Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata

Macleaya cordata produces a variety of benzylisoquinoline alkaloids (BIAs), such as sanguinarine, protopine, and berberine, which are potential anticancer drugs and natural growth promoters. The genes encoding the berberine bridge enzyme (BBE) were isolated from M. cordata and Papaver somniferum, and then the two genes were overexpressed in M. cordata. Through liquid chromatography with triple-quadrupole mass spectrometry analysis, it was determined that McBBE-OX caused higher levels of (S)-norcoclaurine, (S)-coclaurine, (S)-N-cis-methylcoclaurine, (S)-reticuline, (S)-tetrahydrocolumbamine, (S)-tetrahydroberberine, (S)-cheilanthifoline, and (S)-scoulerine than PsBBE-OX, empty vector or control treatments. qRT-PCR analysis demonstrated that the introduced genes in the transgenic lines were all highly expressed. However, the levels of sanguinarine (SAN) and chelerythrine (CHE) in all the transgenic lines were slightly lower than those in the wild-type lines, possibly because the overexpression of McBBE causes feedback-inhibition. This is the first report on the overexpression of potential key genes in M. cordata, and the findings are important for the design of metabolic engineering strategies that target BIAs biosynthesis.

In recent years, many studies have engineered microbes and plants to produce BIAs [11][12][13][14][15][16][17] . Microbial-based production of BIAs requires the introduction of some heterologous plant genes and numerous genetic modifications to increase productivity. However, the advances in microbial-based methods have significantly shortened production time and although microbial methods are not affected by the environment, the production of BIAs still needs an external supply of precursors 15 . In a recent study, researchers combined different enzymes from various species and, through pathway and strain optimization, accomplished full opiate biosynthesis in yeast 16 . This work solved the problem of BIA production needing precursors, but the synthesis step was too long, and the final product concentration was hardly sufficient for industrial production. For this reason, many studies have used transgenic methods to increase production, especially overexpression of potential key enzymes. The most famous examples are the strategies used to increase artemisinin yield in Artemisia annua. Although semi-synthesis of artemisinin was successfully achieved in yeast 18 , many groups still enhance the yield of artemisinin by transgenic methods 19 . In addition, overexpression of synthesis genes has also been used to increase levels of ginsenoside, phytosterols, scopolamine and terpenoid indole alkaloids [20][21][22] . In addition, in a previous study, the content of BIAs in California poppy was increased through overexpression of the P. somniferum BBE gene (PsBBE), and this experiment indicated that BBE is a potential key enzyme to enhance the content of BIAs 12 .
Macleaya cordata is a perennial herb that belongs to the Papaveraceae family and is used for the commercial production of BIAs, including SAN, PRO, BBR; the herb contains many trace BIAs that have beneficial medicinal properties, such as (S)-reticuline and (S)-scoulerine. Recently, the whole genome of M. cordata has been de novo sequenced and validated for SAN biosynthesis 9 . In addition, a transformation and regeneration system to produce transgenic M. cordata has been constructed 23 . These genomics data and transgenic methods provide the opportunity for enhanced production of BIAs through plant metabolic engineering. In this study, we overexpressed BBE genes isolated from M. cordata and P. somniferum (McBBE and PsBBE) in M. cordata. Then, we investigated whether the overexpression of McBBE or PsBBE in M. cordata could affect the biosynthesis of BIAs. Finally, we investigated the metabolite profiles through ESI/QQQ MS analysis and detected the expression levels of 11 genes involved in the synthesis of SAN and CHE. To our knowledge, this is the first study using overexpression of BBE in transgenic M. cordata with increased alkaloid production.

Establishment of Transgenic Plants with the McBBE/PsBBE
Gene. We subjected leaf and stem explants to vacuum infiltration with Agrobacterium tumefaciens harbouring McBBE-or PsBBE-and GUS (β-glucuronidase)-overexpression vectors, respectively for 10 min, and then the explants were placed into co-cultivation medium (100 μM acetosyringone) for 3 days. Then, the explants were transferred to selection medium (75 mg/l kanamycin and 400 mg/l Timentin) 23 . To determine whether the transgenic lines were established successfully, GUS (β-glucuronidase) histochemical analysis was performed. The GUS histochemical results showed the presence of blue colour in the McBBE-OX and PsBBE-OX plants. In contrast, the wild-type lines did not show GUS activity ( Fig. 2A-D). Polymerase chain reaction was performed using GUS, nptII (kanamycin resistance gene) and PsBBE primers for the transgenic lines, one wild type plant (negative control) and pCMBIA2301, pCM-BIA2301 + McBBE and pCMBIA2301 + PsBBE plasmid vectors (positive control). The results showed that the reporter gene GUS and the nptII gene were detected in all three vectors and in all the transgenic lines ( Fig. 2E-G). Only the 2301 + PsBBE vector and the PsBBE-OX line displayed PsBBE-specific fragments. No PCR amplification product was detected for the wild-type plants.  2000bp-

Discussion
One method to enhance target compounds in a secondary metabolic pathway is to overexpress the potential key enzyme at the genetic level. The BBE gene is responsible for a rate-limiting step in the biosynthetic pathway of BIA and affects the final production of BIA 12 . In the present study, the two BBE genes from M. cordata and P. somniferum were integrated into separate plant lines to analyse their impact on the BIA metabolic network.  24 . These unintended consequences often appear when engineering single enzymes to increase flux. For example, overexpression of phytoene synthase in tomato increases lycopene content but downregulates the gibberellin pathway, ultimately resulting in dwarf plants. However, overexpression of BBE in E. californica hairy root culture increases total benzophenanthridines and slightly increases SAN content 12 , indicating that there are difference between species even when the same metabolic pathways are disturbed. On the other hand, the gene expression level of McTDC was significantly decreased in McBBE-OX lines, while the content of (S)-tetrahydroberberine in the McBBE group was the highest among all the groups (Fig. 3). Similarly, there were no differences between transgenic plants and wild-type plants in McCFS and McTNMT mRNA expression, but (S)-cheilanthifoline and (S)-tetrahydroberberine levels were significantly higher in McBBE-OX plants than in WT plants. The possible reason is that the accumulation of a large number of synthetic precursors in the McBBE-OX line resulted in higher levels of downstream products and caused a corresponding impact on metabolites. Through analysis of gene expression, we found that 9 genes were significantly influenced in transgenic plants, which affected the metabolite profiles (Figs 3 and 4). Another unexpected observation in this study was that transformation with PsBBE did not increase any BIAs. However, the overexpression of PsBBE in Eschscholzia californica significantly increases the levels of the end products 12 . Because the focus of this study was to use overexpression of McBBE to enhance BIA content, we will study this phenomenon in the PsBBE group in a future study.
Previous researchers have succeeded in achieving high production of BIAs by overexpressing the key enzyme in Papaveraceae plants [12][13][14] . However, it is difficult to cultivate many Papaveraceae plants; for example, the cultivation of poppy is regulated by the government. In contrast, M. cordata is free of addictive compounds and has a good prospect of cultivation 9 . The alkaloid profile in transgenic M. cordata was clearly made more diverse through overexpression of the key step gene and introduction of the exogenous gene. This article is the first to report the successful overexpression of key genes in M. cordata.

Materials and Methods
cDNA Synthesis and PsBBE synthetic. RNA  Plant Expression Vector Construction. The plant expression vector pCAMBIA2301 (Fig. 5) was purchased from (Miaolingbio, China). McBBE and PsBBE was amplified using the primer listed in Table 1 by  . Asterisks denote the significant changes (* means P < 0.05, ** means P < 0.01, *** means P < 0.005, **** means P < 0.001). EV means empty vector, WT means wild type. Metabolite Extraction and LC-QQQ MS Analysis. The WT and putative transgenic lines were collected after 3-month-old days of culture and ground into a fine powder using liquid nitrogen and then freeze-dried. Then, use ultrasonic extraction for 30 min at room temperature 1 mL of methanol, followed by ultrasonic extraction for 60 min at room temperature to isolate metabolites from 50 mg tissues. After filtration through a 0.22-mm membrane filter (Pall, USA), the solution was quantitative analyzed by LC/triple-quadrupole (QQQ) MS. An ultra-HPLC Agilent 1290 instrument coupled to a QQQ mass spectrometer (6460 A, Agilent) with a BEH C18 column (2.1 3 100 mm, 1.8 mm; Waters, Ireland) was used for the determination of 18 target alkaloids. The quantitative analyzed for metabolite according to our previous research 9 . The LC-QQQ MS data were processed using the Agilent Mass hunter Quantitative Analysis software (B.07.00). For absolute quantification analysis, the method was validated using the mixed standard solution, which was diluted with methanol to produce a at least 5 points and was used to evaluate absolute quantification of the target compound.

NPTII
Gene Expression Analysis by qPCR. Total RNA was isolated from putative transgenic line and wild-type of M. cordata using MiniBEST Plant RNA Extraction Kit (TaKaRa). The quality of RNA was checked by agarose gel electrophoresis and quantity was confirmed by Qubit 2.0. One micrograms of total RNA from each sample was reverse transcribed into cDNA using the PrimeScript RT reagent kit with gDNA eraser (TaKaRa, Dalian, China). The resulting cDNA products were diluted to 100 μL used as templates for subsequent experiments. PCR was performed on an ABI 7300 using FastStart Universal SYBR Green Master (ROX) according to the manufacturer's instructions. The total volume of Quantitative real-time PCR assay was performed in a 20 μL (10 μL of PCR Mix, 0.5 μL of specific primers, 4 μL of cDNA and 5 μL of water). The qPCR cycling conditions were as follows: 95 °C for 15 min; 95 °C, 15 s; 55 °C 15 s; 72 °C 20 s, with 40 cycles. In this method, three replicates were performed in all cases. Relative gene expression was performed using the comparative 2 −ΔΔCT method. We have been proved that only one gene involved in two methylation steps (4OMT and 6OMT) in previous study. Therefore, the primers used in 4OMT and 6OMT are the same. All the primer sequence (Table 3) was checked via blast analysis and the 18S gene as the internal reference.   Statistical Analysis. All the transgenic experiments were carried out in triplicate and each treatment contained 100 explants. All metabolic content and qPCR experiments were analyzed by one-way analysis of variance (ANOVA). The data were statistically analyzed using the GraphPad prism statistical software (version 7.0, USA). Differences between means were determined by analysis of variance with Tukey's test on the level of significance declared at P < 0.05.