Particle Bombardment-mediated Transformation Research Articles

Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3′ splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.

Pretreatment and posttreatment in the biolistic transformation of tea plant (Camellia sinensis) somatic embryos.

The tea plant (Camellia sinensis) contains various metabolic substances, including catechins and caffeine, for which genetic transformation techniques are essential for investigating the associated metabolic pathways. In this study, we sought to optimize the conditions and culture process for particle bombardment-mediated transformation of tea plant somatic embryos. We describe somatic embryo pretreatment for effective transient transformation in biolistic bombardment and the posttreatment conditions of somatic embryos for accelerating differentiation after bombardment. For the purpose of transformation, we used the somatic embryos of C. sinensis var. assamica 'Tingamira normal,' which were cultured in Murashige and Skoog (MS) medium containing 2 mg l-1 indole-3-butyric acid (IBA) and 4 mg l-1 6-benzyladenine (BA) at 25°C ±2°C under a 16-h photoperiod. With respect to the optimization of particle bombardment conditions for tea somatic embryos, we examined the effects of different Au colloid particle diameters and bombardment pressures, and accordingly established bombardment with 1.0-µm-diameter DNA-coated Au colloid at 1,100 psi as optimal conditions for introducing DNA for the transient expression of GUS. Additionally, we found that transplantation of tea somatic embryos from IBA/BA medium to a hormone-free medium prior to bombardment and incubation in the dark post-bombardment increased the frequency of secondary embryo production. Furthermore, osmotic treatment by culturing the somatic embryos in medium supplemented with 0.4 M mannitol improved transient transformation efficiency. After transformation, the culture of somatic embryos on filter papers or Kimwipes soaked in MS medium facilitated rapid and effective development of the somatic embryos.

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger.

A full-length cDNA of phyA gene of Aspergillus niger, encoding phytase enzyme, was cloned and expressed in E. coli BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33U/mL. The phyA gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T0 transgenic plants and their stable inheritance in the T1 progenies. Three transgenic events expressing detectable level of A. niger phytase were characterized by western blot analysis. Phytase activity of 463.158U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5- to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6- to 5-fold higher inorganic phosphorus content.

Efficient particle bombardment-mediated transformation of Cuban soybean (INCASoy-36) using glyphosate as a selective agent

Soybean is highly affected by weeds in tropical countries, causing significant losses in yields. Transgenic herbicide resistant soybeans have been produced in a limited number of varieties and parental lines. This study was conducted to obtain glyphosate herbicide resistant transgenic soybean plants through particle bombardment of embryonic axes in a Cuban variety. Shoot regeneration in 25 mg/L of glyphosate occurred within a short period and plantlets developed roots in a medium without selection pressure, which favored the in vitro growth of plants at a transformation frequency of 3.1–6.0 %. Expression and integration of the cp4epsps gene was confirmed in the progeny by an immune-detection assay, PCR and Southern blot. All greenhouse evaluated transgenic soybean lines (T1) displayed tolerance to 1.25 Kg/ha of glyphosate. Growth and seed development of transformed plants was similar to untransformed plants. The regeneration procedure using embryonic axes combined with the efficient selection of shoots in glyphosate enabled the production of transgenic plants of this Cuban genotype, showing high tolerance to the herbicide, good efficiency and reproducibility.

Production and immunogenicity of Actinobacillus pleuropneumoniae ApxIIA protein in transgenic rice callus

Actinobacillus pleuropneumoniae is a major etiological agent that is responsible for swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA is one of the virulence factors in A. pleuropneumoniae and has been considered as a candidate for developing a vaccine against the bacterial infection. A gene encoding an ApxIIA fragment (amino acids 439–801) was modified based on a plant-optimized codon and constructed into a plant expression vector under the control of a promoter and the 3′ UTR of the rice amylase 3D gene. The plant expression vector was introduced into rice embryogenic callus (Oryza sativa L. cv. Dongjin) via particle bombardment-mediated transformation. The integration and transcription of the ApxIIA439–801 gene were confirmed by using genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis of ApxIIA439–801 antigen protein in transgenic rice callus was confirmed by western blot analysis. The concentration of antigen protein in lyophilized samples of transgenic rice callus was 250 μg/g. Immunizing mice with protein extracts from transgenic plants intranasally elicited secretory IgA. These results demonstrate the feasibility of using a transgenic plant to elicit immune responses against A. pleuropneumoniae.

AN EFFICIENT RECOVERY OF TRANSGENIC PLANTS FROM A TROPICAL INDIAN MAIZE INBRED LINE

Tropical Indian maize inbreds are known for their recalcitrance in tissue culture which is a prerequisite for development of transgenic lines from such cultivars. In the present study, particle bombardment-mediated transformation of a tropical Indian maize inbred line, UMI29 was attempted. Parameters that infulence transforamation efficiency such as size of embryo (1.5 mm and 2.0 mm), micro-carrier flying distance (6 cm and 9 cm) and post-bombardment resting period (2, 7 and 10 days) on transient and stable expression of transgene were investigated. The greatest transformation efficiency of 2.67% was obtained using immature embryos of 1.5 mm at 6 cm of microcarrier flying distance with a resting period of 2 days. Stable inheritance of transgenes, viz., gusA and bar was confirmed in T1 lines.

Dengue virus E glycoprotein production in transgenic rice callus.

Dengue is a disease caused by dengue virus and represents the most important arthropod-borne viral disease in humans. Dengue virus enters host cells via binding of envelope glycoprotein (E) to a receptor. In this study, plant expression vectors containing native and synthetic glycoprotein E genes (sE) modified based on plant-optimized codon usage and fused with an ER retention signal were constructed under control of the rice amylase 3D promoter expression system. Plant expression vectors were introduced into rice callus (Oryza sativa L. cv. Dongin) via particle bombardment-mediated transformation. The integration and expression of target genes were confirmed in the transgenic callus by genomic DNA PCR and Northern blot analyses, respectively. The plant-codon optimized sE gene with an ER retention signal showed high protein production levels based on Western blot analysis of approximately 18.5ug/g dried calli weight by immunoblot-based densitometric analysis. These results suggest that the plant-codon optimized sE gene with an ER retention signal was highly produced in the transgenic rice callus.

Snowdrop lectin (Galanthus nivalis agglutinin: GNA) 유전자 도입에 의한 벼멸구 저항성 형질전환 벼 개발

Transgenic rice plants with increased resistance to rice brown planthopper (Nilaparvata lugens Stal) were generated by particle bombardment-mediated transformation of plants with a gene encoding snowdrop lectin (Galanthus nivalis agglutinin; GNA) under control of the rice Rubisco small subunit (rbcS) promoter.. A large number of transgenic rice plants containing the GNA gene were generated. The integration, expression, and inheritance of this gene in the R1 and R2 generations were demonstrated by Southern and western blot analyses. The plants contained one to five copies of the transgene. The GNA protein comprised approximately 0.01-2.0% of total soluble protein in the R1 and R2 transgenic plants. Insect bioassays and feeding studies showed that the GNA protein expressed in the R2 transgenic rice plants reduced the survival of brown planthoppers. The introduction of GNA into rice plants therefore can help to control insect pests.

Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli.

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE–Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE–Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE–Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE–Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer’s patches from immunized mice. These results indicated that oral immunization of plant-produced COE–Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice.

Expression of a consensus dengue virus envelope protein domain III in transgenic callus of rice

An effective dengue vaccine should elicit immune responses against all four different dengue virus serotypes. This study optimized the codon usage of a gene encoding consensus dengue virus envelope protein domain III (cEDIII) with cross-neutralizing activity against four dengue virus serotypes for plant expression. Then, a plant expression vector was constructed with this gene under the control of the rice amylase 3D promoter (RAmy3D), which is a strong inducible promoter under sugar starvation conditions. The synthetic cEDIII gene was fused with the RAmy3D signal peptide and ER retention signal, SEKDEL, and was introduced into rice callus by particle bombardment-mediated transformation. The integration and expression of cEDIII gene in transgenic rice callus was confirmed by genomic DNA PCR amplification, Northern blot analysis, and western blot analysis, respectively. Densitometric analysis determined that the highest expression level of the cEDIII protein in lyophilized rice callus was approximately 0.45 mg g−1. These results suggest that it is feasible to use transgenic rice callus to produce the consensus dengue virus envelop protein domain III for edible vaccine purposes.

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.

The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP) based detection procedures.

An efficient protocol for particle bombardment-mediated transformation of Centella asiatica callus

In this study, we report an optimization of particle bombardment transformation system for Centella asiatica callus. A total of eight parameters affecting the genetic transformation system were optimized using the synthetic green fluorescent protein (sGFP) as a reporter driven by the CaMV 35S promoter. The results indicated that DNA delivery conditions of 9-cm target distance, 1,100 psi helium pressure, 1.0 μm gold particles size, 27 mmHg chamber vacuum pressure, 2 times number of bombardment, spermidine as precipitation agent, 60 h post-bombardment incubation time and 2 μg plasmid DNA concentration were optimal for C. asiatica callus transformation. The expression of sGFP was monitored using fluorescence microscope and further confirmed using RT-PCR. This optimized genetic transformation system is applicable for rapid transient gene analysis and transgenic C. asiatica production.

Establishment of an efficient in vitro culture and particle bombardment-mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop

Plants belonging to the genus Miscanthus are considered promising bioenergy crops. Here, we report the establishment of tissue culture system through particle bombardment-mediated transformation in Miscanthus sinensis Anderss. Callus was induced efficiently from mature seeds in a medium containing a combination of a relatively high level of 2,4-dichlorophenoxyacetic acid (5 mg L−1) and a relatively low level of 6-benzyladenine (BA) (0.01 mg L−1). Callus induction potential of 18 accessions of M. sinensis, which were collected from various sites in Japan, was compared. Significant correlation was detected between compact (embryogenic) callus induction frequency and average annual air temperature in collection sites. An accession from Tanegashima Island showed the highest production of compact callus. We found that a higher level of BA causes callus browning; the 2 mg L−1 BA is the optimal concentration for regeneration. Both compact and friable calli were suitable for particle bombardment transformation. Through selection under the presence of 50 mg L−1 hygromycin for 3 weeks and further selection under the presence of 150 mg L−1 for 1 month, hygromycin-resistant calli survived, of which 72.2% had been entirely transformed. Plants were regenerated from calli in the presence of hygromycin; transcripts of the hpt and gfp genes, which were cobombarded to the calli, were detected in the regenerated plants. This is the first report on the establishment of the in vitro culture of M. sinensis using mature seeds, the variation of callus formation among accessions collected from various sites in Japan, and particle bombardment-mediated transformation in the genus Miscanthus.

A practical protocol for particle bombardment-mediated transformation of Phyllostachys bamboo suspension cells

We developed a particle bombardment-mediated transformation protocol in Phyllostachys bamboo by optimizing the growth efficiency of a target cell culture system. Under the optimal condition, i.e. Murashige and Skoog medium containing 680 mg l−1 KH2PO4 and 10 μM Picloram, bamboo suspension cells actively proliferated at ca. 80 ml sedimented cell volume per 100 ml medium in 2 weeks. Log phased cells, i.e. 8 to 13-day-old suspension cells, which showed synchronous cell divisions with uniform morphology, were selected for the bombardment. We found that a target distance, 6 cm was much better for the transient gene expression (222 GUS-positive cells/dish/shot in average) than that of 9 cm (38 GUS-positive cells/dish/shot in average) in the target cells. When the bombardment was carried out using lag phased cells, e.g. 5-d-old cells, no or less GUS-positive cells could be seen. A high generation of stable transgenic bamboo cells was achieved with constructs expressing hygromycin phosphotransferase gene and enhanced fluorescent protein genes namely AcGFP1 and mCherry.

High-level production of bioactive heterodimeric protein human interleukin-12 in rice

Human interleukin-12 (hIL-12), a heterodimeric cytokine comprised of p35 and p40 subunits, was generated previously via tobacco cell suspension culture at a maximum concentration of only 175 μg/L. In order to improve the productivity, the rice α-amylase 3D promoter (Ramy3D) was utilized in order to express recombinant protein at high levels under sucrose starvation conditions. The hIL-12 gene was cloned into the rice expression vector under the control of the Ramy3D promoter. Transgenic rice calli were prepared via particle bombardment-mediated transformation and were screened for rhIL-12p70 expression using ELISA. Western blot and bioassay results confirmed that rhIL-12p70 was expressed and secreted into the culture medium as a functionally active heterodimeric form, and the productivity of rhIL-12p70 was estimated as 12.7 mg/L, which is approximately 73-fold higher than was observed with the tobacco cell culture system [Kwon TH, Seo JE, Kim J, Lee JH, Jang YS, Yang MS. Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture. Biotechnol Bioeng 2003;81:870–5]. However, the secreted rhIL-12p70 proved unstable in the medium, and degraded rapidly after day 13. In order to stabilize the secreted rhIL-12p70, three stabilizing polymers were tested (polyethylene glycol, polyvinylpyrrolidone, and gelatin). Gelatin was the most effective in stabilizing the secreted rhIL-12p70. After the addition of 2% (w/v) gelatin, the maximum rhIL-12p70 concentration reached 31.0 mg/L, representing a 2.5-fold increase over the control.

Expression and Immunogenicity of Enterotoxigenic Escherichia coli Heat-Labile Toxin B Subunit in Transgenic Rice Callus

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

Development of a Particle Bombardment-Mediated Transient Transformation System for Taxus spp. Cells in Culture

In developing and developed nations, plant cell culture systems are used to supply desirable compounds in lieu of chemical synthesis or natural extraction. When plant cell culture systems are unable to meet commercial demand, metabolic engineering offers a method to increase yields. However, to benefit from metabolic engineering approaches, effective transient transformation methods are required to rapidly identify and characterize key regulatory genes before intensive, time-consuming stable transformation efforts can proceed. This paper describes a particle bombardment-mediated transient transformation system for Taxus spp. in cell culture. Optimal parameters were established for the T. cuspidata cell line P991 and the T. canadensis cell line CO93D, resulting in reliable, efficient, transient expression of the firefly luciferase gene under control of the constitutive CaMV 35S promoter. Multiple bombardments and larger gold microcarriers (1.6 vs 1.0 microm in diameter) were particularly effective in increasing luciferase activity and in reducing variation among replicates. This particle bombardment-mediated transformation system was also shown to be capable of transiently expressing the DsRed and beta-glucuronidase reporter genes under the control of the maize ubiquitin and CaMV 35S promoters, respectively. With the ability to transiently transform Taxus spp. cell cultures using a variety of promoters and reporters, characterization of genes related to paclitaxel accumulation in culture can now proceed.

Particle bombardment-mediated transformation of barley with an Arabidopsis NDPK2 cDNA

As barley is recalcitrant to transformation with current methods, a new improved system is required to apply genetic transformation in breeding programs. In a previous study, we defined optimal conditions for plant regeneration (PR) using mature embryos. This study was conducted to establish an improved transformation system employing the previously adjusted regeneration conditions. Optimal DNA delivery condition for the embryogenic calli developed from mature embryos was bombardment pressure of 1,100 psi at the target distance of 6 cm. The feasibility of the regeneration and DNA delivery conditions was confirmed by developing transgenic barley plants transformed with the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) cDNA via particle bombardment of embryogenic calli from mature embryos. Stable integration of AtNDPK2 cDNA into barley genome was confirmed by PCR and Southern blot analysis of AtNDPK2 transgene. Transgenic plants showed about 10% reduction in membrane damage caused by methyl viologen, indicating the expression of AtNDPK2 transgene. The results demonstrated that the transformation system developed in this study employing the PR from mature embryo-derived embryonic callus is applicable in transgenic barley production.

Production of transgenic Hypericum perforatum plants via particle bombardment-mediated transformation of novel organogenic cell suspension cultures

We have developed particle bombardment-mediated transformation procedure for Hypericum perforatum L. (St. John's wort), an important medicinal species that remains highly recalcitrant towards Agrobacterium-mediated transformation. Among the major transformation techniques evaluated in the present study ( Agrobacterium tumefaciens-, A. rhizogenes- and biolistics-mediated), particle-bombardment-mediated gene transfer was found to be the most successful one. GUS positive cells were obtained from organogenic nodules bombarded with the plasmid vector pCAMBIA1301 encoding an intron-containing β-glucuronidase ( gusA) and hygromycin phosphotransferase ( hpt) genes. After 3 months of continuous selection of bombarded nodules with 20 mg l −1 hygromycin, transgenic hygromycin-resistant callus cultures and subsequently transgenic plants were produced. PCR analysis of DNA isolated from GUS positive plants showed the presence of both gusA and hpt genes. Southern blot analysis confirmed the transgene integration and revealed diverse copy numbers and insertion sites. The data presented here demonstrate for the first time H. perforatum can be efficiently transformed via particle bombardment of organogenic cell suspension. Our results open the possibility of using particle bombardment-mediated transformation to elucidate biosynthetic pathways and to improve secondary metabolite production in H. perforatum.