the above four primers was used for rapidly growing kanamycin-resistant lines with no bacterial simultaneous detection of pmt and h6h from hairy root samples contamination were used to establish hairy root lines. About 100 derived from transformation (T lines) by using the same PCR mg of fresh roots (3 cm in length) were inoculated into 150-ml conditions for the detection of the h6h gene. conical flasks containing 40 ml of liquid and half-strength B5 medium and cultured on an orbital shaker (100 rpm) at 25°Cin Northern Blot Analysis. Total RNA was isolated from separately the dark. After 28 days of culture, the roots were filtered and generated 4-week-old root lines of culture by using TRIzol washed with 10 ml of sterile distilled water and lyophilized (23). Reagent (GIBCO͞BRL) and subjected to Northern blot analysis Root tissues from three flasks of cultures were collected indi- for the expression of pmt and h6h. Aliquots of total RNA (10 ␮g vidually at days 3, 7, 14, 21, 28, and 35 after inoculation. They per sample) were denatured and separated on a 1.1% formal- were then treated as described above. dehyde-denatured (wt͞vol) agarose gel (27). After electrophore- sis, the RNA was transferred onto a positively charged Hy- ϩ PCR Analysis. Genomic DNA was isolated from hairy root samples bond-N nylon membrane (Amersham Pharmacia) through by using the acetyl trimethyl ammonium bromide (CTAB) capillary transfer (27). The probe was generated by PCR (PCR DIG Probe Synthesis Kit, Roche) with pXI as template using method (24). The DNA was then used in PCR analysis for ͞ ͞ detecting the presence of Agrobacterium rol (B, C), pmt, and h6h primers FPMT RPMT for pmt and FH6H RH6H for h6h. PCR labeling of the probes with digoxigenin (DIG)-dUTP and hy- genes in transgenic hairy root tissues, following the previously bridization (30-min prehybridization at 50°C followed by 16-h described method (25, 26). PCR primers for pmt detection were Ј Ј Ј hybridization at 50°C) were performed according to the manu- FPMT (5 -GCCATTCCCATGAACGGCC-3 ) and RPMT (5 - facturer’s instructions (Roche). Hybridizing bands were detected Ј CCTCCGCCGATGATCAAAACC-3 ) whereas the primers by using the DIG Luminescent Detection Kit (Roche), and FH6H (5Ј-CCGGAATTCGGATCCCAACGTATAGAT- signals were visualized by exposure to Fuji x-ray film at 37°C for TCTTC-3Ј) and RH6H (5Ј-CGGGAATTCGGATCCCAAAC- 10 min. CATCACTGCAAT-3Ј) were used for h6h detection. PCR was carried out in total volumes of 50 ␮l reaction mixtures, contain- Enzyme Activity Assay. PMT enzymatic activity was evaluated by ing 1 ␮l of each primer (10 ␮mol͞liter), 1 ␮l of 10 mmol͞liter using the method of Feth et al. (28) with some modifications.

6788 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401391101 Zhang et al. Downloaded by guest on October 1, 2021 Table 1. Gene constructs and derived root cultures Number of established root lines

T-DNA construct Strain Total Antibiotic-resistant PCR-positive Established root cultures

ϽnptIIϽpmtϽh6h C58C1 25 16 11 T1,T2,T3,T6,T8,T10 ϽnptIIϽpmtϾ C58C1 41 28 28 P4,P5,P18 ϽnptIIϽh6hϾ LBA9402 50 7 3 H4,H11,H20

The antibiotic resistance gene (nptII) is always placed near the left border of the T-DNA; Ͻ and Ͼ indicate the direction of transcription. In addition, two lines transformed with C58C1 (pRiA4) were also established. T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells.

Tissues (0.5–1.0 g of FW) were extracted on ice with 3 vol of 100 25 T hairy root lines were generated, of which 28 P, 7 H, and 16 mmol͞liter potassium phosphate buffer (pH 7.5, buffer A), T lines survived the successive subculture process (Table 1). containing 5 mmol͞liter EDTA, 10 mmol͞liter mercaptoetha- Some transgenic hairy root lines turned brown and aged con- nol, 0.5% sodium ascorbate, and 2% polyethyleneglycol 400, siderably faster than wild-type hairy root lines. These lines were followed by centrifugation at 27,000 ϫ g for 30 min. The discarded, and the remaining hairy root lines were subcultured supernatant was loaded onto a Sephadex G25 prepacked PD-10 for 4–5 weeks in hormone-free, half-strength B5 liquid medium column (Amersham Pharmacia Biotech) equilibrated and eluted (Fig. 3A). with 50 mmol͞liter potassium phosphate buffer (pH 8, buffer B) Significant differences of growth characteristics were detected containing 1 mmol͞liter EDTA and 5 mmol͞liter mercaptoetha- among independent transformed root lines. All of the P and T nol. The reactions were performed by incubating 100 ␮lofthe lines grew fast and vigorously with thick and fewer branches purified supernatant with 20 ␮l of 25 mmol͞liter putrescine whereas H lines grew slowly with slender, dense and white (final concentration: 3.6 mmol͞liter), 8 ␮l of 10 mmol͞liter branches. Significant differences (P Ͻ 0.01) were found between S-adenosylmethionine (final concentration: 0.6 mmol͞liter) and T͞P lines and H lines with respect to growth rate, but no 12 ␮lofbufferBat37°C for 30 min. After stopping the reactions significant difference (P Ͼ 0.05) was detected among H lines and by heating in boiling water, 65 mmol͞liter borate-KOH buffer hairy roots generated by blank transformation (A4, LBA9402) and a solution of dansylchloride (5.4 mg͞ml acetonitrile) were (Fig. 3B). Generally, a fairly high content of secondary metab- added to the incubation mixture. After heating at 60°C for 15 olites in the tissues is associated with poor growth, and the actual min, the dansylated amines were extracted by adding 0.5 ml of total productivity of secondary metabolites therefore remains followed by vortex mixing for 30 s. After an aliquot (400 low (14). We found in this study, however, that root morphology ␮l) of the toluene was removed, the residue was dried and had a considerable influence on produc- resuspended in a fixed volume of acetonitrile, which was then tion whereas the growth rate in the high scopolamine-producing injected into the HPLC. The chromatographic conditions for the lines was not reduced as compared with those with low scopol- separation of dansylated N-methylputrescine were as described amine production. Line T3, for example, which produced the before (29). The retention time of N-methylputrescine was 24 highest level of scopolamine, grew very rapidly. Aging was min. observed along with the generation of brown pigments in the H6H enzymatic activity was assayed by using GLC by mea- roots, most likely due to the accumulation of phenolic com- suring the formation of 6␤-hydroxyhyoscyamine or scopolamine pounds. All types of hairy root lines reached the highest growth (8). Subsequent extraction of the reaction products and the rate at the third week and achieved maximum fresh weight at the conditions for GLC analysis were as described by Hashimoto and fifth week. Yamada (9). Molecular Analysis of Genetically Engineered Hairy Roots. Allofthe Alkaloid Extraction and Analysis. Extraction of tropane alkaloids hairy roots contained the rol genes (rol B, rol C), revealed by (hyoscyamine and scopolamine) was based essentially on the PCR analysis (Fig. 2B). The integration of pmt and h6h in method described by Hashimoto et al. (13) and analyzed by transformed hairy roots was also confirmed by PCR (Fig. 2C). HPLC as described (30). Pyridine alkaloid (nicotine) was ex- In total, the PCR-positive, kanamycin-screened hairy root lines tracted from hairy root cultures with 25 mmol͞liter sodium amounted to 82.35% (42͞51), with 100% (28͞28) for P, 42.86% phosphate buffer (pH 7.8) at 30°C for 24 h with constant (3͞7) for H, and 68.75% (11͞16) for T lines, respectively. The agitation and analyzed by HPLC by using a mobile phase of 40% percent of PCR-positive hairy roots in this study is much higher methanol containing 0.2% phosphoric acid buffered to pH 7.25 than that previously reported in Nicotiana tabacum (13%) (31). with triethylamine. Standards for hyoscyamine, scopolamine, Because both FH6H and RH6H primers were designed to cover and nicotine (Sigma) were prepared in methanol at a final the h6h sequence and the vector sequence, none of the checked concentration of 1 mg͞ml. DNA band was amplified from the control wild-type root samples. Results and Discussion Total RNA was isolated from separately generated root lines Transformation of H. niger with Plasmids Containing pmt and h6h. at the fourth week of cultivation for Northern blot analysis of pmt Three plasmids, pBMI, pLAL21, and pXI, containing the ex- and h6h expression. Our results showed that the pmt and h6h PLANT BIOLOGY pression cassettes of the cDNAs encoding pmt and h6h, sepa- transcripts accumulated at quite variable levels among indepen- rately or together (Fig. 2A), were separately introduced into H. dent transgenic hairy root lines (Fig. 2D). When compared with niger leaf explants by using disarmed A. tumefaciens C58C1 strain the wild-type plants, the h6h transcript in all of the transgenic H and A. rhizogenes LBA9402. The abbreviations P, H, and T refer and T lines (including H4,H11,T1,T2,T3, and T10), except for line to the transgenic hairy root lines generated from pmt single H20, expressed at higher or comparable levels. However, the gene-, h6h single gene-, and pmt͞h6h double gene-transforma- transcript level of h6h in line H20 was lower than that of the wild tions, respectively. Hairy root lines generated from transforma- type, implying the occurrence of cosuppression, which subse- tions with strains A4 and LBA9402 containing no pmt or h6h are quently resulted in the low scopolamine accumulation in this line denoted as A4 and LBA, respectively. In total, 41 P, 50 H, and (see Fig. 2D). The cosuppression phenomenon of pmt was

Zhang et al. PNAS ͉ April 27, 2004 ͉ vol. 101 ͉ no. 17 ͉ 6789 Downloaded by guest on October 1, 2021 Fig. 3. Analyses for the morphology, growth rate, enzyme activities, and alkaloid contents in transgenic H. niger hairy root lines. (A) Phenotype of developed root lines. I, pmt and h6h dual plasmid generated transgenic hairy root lines (T) on solid 1͞2 B5 medium; II, transformed hairy root lines generated through blank transformation with A. rhizogenes strain A4 on solid 1͞2 B5 medium; III, the covalent pmt and h6h dual plasmid generated transgenic hairy root lines (T) in liquid 1͞2 B5 medium; IV, wild-type H. niger root culture in liquid 1͞2 B5 medium. (B) Time courses of growth of five randomly selected hairy root lines. Each value represents the means of three to five determinations ϮSD; g, grams of tissues. (C) PMT activity in the T3 line (n ϭ 32, Ϯ SD) and WT line (n ϭ 16, Ϯ SD). (D) H6H activity in the T3 line (n ϭ 57, ϮSD) and WT line (n ϭ 22, ϮSD). (E) Alkaloid contents in root lines. A4, transformed hairy root lines generated through blank transformation with A. rhizogenes strain A4; H, h6h transgenic hairy root lines; P, pmt transgenic hairy root lines; T, transgenic hairy root lines containing both pmt and h6h genes.

previously observed in transgenic N. sylvestris plant (15). Be- transcripts produced tropane alkaloids in which scopolamine was cause PMT is a common key enzyme catalyzing polyamine the staple compound. However, scopolamine levels in H lines metabolism existing in most Solanaceae species (1, 7), all of the were highly variable, ranging from 25.1 to 184.4 mg͞liter. The transgenic lines showed pmt transcripts at various levels. The H expression levels of the h6h transgene also varied from line to lines (H4,H11, and H20) expressed lower levels of pmt whereas line but showed positive correlation to their corresponding T lines showed higher levels compared with the wild type. scopolamine concentrations. Elevated h6h activity was widely detected in the highly productive H and T lines. The inability of Analysis for Enzyme Activity and Alkaloid Concentration. The PMT line H20, the P lines, A4, and wild-type control lines to accumu- and H6H enzymatic activities in the transgenic hairy roots were late scopolamine may partly result from their low h6h activities. determined by HPLC (28) and GLC (8), respectively. A good These findings suggest that high H6H activity has a positive correlation was observed between mRNA levels and enzymatic influence on the flow of metabolites through the pathway. This activities in transgenic hairy root lines. PMT activity (50.7 positive correlation between H6H activity and scopolamine pkat͞mg Ϯ 4.3, n ϭ 32, ϮSD) and H6H activity (10.2 pkat͞mg Ϯ production has also been found in tissues of A. belladoma (13), 0.45, n ϭ 57, ϮSD) in line T3 were significantly higher (P Ͻ 0.01) H. muticus (14), and N. tabacum (31). These data demonstrate than those in wild-type lines (2.53 pkat͞mg Ϯ 0.25, n ϭ 16, ϮSD the desirability of overexpressing h6h for elevated scopolamine for PMT and 4.03 pkat͞mg Ϯ 0.85, n ϭ 22, ϮSD for H6H) (Fig. production. 3 C and D). All of the transgenic lines achieved maximum PMT In the present study, although having higher pmt transcript activity at the end of culture period (35 days) but achieved peak levels than the control lines (Fig. 2D), the pmt single-gene H6H activity already after 3 wk of culture. transgenic hairy root lines (P lines) produced alkaloids (hyoscy- The profiles of alkaloids (nicotine, hyoscyamine, and scopol- amine, scopolamine, and nicotine) at similar levels to those of amine) of transgenic hairy root lines were determined by HPLC. the control lines (Fig. 3E). This result indicates that enhance- The capacities of transgenic root lines to biosynthesize hyoscy- ment of pmt transcript alone was not sufficient to boost scopol- amine and scopolamine are shown in Fig. 3E. Except for line H4, amine biosynthesis, suggesting that this pathway is mainly down- which showed a low level of nicotine content (Ͻ26 mg͞liter), no stream-limited, rather than limited at the step where putrescine pyridine alkaloid nicotine was detected in any of the hairy root is converted to methylputrescine in H. niger. lines, implying that overexpression of pmt did not promote the Our observation is consistent with the results of pmt single- accumulation of nicotine in H. niger. All lines expressing h6h gene overexpression in A. belladonna (13), N. sylvestris (15) and

6790 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401391101 Zhang et al. Downloaded by guest on October 1, 2021 Duboisia (16), but contrary to those in (7) and product (8), it is suggested that it is completely desirable to hybrid (32). It is conjectured that the response of fundamentally enhance the scopolamine production by simul- Solanaceous species to pmt overexpression is species-related, taneously promoting the hydroxylation activity of H6H and which may be partly due to a different and specific posttrans- directing the flux from the polyamine pool to the tropane lational regulation of the endogenous enzyme in respect to the alkaloid pathway. This result is in accordance with our antici- exogenous one (16). pation that fortification of multiple steps by overexpressing Two of the T lines (T3 and T10) presented the highest multiple biosynthetic genes is crucial for improved production of scopolamine production (411.2 and 224.8 mg͞liter, respectively). useful plant secondary metabolites. Are the high scopolamine levels associated only with h6h This report on engineering pmt and h6h simultaneously into single-gene overexpression, or with a coordinative effect of both scopolamine-producing plant species results in significant en- pmt and h6h transgenes? Northern blot analysis showed that line hancement of scopolamine accumulation in cultured hairy root H11 had a relatively higher h6h transcript level than T3 and T10, lines. Overexpression of multiple biosynthetic genes or transcrip- ͞ but its scopolamine content was not the highest (184.4 mg liter). tion factors that control the expression of genes in the target Whereas H4 and T3 showed similar h6h transcript levels, the bioengineering pathway is a promising strategy to alter the scopolamine concentration in T3 was significantly higher than accumulation of certain secondary metabolic products. When ͞ that in H4. The best line (T3) produced 411.2 mg liter scopol- the steps of the biosynthetic pathway are elucidated and the ͞ amine, more than nine times that in the wild type (43.7 mg liter) respective genes have been cloned, exact regulation toward and over two times that in the highest scopolamine–producing ͞ enhanced productivity of medicinal natural products will be h6h single-gene transgenic line H11 (184.4 mg liter). To our possible. Metabolic engineering, either alone or in combination knowledge, this is the highest scopolamine content achieved in with traditional breeding, provides a practical means to stimulate planta developed through genetic engineering to date. valuable secondary metabolites production. It is concluded that, as in the case of transgenics that include The current study provides an effective approach for commer- both pmt and h6h in H. niger, the pulling force from the cially large-scale production of scopolamine by using the hairy root metabolically downstream enzyme (the faucet enzyme) H6H systems as bioreactors. This work also sheds light on how to plays a more important role in stimulating scopolamine accu- effectively increase the end products of secondary metabolic path- mulation whereas the proportional functioning of the upstream ways by appropriate genetic engineering strategies. enzyme PMT is well coordinated. The expression of pmt and h6h in H. niger is similarly regulated at the transcriptional level. The We thank Professor T. Hashimoto (Nara Institute of Science and first specific precursor of the tropane alkaloid pathway is N- Technology, Japan) for supplying pTVPMT carrying the tobacco pmt methylputrescine, whose formation from putrescine is catalyzed cDNA to make pBMI. Dr. Ping Zhang’s (Fudan University, China) by PMT, which seems to be flux-limiting and hence represents assistance in HPLC and GLC analyses is also acknowledged. This the first committed step in tropane alkaloid biosynthesis (1, 7). research is supported by the China National ‘‘863’’ High-Tech program Because h6h encodes an enzyme catalyzing the final two steps and the China͞United Kingdom Science and Technology Collaborative from hyoscyamine to scopolamine that is not inhibited by end Fund.

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