Production of Multiple triterpenes Including Taraxasterol in Transgenic Tobacco Overexpressing Multifunctional Oxidosqualene Cyclase (TcOSC1) of Taraxacum Coreanum

Taraxasterol and ψ-taraxasterol are pentacyclic triterpenoids, they are commonly found in the family Asteraceae. These two compounds are useful candidates for pharmacologically active triterpenes in dandelion. A multifunctional oxidosqualene cyclase (TcOSC1) of Taraxacum coreanum catalyzes the cyclization of 2,3-oxidosqualene into various triterpenes (taraxasterol, ψ-taraxasterol, δ-amyrin, β-amyrin, α-amyrin, and dammarendiol-II). Here, we established the production of taraxasterol, ψ-taraxasterol, δ-amyrin, β-amyrin, and α-amyrin in transgenic tobacco overexpressing TcOSC1 gene of T. coreanum. Transgenic tobacco overexpressing TcOSC1 gene was induced via Agrobacterium-mediated transformation, and four transgenic lines were selected. Introduction and expression of transgenic genes in tobacco was conrmed by genomic PCR, and qRT-PCR, respectively. All the four transgenic lines of tobacco produced obviously the ve triterpenes, namely taraxasterol, ψ-taraxasterol, δ-amyrin, β-amyrin, and α-amyrin. Organ-specic triterpene accumulation occurred in transgenic tobacco plants (leaf > stem > root). The amount of taraxasterol was found the highest among the ve triterpenes produced in tobacco. The total amount of triterpenes in transgenic line 3 (Tr3) exhibiting the highest amount of triterpenes that was 598 µg g − 1 dry weight. Production of phytosterols (β-sitosterol, campesterol, and stigmasterol) was reduced in transgenic tobacco compared to those of wild-type control. Conclusively, we successfully established the production of taraxasterol and ψ-taraxasterol triterpenes in transgenic tobacco, which can be applied to the cost-effective production for the utilization and as a source of pharmacologically active materials. Bar: Basta resistance gene; GC/MS: Gas chromatography/mass spectrometry, OSC: 2, 3-Oxidosqualene cyclase, TcOSC1: Taraxacum coreanum multifunctional triterpene synthase; PCR: Polymerase chain reaction. qRT-PCR: quantitative real-time polymerase chain reaction

Genetic engineering of plants is useful technology for producing valuable secondary compounds (Verpoorte and Memelink 2002;Wu and Chappell 2008;Kowalczyk et al. 2020). Production of triterpenes from other plant species or microbial hosts via metabolic engineering might be a promising technology for cost-effective production. In this work, we constructed transgenic tobacco overexpressing a multifunctional triterpene synthase (TcOSC1) of Korean dandelion and analyzed the production of taraxasterol, ψ-taraxasterol, α-amyrin, β-amyrin, and δ-amyrin in transgenic tobacco.

Construction of overexpression vector harboring TcOSC1 gene
The full-length sequence of T. coreanum TcOSC1 gene (GenBank accession number, MK351896) was isolated by PCR using primers 5'-ATG TGG AAG CTG AGA ATA GGT GA-3' and 5'-TTA GGT TTC TTG TTT TGG TAA C -3'. The open reading frame of TcOSC1 was cloned via GATEWAY vector pCR8/GW/TOPO (Invitrogen) and transferred into binary destination vector pB7WG2, placing it under the control of the CaMV 35S promoter ( Fig. 2A). Eventually, the overexpression constructs harboring TcOSC1 was inserted into Agrobacterium tumefaciens LBA4404 strain by the heat shock method.
Production of transgenic tobacco overexpressing TcOSC1 gene by A. tumefaciens-mediated transformation A. tumefaciens harboring the overexpression vectors were cultured for 48 h at 25°C in a yeast exact broth (YEB) medium in a gyratory shaker at 200 rpm. The bacterial suspension (absorbance of approximately 0.6 at 600 nm) was re-suspended in hormone-free MS (Murashige and Skoog 1962) liquid medium after centrifugation for 5 min at 5000 rpm.
Leaf segments from the in vitro grown tobacco plants (Nicotiana tabacum, cv. Xanthi) were immersed in the bacterial suspension containing 20 µM acetosyringone for 10 min. They were blotted on sterile lter paper and then transferred onto a co-culture medium (MS medium with 2 mg/l BA and 0.5 mg/l IBA).
After 3 days of co-culture incubation in darkness, leaf segments were transferred onto a medium with 500 mg/l cefotaxime and kept in a culture room at 24 ± 2°C with a 16 h photoperiod of 50 µmol m − 2 s − 1 light. After 4 weeks of culture, leaf pieces containing early developmental stage of adventitious shoots were transferred onto a selection medium containing 300 mg/l cefotaxime and 20 mg/l bialaphos. Bialaphosresistant shoots (approximately height: 1-2 cm) were transferred to a new selection medium and maintained by consecutive subculture by 3 week intervals on 1/2 MS medium containing 20 mg/l bialaphos. Transgenic plantlets with roots were transferred to the greenhouse after acclimatization. qRT-PCR analysis Total RNAs were isolated from leaves of wild-type and transgenic tobacco using the RNeasy plant mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). The primers used for the TcOSC1 gene were 5'-GAA ACC TAA ACT CCA TTT TAG TGA A -3' and 5'-GCC CGT GAT GAG AGT TTG TA-3'. The primers for tobacco β-actin gene were 5'-GCG ACG GTG TCT CAC ATA CA -3' and 5'-ACG TAC ATG GCG GGA ACA TT-3' that was used as the control to check for RNA quality. qPCR data are presented as the mean ± standard error, and the experiments were repeated for three times.

GC-MS analysis of triterpene and phytosterol production in transgenic tobacco
One hundred mg of milled powder from air-dried samples (leaves, stems, roots) was extracted in 100% methanol by sonication for 30 min at a frequency of 20 kHz at 25°C. The supernatant obtained by centrifugation at 15,000 rpm was ltered using a SepPak C-18 cartridge (Waters, Milford, MA, USA). A 10µl aliquot was analyzed by Agilent 7890A gas chromatography system linked to an Agilent 5975C inert MSD system with a Triple-Axis detector, and equipped with a HP-5MS capillary column (30 m×0.25 mm i.d., lm thickness 0.25 µm). The injection port was 250°C, and the column oven temperature program was 150°C for 5 min, followed by a rise to 300°C at a rate of 5°C min − 1 and a hold at 300°C for 20 min. The ow rate of He (as carrier gas) was 1.2 ml min − 1 . The temperature at the interface was 300°C with a split injection (10:1). The ionization chamber was set at 250°C, and electron impact (EI) ionization operated at 70 eV. Identi cation of all GC chromatogram peaks were made by comparison of their retention times and mass fraction patterns with those of authentic standards. The α-amyrin and β-amyrin standards were obtained from Sigma − Aldrich Inc. (Saint Louis, MO, USA), and δ-amyrin, ψ-taraxasterol, and taraxasterol were obtained from Toronto Research Chemicals Inc. (North York, Canada).

Results
Production of transgenic tobacco overexpressing TcOSC1 gene Transgenic tobacco plants overexpressing TcOSC1 gene (MK351896.1) under the control of the CaMV 35S promoter were constructed ( Fig. 2A). Four independent transgenic lines were nally selected. Integration of T-DNA into the tobacco genome was con rmed by PCR of genomic DNA. All the four transgenic lines showed the expected PCR products for the BAR and TcOSC1 genes (Fig. 2B, C). No PCR signal was detected in wild-type tobacco (Fig. 2B,C).
cDNAs extracted from leaves of wild-type and transgenic plants were subjected to qPCR analysis to detect the expression of TcOSC1 gene. All four transgenic lines showed a clear accumulation of TcOSC1 mRNA (Fig. 3). In particular, Tr3 line exhibited the highest transcription of TcOSC1 compared to the other lines (Fig. 3).

Triterpene analysis in the lines of transgenic tobacco overexpressing TcOSC1
To analyze the triterpene production driven by overexpression of TcOSC1 in transgenic tobacco, the third leaves from the top were analyzed by GC/MS. All the four transgenic lines (Tr1, Tr2, Tr3, and Tr4) showed several new triterpene products (taraxasterol, ψ-taraxasterol, α-amyrin, β-amyrin, and δ-amyrin) at the retention times between 35 min to 38 min compared to the wild-type control (Fig. 5B-E). In the wild-type plants, no signal for the ve triterpenes was detected except the phytosterols belonging to the triterpene family (Fig. 5A). Identi cation of triterpenes was con rmed by matching the retention times of triterpene standards (Fig. 5F) with those in transgenic tobacco leaf extracts (Fig. 5B-E). The MS spectra of the ve triterpene products revealed that the fragmentation patterns of each triterpenes product in transgenic tobacco were completely matched to the MS spectra of authentic ve triterpene standards ( Supplementary Fig. 1). In all transgenic tobacco plants, the total amount of triterpenes in the leaves was 401.3 to 598.7 µg g − 1 DW (Fig. 6A). The content of taraxasterol showed the highest amount among other triterpenes (Fig. 6A). The order of triterpene accumulation was taraxasterol > ψ-taraxasterol > β-amyrin > αamyrin > δ-amyrin in all four transgenic lines (Fig. 6A).

Triterpene analysis in different parts of transgenic tobacco overexpressing TcOSC1
Accumulation of triterpenes in different portion (leaf, stem, and root) of transgenic tobacco (line 3) was analyzed. Triterpene accumulation occurred in an organ-speci c manner (leaves > stems > roots) (Fig. 4). Leaves contained the highest amount of triterpenes compared to other roots and stems. In roots, taraxasterol, ψ-taraxasterol, β-amyrin occurred at the detectable amount, but α-amyrin and δ-amyrin existed traceable amount (Fig. 4).

Phytosterol analysis in the lines of transgenic tobacco overexpressing TcOSC1
The production of new triterpenes in transgenic tobacco resulted in the negative relationship in the accumulation of phytosterols (Figs. 5A,6B). GC chromatogram revealed that the peak heights of three phytosterols (β-sitosterol, campesterol, and stigmasterol) in leaves of wild-type tobacco (Fig. 5A) were conspicuously higher than those of transgenic lines (Fig. 5B-E). In all four transgenic lines, accumulation of phytosterols (β-sitosterol, campesterol, and stigmasterol) was clearly decreased compared to those of wild-type (Fig. 6B). However, there was no apparent phenotypic difference in the general plant statures among wild-type and transgenic plants, and all transgenic lines of tobacco successfully set seeds.

Discussion
Functional characterization of OSC genes has been mainly achieved by the expression of the gene in S. cerevisiae. Some OSCs are identi ed as multifunctional enzymes with mixed end-products. However, the mechanism of generating mixed-products is unclear, it is probably due to the deprotonation of numerous sites during the cyclization process (Thimmappa et al. 2014). T. coreanum TcOSC1 enzyme was characterized as multifunctional triterpene synthase by heterologous expression in yeast (Han et al. 2019). The enzyme can be able to produce 6 triterpenes, taraxasterol and ψ-taraxasterol as major compounds, and α-amyrin, β-amyrin, δ-amyrin, and dammarenediol-II as minor ones (Han et al. 2019). In the present work, we constructed the transgenic tobacco overexpressing T. coreanum TcOSC1 and observed the production of triterpenes in transgenic tobacco plants. The transgenic tobacco overexpressing TcOSC1 produced ve triterpenes taraxasterol; Ψ-taraxasterol; α-, β-, and δ-amyrin, but not dammarenediol-II. Taraxasterol; Ψ-taraxasterol; α-, β-, and δ-amyrin are pentacyclic triterpene, but dammarenediol-II is tetracyclic triterpene. It is unclear why dammarenediol-II is not detected in transgenic tobacco overexpressing TcOSC1. In the yeast overexpressing TcOSC1, production of dammarenediol-II is the one of minor production compared to other triterpenes. Undoubtedly, no detection of dammarenediol-II in the transgenic tobacco overexpressing TcOSC1 might result from below the detection limits in the samples.
In the transgenic yeast expressing TcOSC1, taraxasterol was the major compound among other 6 triterpene products (Han et al. 2019). Similarly, transgenic tobacco overexpressing TcOSC1 showed the production taraxasterol as a major triterpene compound. Thus cyclization pattern of oxidosqualene into triterpenes is similar although expression of TcOSC1 occurs in the different host organism.
Triterpene production in transgenic tobacco overexpressing TcOSC1 showed the organ-speci c pattern. The accumulation of triterpenes in roots was shallow compared to leaf and stem. In transgenic tobacco overexpressing P. ginseng triterpene synthase (PgDDS, dammarnediol-II synthase), the order of dammarenediol-II accumulation in organs was roots> stems> leaves> ower buds (Han et al. 2015). In transgenic tobacco overexpressing P. ginseng PgDDS (dammarnediol-II synthase) and CYP716A47 (protopanaxadiol synthase), the proportion of protopanaxadiol to dammarenediol-II varied among the organs, and the accumulation of dammarenediol-II and protopanaxadiol in the transgenic line occurred in an organ-speci c manner (roots>leaves>stems>petioles) (Chun et al. 2015). Interestingly, different pattern of organ-speci c triterpene accumulation occurred in transgenic tobacco overexpressing TcOSC1, although the TcOSC1 was expressed by constitutive CaMV35S promoter. These results indicate that the organ-speci c accumulation of triterpenes is different depended on the produced triterpene-type. The organ-speci c accumulation of triterpenes might be either caused by the different ux of triterpene precursors in tobacco plants and/or the different activity of triterpene carriers in tobacco. It is known that translocation or transport of terpenoids is involved in plant ATP-binding cassette transporters (ABC) transporters (Theodoulou 2000;Hwang et al. 2016). Within the plant, ABC transporter family, pleiotropic drug resistance (PDR) transporters play essential functions, such as in hormone transport or defense against biotic and abiotic stresses (Nuruzzaman et al. 2014). NtPDR1 transports the compounds involved in both diterpenes and sesquiterpenes in plants (Pierman et al. 2017). AaPDR3 is involved in sesquiterpene transport (Fu et al. 2017). Triterpene accumulation in different tissues of transgenic tobacco might be affected by the intrinsic activity of ABC transporters or other types of triterpene carriers in tobacco.

Reduced phytosterols in transgenic tobacco producing multiple triterpenes
In this work, new triterpene production in transgenic tobacco plants resulted in decreased accumulation of phytosterols (β-sitosterol, campesterol, and stigmasterol). A similar reduction of phytosterols occurred in transgenic tobacco producing dammarenediol-II (Han et al. 2014). The reduced phytosterol accumulation in transgenic tobacco might be caused by competition for precursors, as both triterpenes and phytosterols in plants are synthesized from the cyclization of their common precursor, 2,3oxidosqualene (Thimmappa et al. 2014). Thus, cyclization of 2,3-oxidosqualene for the production of multiple pentacyclic triterpenes in transgenic tobacco overexpressing TcOSC1 can restrain the metabolic ow of phytosterol biosynthesis, which might result in the reduced sterol accumulation observed in transgenic tobacco.