a novel udp-glycosyltransferase of rhodiola crenulata

Research ArticleA Novel UDP-Glycosyltransferase of Rhodiola crenulataConverts Tyrosol to Specifically Produce Icariside D2

Zhihua Liao 12 Fei Qiu 2 Junlan Zeng 2 Li Gu 1 BangjunWang2

Xiaozhong Lan 1 andMin Chen 3

1TAAHC-SWUMedicinal Plant Joint RampD Centre Xizang Agricultural and Husbandry College Nyingchi of Tibet 860000 China2Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education)Chongqing Key Laboratory of Plant Ecology and Resources Research in Three Gorges Reservoir RegionSWU-TAAHCMedicinal Plant Joint RampD Centre School of Life Sciences Southwest University Chongqing 400715 China3College of Pharmaceutical Sciences Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Ministry of Education)Southwest University Chongqing 400715 China

Correspondence should be addressed to Xiaozhong Lan lanxiaozhong163com and Min Chen mminchenswueducn

Received 18 October 2017 Accepted 22 May 2018 Published 20 June 2018

Academic Editor Hesham H Ali

Copyright copy 2018 Zhihua Liao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Rhodiola crenulata is a Tibetan native herbal plant belonging to the family of Crassulaceae which produces the pharmaceuticalicariside D2 with the activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells In this study wefunctionally characterized a novel UDP-glycosyltransferase (RcUGT1) that converted tyrosol to specifically produce icariside D2from R crenulata at molecular and biochemical levels RcUGT1 was highly expressed in flowers and roots while the icariside D2content was much higher in stems than that in other organs suggesting the potential translocation of icariside D2 from flowers androots to stems The high production of icariside D2 in stems provided a reasonable suggestion to farmers to harvest stems insteadof roots for icariside D2 production Enzymatic assays of recombinant RcUGT1 indicated that it converted tyrosol to specificallyform icariside D2 with the values of Km 097plusmn010 mM Vmax 286plusmn826 pKatmg Kcat 001552 sminus1 and KcatKm 15955 sminus1 Mminus1Functional identification of RcUGT1 facilitated the icariside D2 production through metabolic engineering in plants or syntheticbiology in microbes

1 Introduction

Rhodiola crenulata a Tibetan native herbal plant belongingto the family of Crassulaceae (Figure 1(a)) has been used asherb and health food to relieve altitude sickness more than1000 years before by the native people in Tibet Plateau [1]This magic plant species grows very slowly due to the harshenvironments in the high mountains more than 5000 meterswhere the ultraviolet radiation is strong the oxygen concen-tration is low the water is precious and the soil is poor [2]Themodern pharmaceutical sciences reveal that the polyphe-nol glucosides such as salidroside in R crenulata are thebiologically active compounds for relieving altitude sicknessreinforcing immunity antifatigue and so forth [3] Recentlyit was found that icariside D2 (the isomer of salidroside)was helpful to inhibit the activity of angiotensin-converting

enzyme [4] and potent to kill leukemia cancer cellsHL-60 [5]Traditionally the perennial roots ofR crenulata are harvestedto produce these pharmaceutical compounds [6] The wildplants of R crenulata are the most important source forproducing these chemicals and unfortunately the artificialcultivation of R crenulata is not available while the demandfor R crenulata has been growing Consequently R crenulatais endangered

It is important to find alternative methods to pro-duce salidroside and icariside D2 Metabolic engineeringas well as synthetic biology might be a promising wayto do this which is dependent on elucidation of theirbiosynthetic pathway Tyrosine decarboxylase (TYDC) isthe first rate-limiting enzyme involved in tyrosol glycosidebiosynthesis TYDC catalyzes tyrosine decarboxylation toform tyramine as a key intermediate for tyrosol glycosides

HindawiBioMed Research InternationalVolume 2018 Article ID 7970590 8 pageshttpsdoiorg10115520187970590

2 BioMed Research International

(a)

OHHO

HO

Tyrosol

O

OHOH OOH

P O P

O

O

O

OH

O

OH

HN

N

O

O

UDP-glucose O

HO OH

OH

HO O

OH

Icariside D2

UDP

P O P

O

O

O

OH

O

OH

HN

NOHO

+

O

HO OH

OH

HO O

OH+

UDP

+

UGT73B6UGT72B14UGT74R1

RcUGT1

Salidroside

P O P

O

O

O

OH

O

OH

HN

NOHO

NH2

O- O-

Na+ Na+

O- O-

O- O-

(b)

Figure 1 Rhodiola crenulata and the biosynthesis of salidroside and its isomer icariside D2 (a)The flowering plant of Rhodiola crenulata (b)UDP-glycosyltransferases (UGTs) convert tyrosol to salidroside and icariside D2

[6] Overexpression of tyrosine decarboxylase enhancedsalidroside and tyrosol accumulation in Rhodiola hairy rootcultures [7] Three specific UDP-glycosyltransferases (UGTs)were reported to convert tyrosol into salidroside (Figure 1(b))in Rhodiola sachalinensis [8] and overexpression of one ofthem (RsUGT73B6) promoted salidroside production [9]RsUGT73B6 was further used to produce salidroside inengineering E coli through the strategy of synthetic biology[10] However the enzyme required for converting tyrosolinto icariside D2 in R crenulata has not been identified Inthis study we characterized a novel UDP-glycosyltransferase(RcUGT1) that converted tyrosol to specifically produceicariside D2 from R crenulata at molecular and biochemicallevels

2 Experimental Procedures

21 Plant Materials and Chemicals Because Rhodiola crenu-lata is an endangered plant species the official approval isnecessary to collect the plant materials The whole plantsof Rhodiola crenulata were collected from the northern areaof Tibet Himalaya Mountain in July of 2011 [6] under theofficial approval of Tibet Sciences and Technology Commit-tee (Lhasa Tibet China) The different organs includingflowers leaves stems and roots were immediately immersedin liquid nitrogen for future use [6] The authentic icarisideD2 was purchased from BioBioPha (Yunnan China) UDP-glucose and salidroside were purchased from Sigma-Aldrich

(Louisiana USA) Tyrosol was purchased from Aladdin(Shanghai China)

22 Gene Cloning and Expression Analysis Total RNAs wereisolated from the different organs of R crenulata usingthe RNAsimple Kit according to the manufacturers pro-tocol (Tiangen Biotech Beijing China) The cDNAs weresynthesized using the FastKing RT Kit (Tiangen BiotechBeijing China) The conserved core sequence of RcUGT1was amplified using a pair of primers dF and dR Basedon the sequence core sequence of RcUGT1 the cDNA endswere cloned using RACE technique The 31015840 RACE and 51015840RACE of RcUGT1 were respectively performed accordingto the manufacturerrsquos protocol (ClonTech CA USA) EachPCR product was subcloned into the pMD-18T vector andsequenced The corresponding fragments of RcUGT1 wereassembled to generate the putative full-length cDNA and thecoding sequence was amplified using a pair of gene-specificprimers pF and pR Real-time quantitative PCR was used toanalyze the tissue profile of RcUGT1 on an iQ5 system (Bio-Rad USA) as described before [6] The primers used in thisstudy were listed in Table 1

23 Bioinformatic Analysis The sequences analysis wasonline performed at the NCBI websites The multiple align-ments of RcUGT1 and other UGTs were aligned using theVector NTI software The phylogenetic tree was constructed

BioMed Research International 3

Table 1 The primers used in this study

Primer Sequence(51015840-31015840) AimdF GGCATCCCGCGGCTNGTNTTYCAYG Cloning of the core sequencedR CGGTCACCAGCTTCTCGTTGWARAAYTGNTC Cloning of the core sequencePrimer 31015840-1 GCCGGACTCGGTCGTGTACGT 31015840 RACEPrimer 31015840-2 GAGAAGTTCCGAGTCAGGAT 31015840 RACEPrimer 51015840-1 GACCATTTCTGCACTCCAACCT 51015840 RACEPrimer 51015840-2 GAACAACGGCCACDTCACCAT 51015840 RACEpF CGCGGATCCATGGATTCTGATTCACGGCCT Vector constructionpR CGCGGTACCCTAGGACAAAGGCACTCTTCT Vector constructionPrimer qF ACTCATTGCGGATGGAAT qPCRPrimer qR CCTTCTCAACCTTCTCCTT qPCR

by MEGA 5 [11] using the Neighbor-joining methods [12]with 10000 repeats

24 Detection of Icariside D2 in Plant Tissues The contentsof icariside D2 were analyzed according to the previouslyreported methods [12] The plant materials were completelydried at 40∘C and then finely powdered The powdered plantmaterials were dipped in distilled water for 15 min and thenautoclaved at 130∘C for 30 min The autoclaved materialswere shaken at 37∘C for 1h at the speed of 200 rpm Thesupernatants were collected after centrifuge (12000 rpm) andthen filtered through 022 120583M film which could be usedfor analysis of icariside D2 using high performance liquidchromatography (HPLC)

25 Purification of the His-Tagged RcUGT1 from EngineeredE coli The coding sequence of RcUGT1 was inserted intoprokaryotic expression vector pQE30 using the restrictionenzymes BamHI and SacI to generate the recombinantvector of pQE30-RcUGT1The recombinant pQE30-RcUGT1vector was introduced into E coli M15 for expression Afterinduction by 05 mM isopropyl 120573-D-1-thiogalactopyranoside(IPTG) for 4 h at 25∘C then the cells were harvested bycentrifugation at 8000 rpm for 5 min About 5 g cell pelletswere resuspended in 50 ml of Buffer A (50 mM sodiumphosphate pH 80 300 mM NaCl and 5 mM imidazole)with lysozyme (1 mgmL) After standing for 30 min onice this cell suspension was lysed by sonication The lysatewas obtained by centrifugation at 12000 rpm for 15 minutesThe recombinant protein could be detected on SDS-PAGEgel with amounts enough for enzymatic assay in the super-natants Then Ni2+-NTA resin was used to bound His-taggedRcUGT1 Next the resin eluted impurity with Buffer B (20mM imidazole 300 mM NaCl 50mM NaH

2PO4 and pH

80) about 50 ml Then the His-tagged RcUGT1 was elutedwith wash buffer (80 mM imidazole 300 mM NaCl 50mMNaH2PO4 and pH 80) Fractions were analyzed by SDS-

PAGE

26 EnzymeAssays of RcUGT1 Thepurified RcUGT1 proteinwas used for enzymatic assay in the 500 120583L reaction bufferscontaining 50 mM Tris-HCl 2 mM tyrosol and 2 mMUDP-glucose [8] 20 120583g of RcUGT1 was used to investigate the

kinetics parameters at pH86The reactionwas performed for30 min at 30∘C and then stopped using 500 120583l methanol in anice box [8] After centrifugation at 15000 g for 10 min at 4∘Cthe reaction products were analyzed byHPLC as the reportedmethods [8] The separation of samples was detected by UVabsorbance at 225 nmThemobile phase contained solvent A(water with 20mM ammonium acetate and 01 formic acid)and solvent B (methanol) The flow rate was 1 mlmin Theboiled RcUGT1 was used as control in enzymatic assay Themass spectrum of icariside D2 was analyzed on the system ofLC-MS 8030 (SHIMADZU Japan) using the Grace AlltechAltima C18 5 120583M column

3 Results and Discussion

31 Molecular Cloning and Sequence Analysis The core frag-ment of RcUGT1was 790 bp in length and the BLASTN resultsuggested that it belonged to the UGT family Based on the790-bp core fragment the 708-bp 31015840 end as well as the 718-bp 51015840 end of RcUGT1 was amplified using RACE techniquerespectivelyThe full-length cDNA of RcUGT1 was generatedthrough assembling the 31015840 end the core fragment and 51015840 endand its physical sequence was further isolated (Figure 2(a))RcUGT1 had a 1425-bp coding sequence encoding a polypep-tide of 474 amino acids with the calculated molecular weightof 53 kDa and isoelectric point of 612 (Figure 2(b)) Thesequence of RcUGT1 was deposited in GenBank (AccessionNumber MH299424) The sequence analysis showed thatRcUGT1 belonged to the glycosyltransferase B-type (GT-B)super family At the amino acid level RcUGT1 exhibited247 to 625 identity to the three reported UGT pro-teins (Figure 3(a)) includingRsUGT72B14 RcUGT74R1 andRsUGT73B6 of R sachalinensis [8] All the three UGTs of Rsachalinensis showed the enzymatic activity of conversion oftyrosol to salidroside [8] According to the results given bysequence comparison (Figure 3(a)) and the phylogenetic tree(Figure 3(b)) RcUGT1 showed relatively close relationshipto RsUGT73B6 The 44-amino-acid C-terminal signaturemotif named PSPG-box (plant secondary product glycosyl-transferase box) was found in RcUGT1 as well as in thethree reported R sachalinensis UGT proteins (Figure 3(a))In the PSPG-box RcUGT1 contained two highly conservedmotifsWAPQ andHCGWNS (Figure 3(a)) present in 95 of


(a)

1 actcgcatccatctcaaccttccacagagaaaaaagatccgacaacctccgacacaaccatagtacatcagaaacagaaaaagtcacagagcg

M SSFLRAIDVMPILHGHAMFPFFFVRLPRSDSD

94 ATGGATTCTGATTCACGGCCTCTACGCGTCTTCTTCTTCCCCTTCATGGCTCACGGCCATCTGATTCCGATGGTCGACATCGCCAGACTCTTCTCTTCG

Q TTIITSHVG P SAQFPLTKISLSTTKSIYNANL

193 CAAGGAGTCCACTCCACCATCATCACCACCCCACTAAACGCCAATTACATCTCCAAAACGACGTCTCTATCCATCAAAACGTTACCGTTTCAAGCTTCA

K PKQLLNAAQFFKFILDPSPLMDVNECGDPLGV

292 AAAGTTGGGCTTCCAGACGGCTGCGAGAATGTCGACATGCTTCCTTCGCCCGATCTCATCTTCAAATTCTTCCAAGCAGCGAATTTACTTCAAAAACCG

F PKELELLNE D RPINFKGASDVSWPFFIDSILC

391 TTCGAGAACCTTCTAGAACTCGAAAAGCCCGATTGCTTAATCTCCGACATCTTCTTCCCCTGGTCAGTCGACTCCGCCGGGAAATTCAACATCCCGAGA

L PKHTKLSEMACMAFFSTGHFV Y FPESDTSVSK

490 CTCGTTTTCCACGGCACGAGCTTCTTCGCCATGTGCGCGATGGAGAGCTTGAAGACCCACAAGCCCTATAAATCGGTAAGTACCGACTCCGAACCGTTC

V ALLKGVGKETDEWADVTFQSKTMKIEDPLNPI

589 GTAATCCCGAATCTCCCTGATGAAATCAAAATGACTAAAAGTCAGTTCACGGTTGACGCTTGGGAAGATACAGAAAAGGGTGTTGGGAAGCTGCTGGCT

D NLVNKYYDAYAPELEYFSNVVVGFSRLGSARA

688 GATGCGAGAGCTTCAGGACTGAGGAGCTTCGGCGTGGTCGTCAACAGCTTCTACGAGCTCGAACCGGCTTACGCGGATTATTACAAGAATGTGTTGAAC

M CEHDDIASKKGRAIKEEDNRYCVSVPGVCWAK

787 ATGAAGGCGTGGTGCGTCGGGCCAGTTTCGGTATGTTACCGAAACGATGAGGAGAAAATTGCAAGAGGGAAGAAATCAGCAATTGATGATCATGAGTGT

L LAIDRLQEDPFSAGSGFCVYVVSDPQKGELWK

880 TTAAAATGGCTGGAGGGAAAGCAGCCGGACTCGGTCGTGTACGTTTGTTTCGGGAGCGGCGCGAGCTTCCCCGATGAGCAGTTGCGCGATATCGCATTG

G VRDEFGEPLYDESGSESSRRIVWIFNVGSDEL

973 GGGCTGGAAGATTCTGGAGTAAATTTCATCTGGGTGATCAGGAGAAGTTCCGAGTCAGGATCAGAAGATTACTTGCCGGAGGGGTTTGAGGACCGGGTG

E QPAWGRIVLGSG V SNWGCHTVFGGVSPHDLIL

1084 GAGGGAAGTGGGCTTGTGATCCGTGGTTGGGCGCCACAGGTATTGATTTTGGACCATCCGTCGGTTGGGGGATTTGTGACTCACTGCGGATGGAATTCG

A LKQNFFQEAFLPWTVMPLGASIGEL I IKLVDT

1183 GCATTGGAGGGGATTTCAGCTGGCTTGCCGATGGTGACGTGGCCGTTGTTCGCCGAGCAGTTTTTCAACCAGAAATTGATTACGGATGTGTTGAAAATT

G EGVMVARVAKEVKEKTVRDEGNRSWKQVGVEV

1282 GGGGTTGAGGTTGGAGTGCAGAAATGGTCTCGGAACGGGGAGGATCGCGTGACGAAGGAGAAGGTTGAGAAGGCGGTGAGGGCTGTTATGGTTGGGGAA

V LNHLDIFSSGDKAAANKALKGLQRARGRREEA

1381 GTGGCGGAGGAGAGGCGCGGCAGAGCTCGTCAGCTTGGGAAATTGGCAAAGAATGCGGCGGCGAAAGATGGGTCTTCTTTTATTGATCTCCACAATTTG

L SLPVRRLKLED

1480 CTTGATGAATTGAAGTTGAGAAGAGTGCCTTTGTCCTAGgttgaatgctctgtatttttcttgtgattcaagtagctgaatggaattctctatatattttcttgcgatgcaagtaacttaaccgaaaaaaaaaaaaa

(b)

Figure 2 Molecular cloning of the full-length cDNA of RcUGT1 (a) RcUGT1 in the agarose gel (b) The cDNA sequence of RcUGT1 ofwhich coding sequence was shown in capital bold letters and the untranslated regions in normal letters The stop codon was marked with anasterisk

UGT sequences [13] which were essential for the enzymaticactivities of glycosyltransferases due to their crucial rolesin the recognition and binding of different aglycones andin the sugar donor specificity [14] These bioinformaticsanalysis results suggested that RcUGT1 might catalyze theglycosylation of tyrosol to given corresponding product

32 Tissue Profile of RcUGT1 Generally the biosynthesisgenes are spatially and temporally expressed in plants toregulate the biosynthesis of targetmetabolites InR crenulataRcTYDC the first committed enzyme gene was highlyexpressed in stems [6]The tissue profile of RcUGT1was ana-lyzed using real-time quantitative PCR The results demon-strated that RcUGT1 was expressed in flowers leaves stemsand roots but at different levels (Figure 4(a)) The RcUGT1transcript level was high in flowers and roots moderate inleaves and low in stems RcUGT1 expression level in flowersleaves and roots was respectively 76- 24- and 45-foldhigher than that in stems These data indicated that RcUGT1mainlyworked in flowers and roots ofR crenulata Accordingto the previous report the salidroside-producing UGT geneof R crenulatawas highly expressed in stems suggesting thatit preferably worked in stems [6] The different tissue profilesbetween RcUGT1 and the salidroside-producing UGT geneimplied their potentially functional difference from eachother

33 Distribution of Icariside D2 in R crenulata Organs Thedistribution pattern of icariside D2 was also investigatedusing HPLC in flowers leaves stems and roots of Rcrenulata The HPLC analysis indicated that the four typesof organs contained icariside D2 at different levels Flowersleaves stems and roots produced icariside D2 at the levelsof 209 mgg dry weight (DW) 097 mgg DW 371 mggDW and 059 mgg DW respectively The highest contentof icariside D2 was found in stems where RcUGT1 wasexpressed at the lowest level among the four types of organs(Figure 4(b)) The discrepancy between RcUGT1 expressionand icariside D2 content in stems strongly suggested thatthe translocation of icariside D2 might exist in this plantand that there might be other uncharacterized UGTs con-verting tyrosol into icariside D2 in R crenulata In fact themetabolite translocation is often found in diverse plants Forexample the tropane alkaloids including hyoscyamine andscopolamine are specially synthesized in secondary roots andconsequently are translocated into airborne parts includingfruits leaves and stems [15] Traditionally the roots ofRhodiola are harvested for salidroside production due tohigh-yield salidroside in roots [8] and obviously this harvestway is not sustainable Since the stems of R crenulata containicariside D2 at a relatively high level (Figure 4(b)) it isreasonable to suggest the farmers to harvest stems alone foricariside D2 production


1 60RcUGT1 (1)

RsUGT73B6 (1)RsUGT72B14 (1)

RsUGT74R1 (1)61 120

RcUGT1 (55)RsUGT73B6 (59)

RsUGT72B14 (56)RsUGT74R1 (46)

121 180RcUGT1 (106)

RsUGT73B6 (114)RsUGT72B14 (110)

RsUGT74R1 (100)181 240


RsUGT72B14 (165)RsUGT74R1 (150)

241 300RcUGT1 (221)

RsUGT73B6 (230)RsUGT72B14 (223)

RsUGT74R1 (210)301 360


RsUGT72B14 (273)RsUGT74R1 (270)

361 420RcUGT1 (327)

RsUGT73B6 (336)RsUGT72B14 (333)

RsUGT74R1 (314)421 480


RsUGT72B14 (392)RsUGT74R1 (374)

481 509RcUGT1 (446)

RsUGT73B6 (455)RsUGT72B14 (447)

RsUGT74R1 (429)

-MDSDSRPLRVFFFPFMAHGHLIPMVDIARLFSSQG-VHSTIITTPLNANYISKTT----

-MGSETRPLSIFFFPFMAHGHMIPMVDMARLFASQG-VRCTIVTTPGNQPLIARSIGKVQ

MAGSGTGAPHIALLPSPGMGHLIPMAEFAKRLVHHH-NFTVTFIIPTDGPPSAAYR----

---MATKKTQILILPYPIQGHINPMLQFAKRLASKSRHLILTLLLP-----TS-------

----SLSIKTLPFQASKVGLPDGCENVDMLPSPD-----LIFKFFQAANLLQKPFENLLE

LLGFEIGVTTIPFRGTEFGLPDGCENLDSVPSPQ-----HVFHFFEAAGSLREPFEQLLE

-----QVLASLPTSISHIFLPP-VDLSDVVPSHPRIETLISLTVVRSLPSLHNTIASLLA

------HARSISSHIGSINVQPISDGADQQGQQFQTAETYLQQFQRAVPGSLDDLIRLER

LEK---PDCLISDIFFPWSVDSAEKFNIPRLVFHGTSFFAMCAMESLKTHKPYKSVSTDS

EHK---PDCVVGDMFFPWSTDSAAKFGIPRLVFHGTSYFALCAGEAVRIHKPYLSVSSDD

SKN---LAALFVDLFGTDAFDPAIDLGVSPYIFFPS--TAMTLSLILHMPELDRSVTCEY

GHDQPQPTILIYDSFFPWALDVAHSNGLAAAPFFTQTCSVSSVYFLFK----------EG

EPFVIPNLPDEIKMTKSQFATDAWEDT--EKGLGKLLAEARASGLRSFGMVVNSFHELEP

EPFVIPGLPDEIKLTKSQLPMHLLEGKK-DSVLAQLLDEVKETEVSSYGVIVNSIYELEP

RHMTDLVRIPGCIPIRGSDLFDPVQDRT-DEAYKRIVHHAKRYPMAEG-IIENSFMELEP

RLSDEMELPHGIPRLEQRDLPSFIQDKENSAHLLELLVDQFSNLDEADYVFFNTFDKLEN

AYADYYKNVLNMKAW-CVGPVSLCNRNVEEKIARGKKSAIDDHE-CLKWLEGKKPDSVVY

AYADYFRNVLKRRAW-EIGPLSLCNRDVEEKAMRGMQAAIDQHE-CLKWLDSKEPDSVVY

GALKYLQSVEPGRPP-VYAVRPLIKMDYEVDSS-GSK--------IIEWLDGQPIGSVLF

QMVEWMARQWQVLTVGPTIPSMYLDKCVKDDRSYGLNLFKPNRESCRDWLCERRASSVIY

VCFGSGASFPDEQLRDIALGLEESGVNFIWVIRK------------SSESRSEDYLPEGF

VCFGSTCKFPDDQLAEIASGLEASGQQFIWVIRR------------MSDDSKEDYLPKGF

ISFGSGGTLSFDQMTELAHGLESSQQRFLWVVRSPSLIPNSAYFSAQSQNDPLAYLPDGF

VSFGSMAILKQEQIEEIAKCLENLQTRFIWVVRET----------------EMAKLPSEF

EDRVKDRGL-VIRGWAPQVLILDHPSVGGFVTHCGWNSSLEGISAGLPMVTWPLFAEQFF

EERVKDRAL-LIRGWAPQVLILDHQSVGGFVSHCGWNSTLEGISAGLPMVTWPVFAEQFY

LNRTSDRGL-VVPNWAPQAQILSHGSTGGFMSHCGWNSILESVVYGVPIIAWPLYAEQKT

VEWNLSSGLGLVVTWCNQLDILAHETVGCFVTHCGWNSVLEALCLGVPMVGVPNWSDQPT

NQKLITDVLKIGVEVGVQKWSRNGEDRVTKEKVEKAVRAVMVGEVAEERRGRARQLGKLA

NEKLLTEVLKIGVAVGARKWRQLVGDFVHKDAIQRAVREIMEGEEAEERRIIARQMGKMA

NSIIVVEDVKVAVRP-----AGVGEGLVKRLEVATAVKALMEGEEGKKVRNRMRDLKDAA

NAKFVEDVWKVGVRA-----KEDEDGIVKSMVLEKCVRAVLEGEKGEVVRRNAGKIKRWA

KNAAAKDGSSFIDLHNLLDELKLRRVPLS

KRAVEKDGSSWTNLNNLLQELKLKKV---

ARAICVDGASTKAIAELAKKWRSSVKH--

LEAVQLGGSSDNNIAKFVTGLALKD----

(a)

TcUGT73B4TcUGT73B3CcUGT1CcUGT2CfUGT1CfUGT2CsUGT73A20CsUGT73A17PgUGTPg44BcUGT7GsUGFT7TcUAGTRcUGT1RsUGT73B6PmUGT14A05PmUGT3RsUGT72B14RsUGT74R1

100

100

99

99

100

80

58100

100

100

8358

40

67

00010203040506

(b)

Figure 3 The multiple alignments and phylogenetic analysis of plant UGTs (a) The multiple alignments of RcUGT1 and the three UGTs ofR sachalinensis The PSPG-box (plant secondary product glycosyltransferase box) was boxed (b) The phylogenetic analysis of plant UGTsThe numbers represented the bootstrap values given by 1000 repeats The scale represented the genetic distance

34 Enzymatic Assays of RcUGT1 In order to study whetherRcUGT1 converted tyrosol to give its derived glucosides suchas icariside D2 and salidroside we expressed RcUGT1 in Ecoli and purified the recombinant His-tagged RcUGT1 pro-tein from engineering E coli When E coli M15 harboringpQE30-RcUGT1 was induced by 05 mM IPTG for 4 hat 25∘C the recombinant protein could be detected onSDS PAGE gel with amounts enough for enzymatic assayin the supernatants On the SDS-page gel 6timesHis-RcUGT1showed the molecular weight of about 53 kDa (Figure 5(a))

consistent with its calculated molecular weight RcUGT1showed very similar molecular weight to the three reportedUGT proteins of R sachalinensis [8] Subsequently thepurified RcUGT1 protein was purified through Ni2+-NTAresin and then used for enzymatic assay A single product wasdetected with the retention time of 10 min in the reactionbuffers consistent with the retention time of the authenticsample of icariside D2 (Figure 5(b)) At the same timeno salidroside was detected In the control (RcUGT1 wasboiled) this specific product was not detected Then this


the r

elat

iv ex

pres

sion

leve

l of R

cUGT1

FlowerStem LeafRoot

5

4

3

2

1

0

A

B

C

D

(a)

the c

onte

nts o

f Ica

risi

de D

2 (m

gg-1

DW

)

AB

C

D

5

4

3

2

1

0FlowerStem LeafRoot

(b)

Figure 4 Tissue profile of RcUGT1 and distribution of icariside D2 in R crenulata organs The bars on columns represented the standarddeviations (n=3) The different letters on column indicated significant difference (plt005) given by t-test (a) The tissue profile of RcUGT1(b) The icariside D2 contents in different organs

(a) (b) (c)

Figure 5 Purification and enzymatic assay of the recombinant His-tagged RcUGT1 (a) Purification of the recombinant His-tagged RcUGT1from E coli (b)The HPLC traces of RcUGT1-mediated reactions 1 represented the authentic icariside D2 and salidroside 2 was the reactionsystemof control inwhich RcUGT1was denatured by boiling 3 represented the product (icariside 2) given by RcUGT1 (c)Themass spectrumof icariside D2 produced by RcUGT1

specific product was identified using LC-MS of which thecharacteristic fragments at the values ofmz 318 andmz 323(Figure 5(c)) were consistent with the reported mz valuesof icariside D2 [16] When these results given by HPLC andMS were considered together it was concluded that RcUGT1converted tyrosol to specifically form icariside D2 which wasdifferent from the three reported R sachalinensis UGTs thatconverted tyrosol to salidroside

35 Enzymatic Kinetics of RcUGT1 The previous publicationstudied the enzymatic activities of the three UGTs of Rsachalinensis at pH 75 alone without optimizing the pHconditions [8] To determine the optimal pH for RcUGT1the enzymatic activity was investigated respectively at pHfrom 58 to 106 RcUGT1 had the maximum activity at pH86 (Figure 6(a)) For example the activity of RcUGT1 was437-fold higher at pH 86 than that at pH 58 respectivelyFinally the kinetic parameters of RcUGT1 were investigatedin the same reaction buffers described above by changing

the concentration of tyrosol from 05 mM to 5 mM at theoptimal pH 86 (Figure 6(b)) The Km value of RcUGT1for tyrosol was 097 plusmn 010 mM (Table 2) According to theprevious reports theKmvalues of the three reportedUGTs ofRhodiola sachalinensis catalyzing tyrosol to form salidrosidewere respectively 47 120583M 1724 120583M and 543 120583M [6] Bycomparison of the Km values of these enzymes RcUGT1exhibited lower affinity for the substrate tyrosol than thereported three enzymes The Vmax value of RcUGT1 fortyrosol was 286plusmn826 pKatmg much higher than that ofRsUGT72B14 (578 pKatmg) and slightly higher than thatof RsUGT73B6 (2498 pKatmg) but at the very similarlevel of RcUGT74R1 (2931 pKatmg) The Vmax analysissuggested that the velocity of RcUGT1 was higher than thoseof RsUGT72B14 and RsUGT73B6 but similar to that ofRcUGT74R1 Although the Kcat values of the three otherUGTs were not reported we detected the Kcat of RcUGT1The Kcat value of RcUGT1 was 001552 sminus1 The KcatKmvalue of RcUGT1 representing the catalytic efficiency was


Table 2 The kinetics parameters of UGT enzymes

Enzyme Substrate Product Km Vmax Kcat KcatKm References(120583M) (pKat mg-1) (s-1) (s-1 M-1)

RcUGT1 Tyrosol Icariside D2 97275plusmn10431 286plusmn826 001552plusmn000044 15955 this studyRsUGT72B14 Tyrosol Salidroside 47plusmn035 578plusmn32 - - Yu et al 2011RsUGT74R1 Tyrosol Salidroside 172plusmn141 2931plusmn14 - - Yu et al 2011RsUGT73B6 Tyrosol Salidroside 543plusmn49 2498plusmn13 - - Yu et al 2011

0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)

Figure 6 The kinetics analysis of RcUGT1 (a) The optimum pH for RcUGT1 (b) The kinetics analysis of RcUGT1 at optimal pH

15955 sminus1Mminus1 The enzymatic assays suggested that RcUGT1might be a good candidate to specifically produce icarisideD2 through metabolic engineering or synthetic biology

4 Conclusion

In summary we have characterized a novel enzyme thatcatalyzes the glycosylation of tyrosol to specifically formicariside D2 which is potentially useful in the productionof icariside D2 through metabolic engineering and syntheticbiology The stems of R crenulata produce icariside D2at higher level than other organs providing a reasonablesuggestion to farmers to harvest stems instead of roots foricariside D2 production

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was financially supported by the NSFC projects(81660628 and 31370317) the Sci-Tech Project of the Educa-tion Bureau of Xizang Autonomous Region (Quality Controland Improvement of Resource Plants for TibetanMedicines)and the PhD funding of Xizang Agricultural andHusbandryCollege (YJS2016-1)

References

[1] S-W Chan ldquoPanax ginseng Rhodiola rosea and Schisandrachinensisrdquo International Journal of Food Sciences and Nutritionvol 63 no 1 pp 75ndash81 2012

[2] Y Zhao W Sun Y Wang P K Saxena and C-Z LiuldquoImproved mass multiplication of Rhodiola crenulata shootsusing temporary immersion bioreactor with forced ventilationrdquoApplied Biochemistry and Biotechnology vol 166 no 6 pp1480ndash1490 2012

[3] Y-N Yang Z-Z Liu Z-M Feng J-S Jiang and P-C ZhangldquoLignans from the root of Rhodiola crenulatardquo Journal ofAgricultural and Food Chemistry vol 60 no 4 pp 964ndash9722012

[4] A Simaratanamongkol K Umehara H Noguchi and PPanichayupakaranant ldquoIdentification of a new angiotensin-converting enzyme (ACE) inhibitor from Thai edible plantsrdquoFood Chemistry vol 165 pp 92ndash97 2014

[5] N T T Hien N X Nhiem D T H Yen et al ldquoChemicalconstituents of the Annona glabra fruit and their cytotoxicactivityrdquo Pharmaceutical Biology vol 53 no 11 pp 1602ndash16072015

[6] X Lan K Chang L Zeng et al ldquoEngineering salidrosidebiosynthetic pathway in hairy root cultures of Rhodiola crenu-lata based onmetabolic characterization of tyrosine decarboxy-laserdquo PLoS ONE vol 8 no 10 Article ID e75459 2013

[7] J-X Zhang L-Q Ma H-S Yu et al ldquoA tyrosine decarboxylasecatalyzes the initial reaction of the salidroside biosynthesispathway in Rhodiola sachalinensisrdquo Plant Cell Reports vol 30no 8 pp 1443ndash1453 2011


[8] H-S Yu L-Q Ma J-X Zhang G-L Shi Y-H Hu and Y-NWang ldquoCharacterization of glycosyltransferases responsible forsalidroside biosynthesis in Rhodiola sachalinensisrdquo Phytochem-istry vol 72 no 9 pp 862ndash870 2011

[9] L-Q Ma B-Y Liu D-Y Gao et al ldquoMolecular cloning andoverexpression of a novel UDP-glucosyltransferase elevatingsalidroside levels in Rhodiola sachalinensisrdquo Plant Cell Reportsvol 26 no 7 pp 989ndash999 2007

[10] Y Bai H Bi Y Zhuang et al ldquoProduction of salidroside inmetabolically engineered Escherichia colirdquo Scientific Reportsvol 4 article 6640 pp 1ndash8 2014

[11] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[12] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[13] H Jugde D Nguy I Moller J M Cooney and R G AtkinsonldquoIsolation and characterization of a novel glycosyltransferasethat converts phloretin to phlorizin a potent antioxidant inapplerdquo FEBS Journal vol 275 no 15 pp 3804ndash3814 2008

[14] K Ghose K Selvaraj J McCallum et al ldquoIdentification andfunctional characterization of a flax UDP-glycosyltransferaseglucosylating secoisolariciresinol (SECO) into secoisolari-ciresinol monoglucoside (SMG) and diglucoside (SDG)rdquo BMCPlant Biology vol 14 no 1 p 82 2014

[15] K Xia X Liu Q Zhang et al ldquoPromoting scopolamine biosyn-thesis in transgenic Atropa belladonna plants with pmt and h6hoverexpression under field conditionsrdquo Plant Physiology andBiochemistry vol 106 pp 46ndash53 2016

[16] Y Bai H Yin H Bi Y Zhuang T Liu and Y Ma ldquoDenovo biosynthesis of Gastrodin in Escherichia colirdquo MetabolicEngineering vol 35 pp 138ndash147 2016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


(a)

OHHO

HO

Tyrosol

O

OHOH OOH

P O P

O

O

O

OH

O

OH

HN

N

O

O

UDP-glucose O

HO OH

OH

HO O

OH

Icariside D2

UDP

P O P

O

O

O

OH

O

OH

HN

NOHO

+

O

HO OH

OH

HO O

OH+

UDP

+

UGT73B6UGT72B14UGT74R1

RcUGT1

Salidroside

P O P

O

O

O

OH

O

OH

HN

NOHO

NH2

O- O-

Na+ Na+

O- O-

O- O-

(b)

Figure 1 Rhodiola crenulata and the biosynthesis of salidroside and its isomer icariside D2 (a)The flowering plant of Rhodiola crenulata (b)UDP-glycosyltransferases (UGTs) convert tyrosol to salidroside and icariside D2

[6] Overexpression of tyrosine decarboxylase enhancedsalidroside and tyrosol accumulation in Rhodiola hairy rootcultures [7] Three specific UDP-glycosyltransferases (UGTs)were reported to convert tyrosol into salidroside (Figure 1(b))in Rhodiola sachalinensis [8] and overexpression of one ofthem (RsUGT73B6) promoted salidroside production [9]RsUGT73B6 was further used to produce salidroside inengineering E coli through the strategy of synthetic biology[10] However the enzyme required for converting tyrosolinto icariside D2 in R crenulata has not been identified Inthis study we characterized a novel UDP-glycosyltransferase(RcUGT1) that converted tyrosol to specifically produceicariside D2 from R crenulata at molecular and biochemicallevels

2 Experimental Procedures

21 Plant Materials and Chemicals Because Rhodiola crenu-lata is an endangered plant species the official approval isnecessary to collect the plant materials The whole plantsof Rhodiola crenulata were collected from the northern areaof Tibet Himalaya Mountain in July of 2011 [6] under theofficial approval of Tibet Sciences and Technology Commit-tee (Lhasa Tibet China) The different organs includingflowers leaves stems and roots were immediately immersedin liquid nitrogen for future use [6] The authentic icarisideD2 was purchased from BioBioPha (Yunnan China) UDP-glucose and salidroside were purchased from Sigma-Aldrich

(Louisiana USA) Tyrosol was purchased from Aladdin(Shanghai China)

22 Gene Cloning and Expression Analysis Total RNAs wereisolated from the different organs of R crenulata usingthe RNAsimple Kit according to the manufacturers pro-tocol (Tiangen Biotech Beijing China) The cDNAs weresynthesized using the FastKing RT Kit (Tiangen BiotechBeijing China) The conserved core sequence of RcUGT1was amplified using a pair of primers dF and dR Basedon the sequence core sequence of RcUGT1 the cDNA endswere cloned using RACE technique The 31015840 RACE and 51015840RACE of RcUGT1 were respectively performed accordingto the manufacturerrsquos protocol (ClonTech CA USA) EachPCR product was subcloned into the pMD-18T vector andsequenced The corresponding fragments of RcUGT1 wereassembled to generate the putative full-length cDNA and thecoding sequence was amplified using a pair of gene-specificprimers pF and pR Real-time quantitative PCR was used toanalyze the tissue profile of RcUGT1 on an iQ5 system (Bio-Rad USA) as described before [6] The primers used in thisstudy were listed in Table 1

23 Bioinformatic Analysis The sequences analysis wasonline performed at the NCBI websites The multiple align-ments of RcUGT1 and other UGTs were aligned using theVector NTI software The phylogenetic tree was constructed







2PO4 and pH


PAGE






(a)






























L SLPVRRLKLED


(b)






1 60RcUGT1 (1)


RsUGT74R1 (1)61 120



121 180RcUGT1 (106)

RsUGT73B6 (114)RsUGT72B14 (110)

RsUGT74R1 (100)181 240


RsUGT72B14 (165)RsUGT74R1 (150)

241 300RcUGT1 (221)

RsUGT73B6 (230)RsUGT72B14 (223)

RsUGT74R1 (210)301 360


RsUGT72B14 (273)RsUGT74R1 (270)

361 420RcUGT1 (327)

RsUGT73B6 (336)RsUGT72B14 (333)

RsUGT74R1 (314)421 480


RsUGT72B14 (392)RsUGT74R1 (374)

481 509RcUGT1 (446)

RsUGT73B6 (455)RsUGT72B14 (447)

RsUGT74R1 (429)





































(a)


100

100

99

99

100

80

58100

100

100

8358

40

67

00010203040506

(b)





the r

elat

iv ex

pres

sion

leve

l of R

cUGT1

FlowerStem LeafRoot

5

4

3

2

1

0

A

B

C

D

(a)

the c

onte

nts o

f Ica

risi

de D

2 (m

gg-1

DW

)

AB

C

D

5

4

3

2

1


(b)


(a) (b) (c)









0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)



4 Conclusion




Acknowledgments


References




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








2PO4 and pH


PAGE






(a)






























L SLPVRRLKLED


(b)






1 60RcUGT1 (1)


RsUGT74R1 (1)61 120



121 180RcUGT1 (106)

RsUGT73B6 (114)RsUGT72B14 (110)

RsUGT74R1 (100)181 240


RsUGT72B14 (165)RsUGT74R1 (150)

241 300RcUGT1 (221)

RsUGT73B6 (230)RsUGT72B14 (223)

RsUGT74R1 (210)301 360


RsUGT72B14 (273)RsUGT74R1 (270)

361 420RcUGT1 (327)

RsUGT73B6 (336)RsUGT72B14 (333)

RsUGT74R1 (314)421 480


RsUGT72B14 (392)RsUGT74R1 (374)

481 509RcUGT1 (446)

RsUGT73B6 (455)RsUGT72B14 (447)

RsUGT74R1 (429)





































(a)


100

100

99

99

100

80

58100

100

100

8358

40

67

00010203040506

(b)





the r

elat

iv ex

pres

sion

leve

l of R

cUGT1

FlowerStem LeafRoot

5

4

3

2

1

0

A

B

C

D

(a)

the c

onte

nts o

f Ica

risi

de D

2 (m

gg-1

DW

)

AB

C

D

5

4

3

2

1


(b)


(a) (b) (c)









0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)



4 Conclusion




Acknowledgments


References




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(a)






























L SLPVRRLKLED


(b)






1 60RcUGT1 (1)


RsUGT74R1 (1)61 120



121 180RcUGT1 (106)

RsUGT73B6 (114)RsUGT72B14 (110)

RsUGT74R1 (100)181 240


RsUGT72B14 (165)RsUGT74R1 (150)

241 300RcUGT1 (221)

RsUGT73B6 (230)RsUGT72B14 (223)

RsUGT74R1 (210)301 360


RsUGT72B14 (273)RsUGT74R1 (270)

361 420RcUGT1 (327)

RsUGT73B6 (336)RsUGT72B14 (333)

RsUGT74R1 (314)421 480


RsUGT72B14 (392)RsUGT74R1 (374)

481 509RcUGT1 (446)

RsUGT73B6 (455)RsUGT72B14 (447)

RsUGT74R1 (429)





































(a)


100

100

99

99

100

80

58100

100

100

8358

40

67

00010203040506

(b)





the r

elat

iv ex

pres

sion

leve

l of R

cUGT1

FlowerStem LeafRoot

5

4

3

2

1

0

A

B

C

D

(a)

the c

onte

nts o

f Ica

risi

de D

2 (m

gg-1

DW

)

AB

C

D

5

4

3

2

1


(b)


(a) (b) (c)









0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)



4 Conclusion




Acknowledgments


References




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



1 60RcUGT1 (1)


RsUGT74R1 (1)61 120



121 180RcUGT1 (106)

RsUGT73B6 (114)RsUGT72B14 (110)

RsUGT74R1 (100)181 240


RsUGT72B14 (165)RsUGT74R1 (150)

241 300RcUGT1 (221)

RsUGT73B6 (230)RsUGT72B14 (223)

RsUGT74R1 (210)301 360


RsUGT72B14 (273)RsUGT74R1 (270)

361 420RcUGT1 (327)

RsUGT73B6 (336)RsUGT72B14 (333)

RsUGT74R1 (314)421 480


RsUGT72B14 (392)RsUGT74R1 (374)

481 509RcUGT1 (446)

RsUGT73B6 (455)RsUGT72B14 (447)

RsUGT74R1 (429)





































(a)


100

100

99

99

100

80

58100

100

100

8358

40

67

00010203040506

(b)





the r

elat

iv ex

pres

sion

leve

l of R

cUGT1

FlowerStem LeafRoot

5

4

3

2

1

0

A

B

C

D

(a)

the c

onte

nts o

f Ica

risi

de D

2 (m

gg-1

DW

)

AB

C

D

5

4

3

2

1


(b)


(a) (b) (c)









0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)



4 Conclusion




Acknowledgments


References




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



the r

elat

iv ex

pres

sion

leve

l of R

cUGT1

FlowerStem LeafRoot

5

4

3

2

1

0

A

B

C

D

(a)

the c

onte

nts o

f Ica

risi

de D

2 (m

gg-1

DW

)

AB

C

D

5

4

3

2

1


(b)


(a) (b) (c)









0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)



4 Conclusion




Acknowledgments


References




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






0012

0010

0008

0006

0004

0002

0000

pH

Activ

ity (u

mol

minmiddotm

g)

111098765

(a)

0020

0016

0012

0008

0004

0000

Tyrosol (mM)6543210

Activ

ity (u

mol

minmiddotm

g)

(b)



4 Conclusion




Acknowledgments


References




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018














Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


a novel udp-glycosyltransferase of rhodiola crenulata

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