Composite

Part:BBa_K2664008

Designed by: Areti-Efremia Mellou   Group: iGEM18_Macquarie_Australia   (2018-10-09)


Complete chlorophyll expression pathway

Complete chlorophyll expression pathway

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1720
    Illegal NotI site found at 3751
    Illegal NotI site found at 9504
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
    Illegal BglII site found at 304
    Illegal BglII site found at 960
    Illegal BglII site found at 1155
    Illegal BglII site found at 4456
    Illegal BglII site found at 4580
    Illegal BglII site found at 6580
    Illegal BamHI site found at 352
    Illegal BamHI site found at 10997
    Illegal BamHI site found at 16476
    Illegal BamHI site found at 17457
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3031
    Illegal NgoMIV site found at 3850
    Illegal NgoMIV site found at 4392
    Illegal NgoMIV site found at 5138
    Illegal NgoMIV site found at 6804
    Illegal NgoMIV site found at 12155
    Illegal AgeI site found at 5784
    Illegal AgeI site found at 7302
    Illegal AgeI site found at 7356
    Illegal AgeI site found at 7575
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 11159
    Illegal BsaI.rc site found at 6906
    Illegal BsaI.rc site found at 7701
    Illegal BsaI.rc site found at 9832
    Illegal BsaI.rc site found at 9988
    Illegal BsaI.rc site found at 10609
    Illegal BsaI.rc site found at 14945
    Illegal SapI.rc site found at 7631




Overview

This series of genes derived from the algae Chlamydomonas reinhardtii have been brought together to express the complete chlorophyll biosynthesis pathway in Escherichia coli. In 2018 Team Macquarie Australia has made rearrangements in existing parts of the pathway, introduced new genes and swapped lac with the trc promoter. The parts have been assembled to allow for gradual transition from Protoporphyrin IX to Chlorophyll and we are confident that our improved plasmid will be more efficient at producing chlorophyll, than its previous versions.


Biology & Literature

The chlorophyll biosynthesis pathway could be divided in three major reactions:
1. Magnesium chelation to protoporphyrin IX give rise to Mg-Protoporphyrin IX
2. Conversion of Mg-Protoporphyrin IX to Protochlorophyllide
3. Final reaction from Protochlorophyllide to Chlorophyll-a

The first set of rections involves the genes ChlI1, ChlI2, ChlD, ChlH and GUN4. To begin with, ChlI1 and ChlI2 form the initial Mg-chelatase complex (Magnesium chelatase subunit I). This complex catalyses the insertion of a magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. This happens through the formation of two different complexes, an ATP dependent hexameric ring complex and a complex with the chlD subunit. Next comes chlH, encoding for the magnesium shelter subunit H. This subunit acts as a chloroplast precursor, catalysing the insertion of a Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX. The GUN4 gene encodes for the genomes-uncoupled-4-protein, which is a tetrapyrrole-binding protein that also controls the production of Mg-protoporhyrin IX, through binding to protoporphyrin IX. Together, the ChlH and GUN4 encoded proteins generate the first activated complex in the pathway. This active complex behaves as a substrate for the motor complex, ChlD, to insert magnesium into the bound Protoporphyrin IX upon ATP hydrolysis.

The second set of reactions involve the genes CTH1, Ycf54, ChlM, FNR and Fdx. Here, Mg-Protoporphyrin IX is converted to Protochlorophyllide. To achieve this, yCF54, works with the CTH1 gene and Plastocyanin within the oxidative cyclase pathway. Deletion studies have detected the importance of yCF54 in maintaining levels of Mg-protoporphyrin IX methyl ester. This indicates that the gene is critical to both the assembly and function of the cyclase complex. The gene CTH1 encodes for the copper target 1 protein, which is a functional variant produced under copper and/or oxygen sufficient conditions. Following these two genes, comes ChlM, which encodes for magnesium protoporhyrin IX methytransferase. This enzyme is involved in the transfer of a methyl group onto one of the rings of magnesium protoporphyrin,forming the magnesium protoporhyrin IX monomethylester . Combined with Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase (Ycf54),it can convert protoporphyrin IX to protochlorophyllide. Increasing transcription of the FNR and FDX genes would improve the efficiency of chlorophyll production. When it comes to FNR and Fdx,they have been reported to aid the chlorophyll biosynthesis pathway by increasing their transcription. This increase has been linked to improving the efficiency of chlorophyll production.

The third and final set of reactions involve POR, ChlP, DVR1 and ChlG, which are responsible for converting protochlorophyllide to chlorophyll-a.
First comes POR, which codes for the enzyme light dependent protochlorophyllide reductase. Its role is to convert protochlorophyllide to chlorophyllide using NADPH and light as the reductants. Formation of the POR protein in plants is crucial, as without it the chloroplasts do not function. The protochlorophyllide is reduced to chlorophyllide through light excitation, functioning as a catalyst. The excitation of the POR encoded molecule and its conversion to chlorophyllide causes the membranes to turn from an inactive state into the active chloroplast. Through the transformation into the active state, the chloroplasts produce that be used by the plant.DVR1 then comes in, which encodes for 3,8-divinyl protochlorophyllide 8-vinyl reductase. This enzyme reduces divinly protochlorophyllide a to monovinyl protochlorophyllide, a molecule ready to be used by ChlP. ChlP encodes for geranylgeranyl reductase, catalysing the addition of the geranylgeranyl pyrophosphate chain into the chlorophyllide molecule. The reaction occurs through hydrogenation of geranylgeranyl diphosphate (GGPP), that reduces the molecule’s (GGPP) double bonds. The last gene in the pathway is ChlG, which encodes the enzyme chlorophyll synthetase. As the name implies, it is the final step in chlorophyll synthesis, which is achieved after chlorophyll synthetase catalyses the esterification of chlorophyllide with GGPP.


Assembly and Design

HydrogenProduction

Figure 1. Graphic representation of the designing process for assembling the complete chlorophyll biosynthesis pathway in E. coli. The process was completed using standard assembly.


Part Verification

Figure 2. Agarose gel electrophoresis (0.5% agarose) with GelRed (2 μl/100 ml) showing single (E) and double (E+P) digests of the complete plasmid for Chlorophyll a biosynthesis pathway (18352bp). The 1kb gene ruler plus was loaded in Lane 1 for size reference.


Protein Information

ChlI1
Mass: 39.95 kDa
Sequence:
MAATEVKAAE GRTEKELGQA RPIFPFTAIV GQDEMKLALI LNVIDPKIGG VMIMGDRGTG KSTTIRALAD LLPEMQVVAN DPFNSDPTDP ELMSEEVRNR VKAGEQLPVS SKKIPMVDLP LGATEDRVCG TIDIEKALTE GVKAFEPGLL AKANRGILYV DEVNLLDDHL VDVLLDSAAS GWNTVEREGI SISHPARFIL VGSGNPEEGE LRPQLLDRFG MHAQIGTVKD PRLRVQIVSQ RSTFDENPAA FRKDYEAGQM ALTQRIVDAR KLLKQGEVNY DFRVKISQIC SDLNVDGIRG DIVTNRAAKA LAAFEGRTEV TPEDIYRVIP LCLRHRLRKD PLAEIDDGDR VREIFKQVFG ME

ChlI2
Mass: 39.56 kDa
Sequence:
MPSTKAAKKP NFPFVKIQGQ EEMKLALLLN VVDPNIGGVL IMGDRGTAKS VAVRALVDML PDIDVVEGDA FNSSPTDPKF MGPDTLQRFR NGEKLPTVRM RTPLVELPLG ATEDRICGTI DIEKALTQGI KAYEPGLLAK ANRGILYVDE VNLLDDGLVD VVLDSSASGL NTVEREGVSI VHPARFIMIG SGNPQEGELR PQLLDRFGMS VNVATLQDTK QRTQLVLDRL AYEADPDAFV DSCKAEQTAL TDKLEAARQR LRSVKISEEL QILISDICSR LDVDGLRGDI VINRAAKALV AFEGRTEVTT NDVERVISGC LNHRLRKDPL DPIDNGTKVA ILFKRMTDPE IMKREEEAKK

ChlD
Mass: 63.84 kDa
Sequence:
MRAMKVSEED SKGFDADVST RLARSYPLAA VVGQDNIKQA LLLGAVDTGL GGIAIAGRRG TAKSIMARGL HALLPPIEVV EGSICNADPE DPRSWEAGLA EKYAGGPVKT KMRSAPFVQI DGVNVVEREG ISISHPCRPL LIATYNPEEG PLREHLLDRI AIGLSADVPS TSDERVKAID AAIRFQDKPQ DTIDDTAELT DALRTSVILA REYLKDVTIA PEQVTYIVEE ARRGGVQGHR AELYAVKCAK ACAALEGRER VNKDDLRQAV QLVILPRATI LDQPPPEQEQ PPPPPPPPPP PPPQDQMEDE DQEEKEDEKE EEEKENEDQD EPEIPQEFMF ESEGVIMDPS ILMFAQQQQR AQGRSGRAKT LIFSDDRGRY IKPMLPKGDK VKRLAVDATL RAAAPYQKIR RQQAISEGKV QRKVYVDKPD MRSKKLARKA GALVIFVVDA SGSMALNRMS AAKGACMRLL AESYTSRDQV VMMVLITDGR ANVSLAKSNE DPEALKPDAP KPTADSLKDE VRDMAKKAAS AGINVLVIDT ENKFVSTGFA EEISKAAQGK YYYLPNASDA AIAAAASGAM AAAKGGY

ChlH
Mass:144.14 kDa
Sequence:
MCNVATGPRP PMTTFTGGNK GPAKQQVSLD LRDDGAGMFT STSPEMRRVV PDDVKGRVKV KVVYVVLEAQ YQSAISAAVK NINAKNSKVC FEVVGYLLEE LRDQKNLDML KEDVASANIF IGSLIFIEEL AEKIVEAVSP LREKLDACLI FPSMPAVMKL NKLGTFSMAQ LGQSKSVFSE FIKSARKNND NFEEGLLKLV RTLPKVLKYL PSDKAQDAKN FVNSLQYWLG GNSDNLENLL LNTVSNYVPA LKGVDFSVAE PTAYPDVGIW HPLASGMYED LKEYLNWYDT RKDMVFAKDA PVIGLVLQRS HLVTGDEGHY SGVVAELESR GAKVIPVFAG GLDFSAPVKK FFYDPLGSGR TFVDTVVSLT GFALVGGPAR QDAPKAIEAL KNLNVPYLVS LPLVFQTTEE WLDSELGVHP VQVALQVALP ELDGAMEPIV FAGRDSNTGK SHSLPDRIAS LCARAVNWAN LRKKRNAEKK LAVTVFSFPP DKGNVGTAAY LNVFGSIYRV LKNLQREGYD VGALPPSEED LIQSVLTQKE AKFNSTDLHI AYKMKVDEYQ KLCPYAEALE ENWGKPPGTL NTNGQELLVY GRQYGNVFIG VQPTFGYEGD PMRLLFSKSA SPHHGFAAYY TFLEKIFKAD AVLHFGTHGS LEFMPGKQVG MSGVCYPDSL IGTIPNLYYY AANNPSEATI AKRRSYANTI SYLTPPAENA GLYKGLKELK ELISSYQGMR ESGRAEQICA TIIETAKLCN LDRDVTLPDA DAKDLTMDMR DSVVGQVYRK LMEIESRLLP CGLHVVGCPP TAEEAVATLV NIAELDRPDN NPPIKGMPGI LARAIGRDIE SIYSGNNKGV LADVDQLQRI TEASRTCVRE FVKDRTGLNG RIGTNWITNL LKFTGFYVDP WVRGLQNGEF ASANREELIT LFNYLEFCLT QVVKDNELGA LVEALNGQYV EPGPGGDPIR NPNVLPTGKN IHALDPQSIP TQAALKSARL VVDRLLDRER DNNGGKYPET IALVLWGTDN IKTYGESLAQ VMMMVGVKPV ADALGRVNKL EVIPLEELGR PRVDVVVNCS GVFRDLFVNQ AVENSSWSDE SQLQEMYLKR KSYAFNSDRP GAGGEMQRDV FETAMKTVDV TFQNLDSSEI SLTDVSHYFD SDPTKLVASL RNDGRTPNAY IADTTTANAQ VRTLGETVRL DARTKLLNPK WYEGMLASGY EGVREIQKRM TNTMGWSATS GMVDNWVYDE ANSTFIEDAA MAERLMNTNP NSFRKLVATF LEANGRGYWD AKPEQLERLR QLYMDVEDKI EGVE

GUN4
Mass: 24.36 kDa
Sequence:
MAMRVTVAAG KLDSVSLFGG DTASLMGGSQ TVEKKKSGKE AVMEVQLSST AGIDYTVLRD HLANGEFREA EDETRALLIK LAGPEAVKRN WVYFTEVKNI SVTDFQTLDN LWKASSNNKF GYSVQKEIWV QNQKRWPKFF KQIDWTQGEN NNYRKWPMEF IYSMDAPRGH LPLTNALRGT QLFQAIMEHP AFEKSSTAKT LDQKAAEAAG RTQSLF

CTH1
Mass: 43.87 kDa
Sequence:
MVAATAAPQE VEGFKVMRDG IKVASDETLL TPRFYTTDFD EMERLFSLEL NKNMDMEEFE AMLNEFKLDY NQRHFVRNET FKEAAEKIQG PTRKIFIEFL ERSCTAEFSG FLLYKELGRR LKATNPVVAE IFTLMSRDEA RHAGFLNKAM SDFNLALDLG FLTKNRKYTF FKPKFIFYAT YLSEKIGYWR YISIYRHLQR NPDNQLYPLF EYFENWCQDE NRHGDFFTAV LKARPEMVND WAAKLWSRFF CLSVYITMYL NDHQRDAFYS SLGLNTTQFN QHVIIETNKS TERIFPAVPD VENPEFFRRM DLLVKYNAQL VNIGSMNLPS PIKAIMKAPI LERMVAEVFQ VFIMTPKESG SYDLDANKTA LVY

YCF54
Mass: 17.07 kDa
Sequence:
MAPAAASADK ATAAEYYALV CNAEWFFMDP QNESVAEQLR EKVRFFKEQN KERDFFIVPN PKWLDAKFPE QAKQVKRPCV ALVSTDKMWI TFMKLRLDRV LKIDLKSMPA SEVLAAGEAL PDFKPDGKWT APYARYTPGW WNVFLPNH

ChlM
Mass: 30.44 kDa
Sequence:
MASEIAQTAD VGSLTFAVGG VGAVVGLGAL LVATDHQKRR SEQMKSFDGD EKEAVKDYFN TAGFERWRKI YGETDEVNKV QLDIRTGHAQ TVDKVLRWVD EEGSVQGITV ADCGCGTGSL AIQLALRGAA VSASDISAAM ASEAEQRYQQ AVAAGQGKAP KVAPKFEALD LESVKGKYDT VTCLDVMIHY PQDKVDAMIT HLAGLSDRRL IISFAPKTLS YSILKRIGEL FPGPSKATRA YLHREEDVEA ALKRAGFKVT KREMTATSFY FSRLLEAIRE

Ferredoxin
Mass: 13.0 kDa
Sequence:
MAMRSTFAARVGAKPAVRGARPASRMSCMAYKVTLKTPSGDKTIECPADTYILDAAEEAGLDLPYSCRAGACSSCAGKVAAGTVDQSDQSFLDDAQMGNGFV LTCVAYPTSDCTIQTHQEEALY

Ferredoxin NADP+ reductase (FNR)
Mass: 38.27 kDa
Sequence:
MQTVRAPAASGVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPP GTKINSKGKEVPHGTRLYSIASSRYGDDFDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATGTGIAPFRS FWRRCFIENVPSYKFTGLFWLFMGVANSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQD MLERVAKEKGLNYEEWVEGLKHKNQWHVEVY

POR
Mass: 41.87 kDa
Sequence:
MVVCAATATAPSPSLADKFKPNAIARVPATQQKQTAIITGASSGLGLNAAKALAATGEWHVVMACRDFLKAEQAAKKVGMPAGSYSILHLDLSSLESVRQFVQNFKASGR RLDALVCNAAVYLPTAKEPRFTADGFELSVGTNHLGHFLLTNLLLDDLKNAPNKQPRCIIVGSITGNTNTLAGNVPPKANLGDLSGLAAGVPAANPMMDGQEFNGAKAYK DSKVACMMTV RQMHQRFHDATGITFASLYPGCIAETGLFREHVPLFKTLFPPFQKYITKGYVSEEEAGRRLAAVISDPKLNKSGAYWSWSSTTGSFDNK

ChlP
Mass: 47 kDa
Sequence:
MVIGGGPSGACAAETLAKGGVETFLLERKLDNCKPCGGAIPLCMVEEFDLPMEIIDRRVTKMKMISPSNREVDVGKTLSETEWIGMCRREVFDDYLRNRAQKLGANIVNGL FMRSEQQSAEGPFTIHYNSYEDGSKMGKPATLEVDMIIGADGANSRIAKEIDAGEYDYAIAFQERIRIPDDKMKYYENLAEMYVGDDVSPDFYGWVFPKYDHVAVGTGTVVN KTAIKQYQQATRDRSKVKTEGGKIIRVEAHPIPEHPRPRRCKGRVALVGDAAGYVTKCSGEGIYFAAKSGRMAAEAIVEGSANGTKMCGEDAIRVYLDKWDRKYWTTYKVLD ILQKVFYRSNPAREAFVELCEDSYVQKMTFDSYLYKTVVPGNPLDDVKLLVRTVSSILRSNALRSVNSKSVNVSFGSKANEERVMAA

DVR1
Mass: 37 kDa
Sequence:
MAMAASRQAVRVAAAVDADYRKREPKDVRVLVVGPTGYIGKFVVKELVSRGYNVVAFARENAGIKGKMGREDIVKEFHGAEVRFGSVLDPASLRDVAFKDPVDVVVSCLA SRTGGKKDSWLIDYTATKNSLDVARASGAKHFVLLSAICVQKPLLEFQKAKLQFESDLQAAGDITYSIVRPTAFFKSIAGQIDIVKKGNPYVMFGDGNLAACKPISEADLASF IADCVTEQNKVNKVLPIGGPSKAFTAKQQADLLFNITGLPPKYFPVPVALMDGMIGLFDSLAKLFPQLEDSAEFARIGKYYATESMLVYDEARGVYRKTKRLVTARTRWKTS SLVQ

ChlG
Mass: 36.84 kDa
Sequence:
MNQQATEEKSDTNSAARQMLGMKGAALETDIWKIRVQLTKPVTWIPLIWGVACGAAASGHYQWNNPTQIAQLLTCMMMSGPFLTGYTQTINDWYDREIDAINEPYRPIPS GRISERDVIVQIWVLLLGGIGLAYTLDQWAGHTTPVMLQLTIFGSFISYIYSAPPLKLKQSGWAGNYALGSSYIALPWWAGQALFGTLTLDVMALTIAYSLAGLGIAIVNDFKSI EGDRQ MGLQSLPVAFGVDTAKWICVSTIDVTQLGVAAYLAWGLHEELYGAVLLALILPQIYFQYKYFLPDPIANDVKYQASAQPFLVFGLLTAGLACGHHVNAVAA AASAAGAL


References

[1] Adhikari ND, Froehlich JE, Strand DD, Buck SM, Kramer DM, Larkin RM. gUN4-Porphyrin Complexes Bind the chlH/gUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis. Plant Cell. 2013; 23: 1449-1467.
[2] Tabrizi S, Sawicki A, Zhou S, Luo M, Willows R. GUN4-Protoporphyrin IX Is a Singlet Oxygen Generator with Consequences for Plastid Retrograde Signaling. Journal of Biological Chemistry. 2016;291(17): 8978-8984.
[3] Brzezowski, P., et al. (2014). "The GUN4 protein plays a regulatory role in tetrapyrrole biosynthesis and chloroplast-to-nucleus signalling in Chlamydomonas reinhardtii." Plant J. 79(2): 285-98
[4] Benton, C. M., Lim, C. K., Moniz, C., & Jones, D. J. (2012). Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation. Biomed Chromatogr, 26(6), 714-719. doi: 10.1002/bmc.1720
[5] Allen, M. D., et al. (2008). "Regulation and localization of isoforms of the aerobic oxidative cyclase in Chlamydomonas reinhardtii." Photochem Photobiol 84(6): 1336-1342.
[6] Walker, C. J., et al. (1991). "Synthesis of divinyl protochlorophyllide. Enzymological properties of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system." Biochem J 276 ( Pt 3): 691-697.
[7] Hollingshead, S., et al. (2012). "Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803." J Biol Chem 287(33): 27823-27833.
[8]Chen, X., Pu, H., Fang, Y., Wang, X., Zhao, S., Lin, Y., Zhang, M., Dai, H-E., Gong, W., Liu, L. (2015). Crystal Structure of the catalytic subunit of magnesium chelatase. Nature Plants, 1, doi:10.1038/nplants.2015.125.
[9] Sirijovski, N., Lunqvist, J., Rosenback, M., Elmlund, H., Al-Karadaghi, S., Willows, R.D., Hansson, M. (2008). Substrate-binding Model of the Chlorophyll Biosynthetic Magnesium Chelatase BchH Subunit. Journal of Biological Chemistry, 283, 11652-11660.
[10] Tegel, H., Ottosson, J. and Hober, S., 2011. Enhancing the protein production levels in Escherichia coli with a strong promoter. The FEBS journal, 278(5), pp.729-739.
[11] Herbst, J., Girke, A., Hajirezaei, M.R., Hanke, G. and Grimm, B., 2018. Potential roles of YCF 54 and ferredoxin‐NADPH reductase for magnesium protoporphyrin monomethylester cyclase. The Plant Journal, 94(3), pp.485-496.
[12] Fujita Y. Protochlorophyllide reduction: a key step in the greening of plants. Plant and cell physiology. 1996 Jun 1;37(4):411-21.
[13] Darrah PM, Kay SA, Teakle GR, Griffiths WT. Cloning and sequencing of protochlorophyllide reductase. Biochemical Journal. 1990 Feb 1;265(3):789-98.
[14] Griffiths WT. Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations. Biochemical Journal. 1975 Dec 1;152(3):623-55.
[15] Shpilyov AV, Zinchenko VV, Shestakov SV, Grimm B, Lokstein H. Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2005 Feb 17;1706(3):195-203.
[16] Davies K, editor. Annual plant reviews, plant pigments and their manipulation. John Wiley & Sons; 2009 Feb 12.



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