CN109666595B - Microorganism and method for improving fermentation yield of hydrophobic compound of microorganism - Google Patents

Microorganism and method for improving fermentation yield of hydrophobic compound of microorganism Download PDF

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CN109666595B
CN109666595B CN201710955112.6A CN201710955112A CN109666595B CN 109666595 B CN109666595 B CN 109666595B CN 201710955112 A CN201710955112 A CN 201710955112A CN 109666595 B CN109666595 B CN 109666595B
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CN109666595A (en
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马田
刘然
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Wuhan Hesheng Technology Co ltd
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Priority to PCT/CN2018/106928 priority patent/WO2019072081A1/en
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Abstract

The invention provides a microorganism. The microorganisms include: overexpression comprises at least one of the following genes: PAH1, DGA1, OLE1, ACC1 x, ACCA2, ACCB, ACCE, DGAT, LPP β, OLE1A, OLE1B, OLE1C, OLE1D, ecaacca, ecACCB, ecacc, ecacccd, pgpB, atfA, fabA, fabB; and silencing comprises at least one selected from FLD1, TGL3, wherein the microorganism is a microorganism having the potential to synthesize hydrophobic compounds. According to the embodiment of the invention, the yield of the hydrophobic compound of the microorganism can be improved by more than 10 percent, for example, the yield of lycopene is improved by more than 40 percent, the yield of natamycin is improved by 14 percent, the yield of spinosad is improved by 20 percent, the yield of astaxanthin is improved by 50 percent, and the product has low toxicity to cell growth.

Description

Microorganism and method for improving fermentation yield of hydrophobic compound of microorganism
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving the yield of a hydrophobic product through lipid synthesis.
Background
The production of compounds by microbial fermentation is a well-established technology, for example, saccharomyces cerevisiae (Saccharomyces cerevisiae) is used as a mature food-safe strain capable of large-scale fermentation, has a clear genetic background, and a mature genetic operation system, is a research and modification object of a plurality of food-grade products, and is also a model strain in industrial production. Mature fermentation platforms and peripheral industries also create convenience for subsequent downstream product popularization.
However, how to further improve the microbial fermentation products is still a key problem to be solved by researchers.
Disclosure of Invention
The present application was made based on the findings of the following problems and facts:
most hydrophobic compounds are synthesized in microorganisms, so that the yield cannot be accumulated in a large amount due to low solubility, for example, lycopene is a typical hydrophobic compound, and due to poor water solubility, a product is positioned on a cell membrane after being synthesized, so that the accumulation of the product is limited, the product is toxic to cells, the accumulation of the product is limited to a certain level, and the growth of the cells is also limited. This poses a huge obstacle to the scale-up of the engineered strains. The inventors have found experimentally unexpectedly that the yield of hydrophobic compounds in microorganisms is significantly increased if the lipid content in the microorganism is increased. Through further research by the inventor, the lipid content in the microorganism is improved, a bearing environment is provided for the accumulation of hydrophobic products such as lycopene, astaxanthin, natamycin, spinosad and the like in the body, the toxicity of the hydrophobic products in cells is reduced as little as possible, and the influence of the accumulation of the hydrophobic products on the cell growth can be effectively reduced while the accumulation of the hydrophobic products is improved.
Based on this, in a first aspect of the invention, a microorganism is presented. According to an embodiment of the invention, the microorganism comprises: overexpression comprises at least one of the following genes: PAH1, DGA1, OLE1, ACC1 ×, ACCA2, ACCB, ACCE, DGAT, LPP β, OLE1A, OLE1B, OLE1C, OLE1D, ecACCA, ecacccb, ecacc, ecacccd, pgpB, atfA, fabA, fabB; and silencing comprises at least one selected from FLD1, TGL3, wherein the microorganism is a microorganism having the potential to synthesize hydrophobic compounds. According to the embodiment of the invention, the yield of the hydrophobic compound of the microorganism can be improved by more than 10 percent, for example, the yield of lycopene is improved by more than 40 percent, the yield of natamycin is improved by 14 percent, the yield of spinosad is improved by 20 percent, the yield of astaxanthin is improved by 50 percent, and the product has low toxicity to cell growth.
According to an embodiment of the present invention, the microorganism may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, said PAH1, DGA1, OLE1, ACC1 is derived from saccharomyces cerevisiae, preferably said ACCA2, ACCB, ACCE, DGAT, LPP β, OLE1A, OLE1B, OLE1C, OLE1D is derived from streptomyces, preferably said ecaacca, ecACCB, ecacc, ecACCC, pbg, atfA, fabA, fabB are derived from escherichia coli.
According to the embodiment of the invention, the amino acid sequence of the polypeptide coded by the gene is shown as SEQ ID NO:1,6 to 27.
MSEESLFESSPQKMEYEITNYSERHTELPGHFIGLNTVDKLEESPLRDFVKSHGGHTVISKILIANNGIAAVKEIRSVRKWAYETFGDDRTVQFVAMATPEDLEANAEYIRMADQYIEVPGGTNNNNYANVDLIVDIAERADVDAVWAGWGHASENPLLPEKLSQSKRKVIFIGPPGNAMRSLGDKISSTIVAQSAKVPCIPWSGTGVDTVHVDEKTGLVSVDDDIYQKGCCTSPEDGLQKAKRIGFPVMIKASEGGGGKGIRQVEREEDFIALYHQAANEIPGSPIFIMKLAGRARHLEVQLLADQYGTNISLFGRDCSVQRRHQKIIEEAPVTIAKAETFHEMEKAAVRLGKLVGYVSAGTVEYLYSHDDGKFYFLELNPRLQVEHPTTEMVSGVNLPAAQLQIAMGIPMHRISDIRTLYGMNPHSASEIDFEFKTQDATKKQRRPIPKGHCTACRITSEDPNDGFKPSGGTLHELNFRSSSNVWGYFSVGNNGNIHSFSDSQFGHIFAFGENRQASRKHMVVALKELSIRGDFRTTVEYLIKLLETEDFEDNTITTGWLDDLITHKMTAEKPDPTLAVICGAATKAFLASEEARHKYIESLQKGQVLSKDLLQTMFPVDFIHEGKRYKFTVAKSGNDRYTLFINGSKCDIILRQLADGGLLIAIGGKSHTIYWKEEVAATRLSVDSMTTLLEVENDPTQLRTPSPGKLVKFLVENGEHIIKGQPYAEIEVMKMQMPLVSQENGIVQLLKQPGSTIVAGDIMAIMTLDDPSKVKHALPFEGMLPDFGSPVIEGTKPAYKFKSLVSTLENILKGYDNQVIMNASLQQLIEVLRNPKLPYSEWKLHISALHSRLPAKLDEQMEELVARSLRRGAVFPARQLSKLIDMAVKNPEYNPDKLLGAVVEPLADIAHKYSNGLEAHEHSIFVHFLEEYYEVEKLFNGPNVREENIILKLRDENPKDLDKVALTVLSHSKVSAKNNLILAILKHYQPLCKLSSKVSAIFSTPLQHIVELESKATAKVALQAREILIQGALPSVKERTEQIEHILKSSVVKVAYGSSNPKRSEPDLNILKDLIDSNYVVFDVLLQFLTHQDPVVTAAAAQVYIRRAYRAYTIGDIRVHEGVTVPIVEWKFQLPSAAFSTFPTVKSKMGMNRAVAVSDLSYVANSQSSPLREGILMAVDHLDDVDEILSQSLEVIPRHQSSSNGPAPDRSGSSASLSNVANVCVASTEGFESEEEILVRLREILDLNKQELINASIRRITFMFGFKDGSYPKYYTFNGPNYNENETIRHIEPALAFQLELGRLSNFNIKPIFTDNRNIHVYEAVSKTSPLDKRFFTRGIIRTGHIRDDISIQEYLTSEANRLMSDILDNLEVTDTSNSDLNHIFINFIAVFDISPEDVEAAFGGFLERFGKRLLRLRVSSAEIRIIIKDPQTGAPVPLRALINNVSGYVIKTEMYTEVKNAKGEWVFKSLGKPGSMHLRPIATPYPVKEWLQPKRYKAHLMGTTYVYDFPELFRQASSSQWKNFSADVKLTDDFFISNELIEDENGELTEVEREPGANAIGMVAFKITVKTPEYPRGRQFVVVANDITFKIGSFGPQEDEFFNKVTEYARKRGIPRIYLAANSGARIGMAEEIVPLFQVAWNDAANPDKGFQYLYLTSEGMETLKKFDKENSVLTERTVINGEERFVIKTIIGSEDGLGVECLRGSGLIAGATSRAYHDIFTITLVTCRSVGIGAYLVRLGQRAIQVEGQPIILTGAPAINKMLGREVYTSNLQLGGTQIMYNNGVSHLTAVDDLAGVEKIVEWMSYVPAKRNMPVPILETKDTWDRPVDFTPTNDETYDVRWMIEGRETESGFEYGLFDKGSFFETLSGWAKGVVVGRARLGGIPLGVIGVETRTVENLIPADPANPNSAETLIQEPGQVWHPNSAFKTAQAINDFNNGEQLPMMILANWRGFSGGQRDMFNEVLKYGSFIVDALVDYKQPIIIYIPPTGELRGGSWVVVDPTINADQMEMYADVNARAGVLEPQGMVGIKFRREKLLDTMNRLDDKYRELRSQLSNKSLAPEVHQQISKQLADRERELLPIYGQISLQFADLHDRSSRMVAKGVISKELEWTEARRFFFWRLRRRLNEEYLIKRLSHQVGEASRLEKIARIRSWYPASVDHEDDRQVATWIEENYKTLDDKLKGLKLESFAQDLAKKIRSDHDNAIDGLSEVIKMLSTDDKEKLLKTLK(SEQ ID NO：1)。
MVDKRESYTKEDLLASGRGELFGAKGPQLPAPNMLMMDRVVKMTETGGNFDKGYVEAELDINPDLWFFGCHFIGDPVMPGCLGLDAMWQLVGFYLGWLGGEGKGRALGVGEVKFTGQVLPTAKKVTYRIHFKRIVNRRLIMGLADGEVLVDGRLIYTASDLKVGLFQDTSAF(SEQ ID NO：6)。
MPTSGTTIELIDDQFPKDDSASSGIVDEVDLTEANILATGLNKKAPRIVNGFGSLMGSKEMVSVEFDKKGNEKKSNLDRLLEKDNQEKEEAKTKIHISEQPWTLNNWHQHLNWLNMVLVCGMPMIGWYFALSGKVPLHLNVFLFSVFYYAVGGVSITAGYHRLWSHRSYSAHWPLRLFYAIFGCASVEGSAKWWGHSHRIHHRYTDTLRDPYDARRGLWYSHMGWMLLKPNPKYKARADITDMTDDWTIRFQHRHYILLMLLTAFVIPTLICGYFFNDYMGGLIYAGFIRVFVIQQATFCINSLAHYIGTQPFDDRRTPRDNWITAIVTFGEGYHNFHHEFPTDYRNAIKWYQYDPTKVIIYLTSLVGLAYDLKKFSQNAIEEALIQQEQKKINKKKAKINWGPVLTDLPMWDKQTFLAKSKENKGLVIISGIVHDVSGYISEHPGGETLIKTALGKDATKAFSGGVYRHSNAAQNVLADMRVAVIKESKNSAIRMASKRGEIYETGKFF(SEQ ID NO：7)。
MQYVGRALGSVSKTWSSINPATLSGAIDVIVVEHPDGRLSCSPFHVRFGKFQILKPSQKKVQVFINEKLSNMPMKLSDSGEAYFVFEMGDQVTDVPDELLVSPVMSATSSPPQSPETSILEGGTEGEGEGENENKKKEKKVLEEPDFLDINDTGDSGSKNSETTGSLSPTESSTTTPLDSVEERKLVEQRTKNFQQKLNKKLTEIHIPSKLDNNGDLLLDTEGYKPNKNMMHDTDIQLKQLLKDEFGNDSDISSFIKEDKNGNIKIVNPYEHLTDLSPPGTPPTMATSGSVLGLDAMESGSTLNSLSSSPSGSDTEDETSFSKEQSSKSEKTSKKGTAGSGETEKRYIRTIRLTNDQLKCLNLTYGENDLKFSVDHGKAIVTSKLFVWRWDVPIVISDIDGTITKSDALGHVLAMIGKDWTHLGVAKLFSEISRNGYNILYLTARSAGQADSTRSYLRSIEQNGSKLPNGPVILSPDRTMAALRREVILKKPEVFKIACLNDIRSLYFEDSDNEMDTEEKSTPFFAGFGNRITDALSYRTVGIPSSRIFTINTEGEVHMELLELAGYRSSYIHINELVDHFFPPVSLDSVDLRTNTSMVPGSPPNRTLDNFDSEITSGRKTLFRGNQEEKFTDVNFWRDPLVDIDNLSDISNDDSDNIDEDTDVSQQSNVSRNRANSVKTAKVTKAPQRNVSGSTNNNEVLAASSDVENASDLVGSHSSSGSTPNKSTMSKGDIGKQIYLELGSPLASPKLRYLDDMDDEDSNYNRTKSRRASSAAATSIDKEFKKLSVSKAGAPTRIVSKIDVSNDVHSLGNSDTESRREQSVNETGRNQLPHNSMDDKDLDSRVSDEFDDDEFDEDEFED(SEQ ID NO：8)。
MSGTFNDIRRRKKEEGSPTAGITERHENKSLSSIDKREQTLKPQLESCCPLATPFERRLQTLAVAWHTSSFVLFSIFTLFAISTPALWVLAIPYMIYFFFDRSPATGEVVNRYSLRFRSLPIWKWYCDYFPISLIKTVNLKPTFTLSKNKRVNEKNYKIRLWPTKYSINLKSNSTIDYRNQECTGPTYLFGYHPHGIGALGAFGAFATEGCNYSKIFPGIPISLMTLVTQFHIPLYRDYLLALGISSVSRKNALRTLSKNQSICIVVGGARESLLSSTNGTQLILNKRKGFIKLAIQTGNINLVPVFAFGEVDCYNVLSTKKDSVLGKMQLWFKENFGFTIPIFYARGLFNYDFGLLPFRAPINVVVGRPIYVEKKITNPPDDVVNHFHDLYIAELKRLYYENREKYGVPDAELKIVG(SEQ ID NO：9)。
MKINVSRPLQFLQWSSYIVVAFLIQLLIILPLSILIYHDFYLRLLPADSSNVVPLNTFNILNGVQFGTKFFQSIKSIPVGTDLPQTIDNGLSQLIPMRDNMEYKLDLNLQLYCQSKTDHLNLDNLLIDVYRGPGPLLGAPGGSNSKDEKIFHTSRPIVCLALTDSMSPQEIEQLGPSRLDVYDEEWLNTIRIEDKISLESSYETISVFLKTEIAQRNLIIHPESGIKFRMNFEQGLRNLMLRKRFLSYIIGISIFHCIICVLFFITGCTAFIFVRKGQEKSKKHS(SEQ ID NO：10)。
MKETAQEYKVSAVIPTLLKNWILRVVYATLDHIPPFVWEILHVITDIYFFWVQKLINYVRPHSRVIYYNAIKKLDECDTYQMWCQQASVVDEITGANLWRRNFFSRRYDFNSVIEQYSILENMLREEKYDVVKEKFSTTGPCMLRNFAGIGDKKLFTKSLMGTKLLIEQYLTRILEGLDILNNQTLTPTSFFQRCKLSLGTTALILQGGSLFGLFHLGVIRGLLLQDLMPNIISGSSMGACVASLFGCLSNEQLKQLLTDDNLLNIIKNDVDLLKSCGYGNLEQHLNLGTLIQNLIHHGYSQDVYLFIRFVMKYIVKEKTFEEVYQITGKVFNIVIHPTDKSCPNLLNYVTTPNVLIKSAIECSLGSGVISEDTSLLCKNLENEIEPFLNINKNKQVKFLTPENANNPSITESPYTRLTELFNVNNFIVSLARPYLAPLVVNDLKHEIKTSKYYYYKHYPNMPPINANTVRKTQRSSSQSPIKAGTVEDLEPEPLMSPVPPSSAVNDSAEYIIPELGIPQLNFTEMEPLAFKFKYHLERKLKNIATMEFRHRMEVLDNLGLLCSLIKRLIIDEKTPRSATEIAVVPRMKSLSLTRIIEGQLNNIPYWIKSGERSTWPALALIKTRCAVEFKLDDIIRARRSR(SEQ ID NO：11)。
MRKVLIANRGEIAVRVARACRDAGIASVAVYADPDRDALHVRAADEAFALGGDTPATSYLDIAKVLKAARESGADAIHPGYGFLSENAEFAQAVLDAGLIWIGPPPHAIRDLGDKVAARHIAQRAGAPLVAGTPDPVSGADEVVAFAKEHGLPIAIKAAFGGGGRGLKVARTLEEVPELYDSAVREAVAAFGRGECFVERYLDKPRHVETQCLADTHGNVVVVSTRDCSLQRRHQKLVEEAPAPFLSEAQTEQLYSSSKAILKEAGYVGAGTVEFLVGMDGTISFLEVNTRLQVEHPVTEEVAGIDLVREMFRIADGEELGYDDPALRGHSFEFRINGEDPGRGFLPAPGTVTLFDAPTGPGVRLDAGVESGSVIGPAWDSLLAKLIVTGRTRAEALQRAARALDEFTVEGMATAIPFHRTVVRDPAFAPELTGSTDPFTVHTRWIETEFVNEIKPFTTPADTETDEESGRETVVVEVGGKRLEVSLPSSLGMSLARTGLAAGARPKRRAAKKSGPAASGDTLASPMQGTIVKIAVEEGQEVQEGDLIVVLEAMKMEQPLNAHRSGTIKGLTAEVGASLTSGAAICEIKD(SEQ ID NO：12)。
MTVLDEAPGEPTDARGRVAELHGIRAAALAGPSEKATAAQHAKGKLTARERIELLLDPGSFREVEQLRRHRATGFGLEAKKPYTDGVITGWGTVEGRTVFVYAHDFRIFGGALGEAHATKIHKIMDMAIAAGAPLVSLNDGAGARIQEGVSALAGYGGIFQRNTKASGVIPQISVMLGPCAGGAAYSPALTDFVFMVRDTSQMFITGPDVVKAVTGEEITQNGLGGADVHAETSGVCHFAYDDEETCLAEVRYLLSLLPQNNRENPPRAESSDPVDRRSDTLLDLVPADGNRPYDMTKVIEELVDEGEYLEVHERWARNIICALARLDGRVVGIVANQPQALAGVLDIEASEKAARFVQMCDAFNIPIITLLDVPGFLPGVDQEHGGIIRHGAKLLYAYCNATVPRISLILRKAYGGAYIVMDSQSIGADLTYAWPTNEIAVMGAEGAANVIFRRQIADAEDPEAMRARMVKEYKSELMHPYYAAERGLVDDVIDPAETREVLITSLAMLHTKHADLPSRKHGNPPQ(SEQ ID NO：13)。
MSPADIRVEKGHAEPEEVAAITALLLARAAARPAEIAPTHGGGRARAGWRRLEREPGFRAPHSWR(SEQ ID NO：14)。
MTPDPLAPLDLAFWNIESAEHPMHLGALGVFEADSPTAGALAADLLAARAPAVPGLRMRIRDTWQPPMALRRPFAFGGATREPDPRFDPLDHVRLHAPATDFHARAGRLMERPLERGRPPWEAHVLPGADGGSFAVLFKFHHALADGLRALTLAAGVLDPMDLPAPRPRPEQPPRGLLPDVRALPDRLRGALSDAGRALDIGAAAALSTLDVRSSPALTAASSGTRRTAGVSVDLDDVHHVRKTTGGTVNDVLIAVVAGALRRWLDERGDGSEGVAPRALIPVSRRRPRSAHPQGNRLSGYLMRLPVGDPDPLARLGTVRAAMDRNKDAGPGRGAGAVALLADHVPALGHRLGGPLVSGAARLWFDLLVTSVPLPSLGLRLGGHPLTEVYPLAPLARGHSLAVAVSTYRGRVHYGLLADAKAVPDLDRLAVAVAEEVETLLTACRP(SEQ ID NO：15)。
MRTERKPTRLDRVFARLDREPERPALLDVPEMSRHRIALFAGTLAFYIAIVWAVVITSWLVRLDWQVMFFRPYQQWPEIHAFVDYYVVLGQRGPTAVMVAAWLGWRSWRQHTLRPLLALGVSLLLLNVTVGAAKYGMGRLGPHYATTIGANEMWLGGDIFPSGHTANAVVTWGILAYLASTHRTRRWLSAISAVTSLGVGMSTVYLGTHWLSDVLLGWVAGLLILLALPWFEPLITRAEAWILGLRDRWYTRRDRRSTTRPPLGPPVPVSPPGSGSRPQAPAREPVAAPRTARAPAHLAPGPHTARSDRTPVTPAGSRRPPHSDRHARNTAPTARPLSGG(SEQ ID NO：16)。
MTTSSDVIPDAPQPAGDAAGPSATLGGEQKRSIEQITLLLFITLPFLALVAAVPLAWGWGVSWLDLGLLVFFYFLGCHGITIGFHRHFTHGSFKAKRPLKIALAIAGSMAVEGPLVRWVADHRKHHKFSDDEGDPHSPWRYGETVPALIKGLWWAHIAWMFDEEQTPQEKYAPDLIKDPALRAVSRQFILWTVVSLALPALIGGLVTMSWWGAFTGFFWGSLVRVALLHHVTWSINSICHAVGKRPFKSRDRSGNVWWLAILSCGESWHNLHHADPTSARHGVMRGQLDSSARLIRWFEQLGWAYDVRWPSRSRIDSRRNTDQDGARRRKETAKAA(SEQ ID NO：17)。
MTMATTATRSDTPGSDFARLSKKVADAGLLGRRPGYYTLRITAVTGLYAAGWAAFVLVGASWWTLAIAAFLAVMYGQVALVAHDMAHRQVFRRRRASELSGRIAGASIGMSYGWWQDKHTRHHANPNTEDLDPDIGPDLLVWSPDQARAATGLPRLLGRWQAFLFFPLLTLEGFNLHVASGRAMANRRLKRRALDGALLLAHCAVYLTALFWVLPPGMAIAFLAVHQCLFGVYLGSAFAPNHKGMPILTADDRPDFLRRQVLTSRNVNGGLFTDLALGGLNHQIEHHLFPSMPSPNLRKARAIVRRYCRDLGVDYAETGLVASYRLALTSLHDAGTPLRRTRVRA(SEQ ID NO：18)。
MPLPRETLPPDTGGSREGSEFTPLLRDVREQQLLERRTGWYARTIAVNALGLAAVGTGMALLGDSWWVLALAPVLAVLCARTAFIGHDAGHAQISGSRAVNRRIGLVHGNLLLGMSYAWWNDKHNRHHANPNHIDKDPDVAADVLVFTSGQAATRTGFRGRLTRHQAWLFFPLTLLEGLALKLHGFQHLRRQRGRARLVEGALLVAHVAGYVTLLLATMPLAHALVFAALHQALFGLHLGMAFAPNHKGMDMPDPDSEAEKWGHLRRQVLTSRNVRGGFLTDWFLGGLNYQIEHHLFPSMPRPHLGLAQAAVKAHCRDLGIPYAETGLVDSYRQALRHMHEVGEPLRADI(SEQ ID NO：19)。
MLVESLPTPAQEKDRERGSDFSELSRRIAGAGLLRRRPLYYTVRFGAVALALAGGVAAFVALGDSWSQLFVAVALAVVFGQLGLAAHDLAHRQVFTRRRPSEAGGLLTANLLLGMSYGWWMNKHTRHHANPNHEEKDPDVSPDILVWSRGQASRATGLPRFVGRHQAALFFPLLTLEGLNLSFNSFKALGSRAVKRPVLEGTLLVAHFAVYFGGLFTVLSPGKALVFLAVHQGLFGIYLGSVFAPNHKGMPMIEEGMRLDFLRRQVLTSRNVRGGALVDAFMGGLNYQIEHHLFPSMPTPALGRAQAITEAYCAELGVPYHQTGLLASHREALRHMRSVGEPLRAAR(SEQ ID NO：20)。
MSLNFLDFEQPIAELEAKIDSLTAVSRQDEKLDINIDEEVHRLREKSVELTRKIFADLGAWQIAQLARHPQRPYTLDYVRLAFDEFDELAGDRAYADDKAIVGGIARLDGRPVMIIGHQKGRETKEKIRRNFGMPAPEGYRKALRLMQMAERFKMPIITFIDTPGAYPGVGAEERGQSEAIARNLREMSRLGVPVVCTVIGEGGSGGALAIGVGDKVNMLQYSTYSVISPEGCASILWKSADKAPLAAEAMGIIAPRLKELKLIDSIIPEPLGGAHRNPEAMAASLKAQLLADLADLDVLSTEDLKNRRYQRLMSYGYA(SEQ ID NO：21)。
MDIRKIKKLIELVEESGISELEISEGEESVRISRAAPAASFPVMQQAYAAPMMQQPAQSNAAAPATVPSMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAFIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPLVVIE(SEQ ID NO：22)。
MLDKIVIANRGEIALRILRACKELGIKTVAVHSSADRDLKHVLLADETVCIGPAPSVKSYLNIPAIISAAEITGAVAIHPGYGFLSENANFAEQVERSGFIFIGPKAETIRLMGDKVSAIAAMKKAGVPCVPGSDGPLGDDMDKNRAIAKRIGYPVIIKASGGGGGRGMRVVRGDAELAQSISMTRAEAKAAFSNDMVYMEKYLENPRHVEIQVLADGQGNAIYLAERDCSMQRRHQKVVEEAPAPGITPELRRYIGERCAKACVDIGYRGAGTFEFLFENGEFYFIEMNTRIQVEHPVTEMITGVDLIKEQLRIAAGQPLSIKQEEVHVRGHAVECRINAEDPNTFLPSPGKITRFHAPGGFGVRWESHIYAGYTVPPYYDSMIGKLICYGENRDVAIARMKNALQELIIDGIKTNVDLQIRIMNDENFQHGGTNIHYLEKKLGLQEK(SEQ ID NO：23)。
MSWIERIKSNITPTRKASIPEGVWTKCDSCGQVLYRAELERNLEVCPKCDHHMRMTARNRLHSLLDEGSLVELGSELEPKDVLKFRDSKKYKDRLASAQKETGEKDALVVMKGTLYGMPVVAAAFEFAFMGGSMGSVVGARFVRAVEQALEDNCPLICFSASGGARMQEALMSLMQMAKTSAALAKMQERGLPYISVLTDPTMGGVSASFAMLGDLNIAEPKALIGFAGPRVIEQTVREKLPPGFQRSEFLIEKGAIDMIVRRPEMRLKLASILAKLMNLPAPNPEAPREGVVVPPVPDQEPEA(SEQ ID NO：24)。
MRSIARRTAVGAALLLVMPVAVWISGWRWQPGEQSWLLKAAFWVTETVTQPWGVITHLILFGWFLWCLRFRIKAAFVLFAILAAAILVGQGVKSWIKDKVQEPRPFVIWLEKTHHIPVDEFYTLKRAERGNLVKEQLAEEKNIPQYLRSHWQKETGFAFPSGHTMFAASWALLAVGLLWPRRRTLTIAILLVWATGVMGSRLLLGMHWPRDLVVATLISWALVAVATWLAQRICGPLTPPAEENREIAQREQES(SEQ ID NO：25)。
MRPLHPIDFIFLSLEKRQQPMHVGGLFLFQIPDNAPDTFIQDLVNDIRISKSIPVPPFNNKLNGLFWDEDEEFDLDHHFRHIALPHPGRIRELLIYISQEHSTLLDRAKPLWTCNIIEGIEGNRFAMYFKIHHAMVDGVAGMRLIEKSLSHDVTEKSIVPPWCVEGKRAKRLREPKTGKIKKIMSGIKSQLQATPTVIQELSQTVFKDIGRNPDHVSSFQAPCSILNQRVSSSRRFAAQSFDLDRFRNIAKSLNVTINDVVLAVCSGALRAYLMSHNSLPSKPLIAMVPASIRNDDSDVSNRITMILANLATHKDDPLQRLEIIRRSVQNSKQRFKRMTSDQILNYSAVVYGPAGLNIISGMMPKRQAFNLVISNVPGPREPLYWNGAKLDALYPASIVLDGQALNITMTSYLDKLEVGLIACRNALPRMQNLLTHLEEEIQLFEGVIAKQEDIKTAN(SEQ ID NO：26)。
MKRAVITGLGIVSSIGNNQQEVLASLREGRSGITFSQELKDSGMRSHVWGNVKLDTTGLIDRKVVRFMSDASIYAFLSMEQAIADAGLSPEAYQNNPRVGLIAGSGGGSPRFQVFGADAMRGPRGLKAVGPYVVTKAMASGVSACLATPFKIHGVNYSISSACATSAHCIGNAVEQIQLGKQDIVFAGGGEELCWEMACEFDAMGALSTKYNDTPEKASRTYDAHRDGFVIAGGGGMVVVEELEHALARGAHIYAEIVGYGATSDGADMVAPSGEGAVRCMKMAMHGVDTPIDYLNSHGTSTPVGDVKELAAIREVFGDKSPAISATKAMTGHSLGAAGVQEAIYSLLMLEHGFIAPSINIEELDEQAAGLNIVTETTDRELTTVMSNSFGFGGTNATLVMRKLKD(SEQ ID NO：7)。
The inventors have found that overexpression of a gene derived from the above-mentioned microorganism in a microorganism having the potential to synthesize a hydrophobic compound can significantly promote accumulation of the hydrophobic compound in the microorganism.
According to an embodiment of the present invention, the microorganism includes at least one selected from the group consisting of yeast, escherichia coli, actinomycetes, bacillus subtilis, corynebacterium glutamicum, aspergillus niger, aspergillus oryzae, trichoderma viride, and trichoderma reesei. The inventors have found that overexpression and silencing of at least one of the genes in the microorganism comprises at least one of FLD1, TGL3, and that the accumulation of hydrophobic compounds in the microorganism is further significantly increased.
According to an embodiment of the invention, the hydrophobic compound comprises at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosyn polyketides, sesterterpenes, triterpenes and tetraterpenoids. The yield of the above hydrophobic compounds is higher in the microorganism according to the embodiment of the present invention.
In a second aspect of the invention, a method is provided for increasing the fermentation yield of a hydrophobic compound from a microorganism. According to an embodiment of the invention, the compound comprises: increasing the lipid content in the microorganism, wherein the microorganism is a microorganism having the potential to synthesize hydrophobic compounds. The inventor unexpectedly finds that the fermentation yield of the hydrophobic compound of the microorganism can be remarkably improved by improving the lipid content in the microorganism with the potential of synthesizing the hydrophobic compound, the fermentation yield of the hydrophobic compound of the microorganism can be improved by more than 10 percent, for example, the yield of lycopene is improved by more than 40 percent, the yield of natamycin is improved by 14 percent, the yield of spinosad is improved by 20 percent, the yield of astaxanthin is improved by 50 percent, and the product has little toxicity to cell growth.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to an embodiment of the invention, the lipid is a triglyceride. The inventors found that increasing the triglyceride content in the microorganism has a more significant effect on the promotion of the fermentation yield of hydrophobic compounds.
According to an embodiment of the present invention, the increase of the lipid content in the microorganism is achieved by increasing at least one of the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids. Further, the fermentation yield of the hydrophobic compound is further improved.
According to an embodiment of the invention, said increasing the lipid content in a microorganism is by overexpression in said microorganism of at least one gene comprising a sequence selected from the group consisting of: PAH1, DGA1, OLE1, ACC1 ×, ACCA2, ACCB, ACCE, DGAT, LPP β, OLE1A, OLE1B, OLE1C, OLE1D, ecACCA, ecacccb, ecacc, ecacccd, pgpB, atfA, fabA, fabB; and silencing comprises at least one of FLD1 and TGL 3. By the mode, the lipid content in the microorganism can be effectively improved, a bearing environment is further provided for the accumulation of the hydrophobic product in vivo, the toxicity of the hydrophobic product in cells is reduced as little as possible, and the influence of the accumulation of the hydrophobic product on the growth of the cells can be further effectively reduced while the accumulation of the hydrophobic product is further improved.
According to an embodiment of the invention, said overexpression is achieved by introducing into said microorganism a construct comprising a gene of interest to be overexpressed and a regulated expression promoter operably linked to said gene of interest. And the target gene can be overexpressed in a controllable manner, the target gene is overexpressed in the cell growth phase, and the expression is stopped in the product accumulation phase.
According to an embodiment of the present invention, the expression-regulating promoter is pHXT1.pHXT1 is a glucose control type promoter, and further controllable expression of a target gene more conveniently and efficiently can be realized.
According to an embodiment of the present invention, pHXT1 has the nucleotide sequence shown in SEQ ID NO 28.
TGCAGGTCTCATCTGGAATATAATTCCCCCCTCCTGAAGCAAATTTTTCCTTTGAGCCGGAATTTTTGATATTCCGAGTTCTTTTTTTCCATTCGCGGAGGTTATTCCATTCCTAAACGAGTGGCCACAATGAAACTTCAATTCATATCGACCGACTATTTTTCTCCGAACCAAAAAAATAGCAGGGCGAGATTGGAGCTGCGGAAAAAAGAGGAAAAAATTTTTTCGTAGTTTTCTTGTGCAAATTAGGGTGTAAGGTTTCTAGGGCTTATTGGTTCAAGCAGAAGAGACAACAATTGTAGGTCCTAAATTCAAGGCGGATGTAAGGAGTATTGGTTTCGAAAGTTTTTCCGAAGCGGCATGGCAGGGACTACTTGCGCATGCGCTCGGATTATCTTCATTTTTGCTTGCAAAAACGTAGAATCATGGTAAATTACATGAAGAATTCTCTTTTTTTTTTTTTTTTTTTTTTTTTTACCTCTAAAGAGTGTTGACCAACTGAAAAAACCCTTCTTCAAGAGAGTTAAACTAAGACTAACCATCATAACTTCCAAGGAATTAATCGATATCTTGCACTCCTGATTTTTCTTCAAAGAGACAGCGCAAAGGATTATGACACTGTTGCATTGAGTCAAAAGTTTTTCCGAAGTGACCCAGTGCTCTTTTTTTTTTTCCGTGAAGGACTGACAAATATGCGCACAAGATCCAATACGTAATGGAAATTCGGAAAAACTAGGAAGAAATGCTGCAGGGCATTGCCGTGCCGATCTTTTGTCTTTCAGATATATGAGAAAAAGAATATTCATCAAGTGCTGATAGAAGAATACCACTCATATGACGTGGGCAGAAGACAGCAAACGTAAACATGAGCTGCTGCGACATTTGATGGCTTTTATCCGACAAGCCAGGAAACTCCACCATTATCTAATGTAGCAAAATATTTCTTAACACCCGAAGTTGCGTGTCCCCCTCACGTTTTTAATCATTTGAATTAGTATATTGAAATTATATATAAAGGCAACAATGTCCCCATAATCAATTCCATCTGGGGTCTCATGTTCTTTCCCCACCTTAAAATCTATAAAGATATCATAATCGTCAACTAGTTGATATACGTAAAATC(SEQ ID NO:28)。
According to an embodiment of the present invention, the microorganism includes at least one selected from yeast, escherichia coli, actinomycetes, bacillus subtilis, corynebacterium glutamicum, aspergillus niger, aspergillus oryzae, trichoderma viride, trichoderma reesei, and the like. The lipid content in the microorganism is increased, and the accumulation of hydrophobic compounds in the microorganism is further significantly increased.
According to an embodiment of the invention, the hydrophobic compound comprises at least one selected from the group consisting of lycopene, carotene, astaxanthin, natamycin, spinosad, polyketide, sesterterpene, triterpene and tetraterpenoid. The yield of the above hydrophobic compounds is higher with the method according to an embodiment of the present invention.
In a third aspect of the invention, the invention proposes the use of a microorganism as described above for increasing the fermentative production of hydrophobic compounds. As described above, the microorganisms according to the embodiments of the present invention have the property of producing hydrophobic compounds with high yield, and the microorganisms according to the embodiments of the present invention can be effectively used in the fermentative production of hydrophobic compounds with high yield of hydrophobic compounds and low toxicity to the microorganisms.
It should be noted that the amino acid sequence of the encoded polypeptide disclosed in the gene of the present application means that any nucleotide sequence encoding the polypeptide having the amino acid sequence is within the scope of the present application.
It is noted that the amino acid sequence of the polypeptide encoded by the gene disclosed in the present application means a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the disclosed amino acid sequence; or
Polypeptides having one or more amino acid substitutions, deletions and/or additions to the disclosed amino acid sequences are within the scope of the present application.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a graph showing the results of different degrees of improvement in the yield of lycopene-producing bacteria overexpressing triglycerides compared to the yield of the original lycopene-producing bacteria TM606, according to an embodiment of the present invention.
Detailed Description
The examples of the present invention are described in detail below, and are represented by Saccharomyces cerevisiae, which produces lycopene, escherichia coli, which produces astaxanthin, streptomyces, which produces natamycin, and Saccharopolyspora, which produces spinosad. The following examples are illustrative only and are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the following examples, the amino acid sequences of the polypeptides encoded by the PAH1, DGA1, OLE1, ACCA2, ACCB, ACCE, DGAT, LPP β, OLE1A, OLE1B, OLE1C, OLE1D, ecACCA, ecACCB, ecACCC, ecACCD, pgpB, atfA, fabA, fabB genes are shown in the sequence listing.
EXAMPLE 1 construction of plasmids required by lycopene-producing bacteria
The primers used in the examples are shown in Table 1.
Table 1: primers for constructing lycopene engineering strain
Figure BDA0001433789650000081
Figure BDA0001433789650000091
Figure BDA0001433789650000101
Figure BDA0001433789650000111
Figure BDA0001433789650000121
Figure BDA0001433789650000131
Plasmids constructed in the examples and fragments used in plasmid construction were PCR amplified using the corresponding primers and template, as detailed in Table 2.
Table 2: plasmid constructed in this example
Figure BDA0001433789650000132
Figure BDA0001433789650000141
Figure BDA0001433789650000151
Figure BDA0001433789650000161
And (3) PCR reaction system: 30.5 μ L H 2 O, 10. Mu.L of 5 × reaction buffer, 4. Mu.L of 2.5mM dNTPs, 2. Mu.L of 10mM forward primer, 2. Mu.L of 10mM reverse primer, 1. Mu.L of template DNA (1-100 ng), 0.5. Mu.L of Phusion High-Fidelity DNA Polymerase. PCR reaction procedure: pre-denaturation at 98 ℃ for 30s; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s/K, for 30 cycles; finally, extension was carried out at 72 ℃ for 10min.
The plasmids constructed in the examples were constructed by the yeast assembly method: the different plasmids were assembled according to the fragments listed in table 2.
PCR fragments were cut and recovered, and the volume used was calculated as 300ng per fragment.
According to the requirement of each plasmid, taking corresponding fragments, mixing uniformly, calculating the volume, adding 10% of 3M NaAc and 2% of 10mg/mL glycogen, mixing uniformly, adding 2 times of absolute ethyl alcohol, mixing uniformly, placing at-80 ℃ for 2h,13, 000rpm, centrifuging at 4 ℃ for 20min, and removing supernatant. Washing with 500 μ L70% ethanol, centrifuging at room temperature of 13 rpm and 200rpm for 3min, discarding supernatant, air drying, adding 4 μ L ddH 2 And (4) re-dissolving the O for later use.
Transforming the treated mixed fragment into Saccharomyces cerevisiae by lithium acetate method, coating corresponding YPD resistant plate, and culturing at 30 deg.C. (the colony growth takes about 3 days). And when the colony grows to a proper size, selecting a monoclonal to a liquid culture medium with YPD corresponding resistance, culturing at 30 ℃ and 220rpm for 20h, upgrading the plasmid, transforming E.coli DH10B, coating an LB solid plate (Ampicillin resistance), culturing at 37 ℃ for about 2 days, selecting a monoclonal to an LB-Amp liquid culture medium, culturing at 37 ℃ and 220rpm for 16h, and carrying out enzyme digestion verification.
Example 2 construction of Saccharomyces cerevisiae lycopene Strain
The construction method of the saccharomyces cerevisiae lycopene strain TM606 comprises the following steps:
expression of tmgh 1 increased the conversion rate of the MVA pathway; silencing GAL1,7, 10 uses galactose to induce lycopene production; the construction method comprises the steps of carrying out enzyme digestion on plasmid pZY141 by NotI, then recovering a target fragment, transforming and integrating the plasmid pZY141 into a Saccharomyces cerevisiae (CEN. PK2-1D) strain by a lithium acetate method according to the using amount of 200ng, screening by using a plate containing corresponding nutritional defects to obtain a target strain, and carrying out PCR verification.
PaCrtB (SEQ ID NO: 3) derived from pantococcus agglomerans, tmCrtE (SEQ ID NO: 2) derived from Taxus plant, btCrtI (SEQ ID NO: 4) derived from Nostospora trispora were heterologously synthesized into lycopene, and the copy number was adjusted.
MAYTAMAAGTQSLQLRTVASYQECNSMRSCFKLTPFKSFHGVNFNVPSLGAANCEIMGHLKLGSLPYKQCSVSSKSTKTMAQLVDLAETEKAEGKDIEFDFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPALCIAACELVGGSQDLAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKPTNHKVFGEDTAVLAGDALLSFAFEHIAVATSKTVPSDRTLRVISELGKTIGSQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILGGATEDEIARIRRYARCVGLLFQVVDDILDVTKSSEELGKTAGKDLLTDKATYPKLMGLEKAKEFAAELATRAKEELSSFDQIKAAPLLGLADYIAFRQN(SEQ ID NO：2)。
MSQPPLLDHATQTMANGSKSFATAAKLFDPATRRSVLMLYTWCRHCDDVIDDQTHGFASEAAAEEEATQRLARLRTLTLAAFEGAEMQDPAFAAFQEVALTHGITPRMALDHLDGFAMDVAQTRYVTFEDTLRYCYHVAGVVGLMMARVMGVRDERVLDRACDLGLAFQLTNIARDIIDDAAIDRCYLPAEWLQDAGLTPENYAARENRAALARVAERLIDAAEPYYISSQAGLHDLPPRCAWAIATARSVYREIGIKVKAAGGSAWDRRQHTSKGEKIAMLMAAPGQVIRAKTTRVTPRPAGLWQRPV(SEQ ID NO：3)。
MSDQKKHIVVIGAGIGGTATAARLAREGFRVTVVEKNDFSGGRCSFIHHDGHRFDQGPSLYLMPKLFEDAFADLDERIGDHLDLLRCDNNYKVHFDDGDAVQLSSDLTKMKGELDRIEGPLGFGRFLDFMKETHVHYEQGTFIAIKRNFETIWDLIRLQYVPEIFRLHLFGKIYDRASKYFQTKKMRMAFTFQTMYMGMSPYDAPAVYSLLQYTEFAEGIWYPRGGFNMVVQKLESIASKKYGAEFRYQSPVAKINTVDKDKRVTGVTLESGEVIEADAVVCNADLVYAYHHLLPPCNWTKKTLASKKLTSSSISFYWSMSTKVPQLDVHNIFLAEAYKESFDEIFNDFGLPSEASFYVNVPSRIDESAAPPNKDSIIVLVPIGHMKSKTGNSAEENYPELVNRARKMVLEVIERRLGVNNFANLIEHEEVNDPSVWQSKFNLWRGSILGLSHDVFQVLWFRPSTKDSTNRYDNLFFVGASTHPGTGVPIVLAGSKLTSDQVCKSFGQNPLPRKLQDSQKKYAPEQTRKTESHWIYYCLACYFVTFLFFYFFPRDDTTTPASFINQLLPNVFQGQNSNDIRI(SEQ ID NO：4)。
The construction method comprises the steps of digesting plasmids pZY151, pZY184 and pZY196 by NotI, recovering a target fragment, continuously integrating the target fragment into the strains through the transformation of the yeast by a lithium acetate method, screening by using a plate containing corresponding auxotrophy to obtain a target strain, and verifying by PCR.
Expressing POS5 gene to balance the reducing power in the yeast; the construction method comprises the steps of digesting the plasmid pTM206 by NotI, recovering a target fragment, transforming and integrating the target fragment into the strain, screening by using a plate containing corresponding resistance to obtain a target strain, and verifying by PCR.
Expression of ADH2, ACS6 (SEQ ID NO: 5), ALD6 gene increased the supply of precursors for lycopene synthesis.
MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTFWGEQGKILDWITPYQKVKNTSFAPGNVSIKWYEDGTLNLAANCLDRHLQENGDRTAIIWEGDDASQSKHISYRELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAVAMLACARIGAVHSVIFGGFSPEAIAGRIIDSSSRLVITADEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRTGNDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPLFILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYHPGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEGVPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEGDKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKEKCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFFGVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLFGDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITGRVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIPHAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIGPLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNLGDTSTLADPGVVEKPLEEKQAIAMPS(SEQ ID NO：5)。
The construction method comprises the steps of digesting the plasmid pTM303 by NotI enzyme, recovering a target fragment, transforming and integrating the target fragment into the strain, screening by using a plate containing corresponding resistance to obtain a target strain, and verifying by PCR.
Silencing Ypl062W, exg1 gene, regulates the yeast synthetic lycopene system as a whole. The construction method comprises the following steps: constructing a corresponding knockout box fragment by taking a hygromycin resistance gene as a marker in a Ypl062W gene to be inactivated, constructing a corresponding knockout box fragment by taking a G418 resistance gene as a marker in an Exg1 gene, respectively integrating the fragments into the engineering bacteria through lithium acetate yeast transformation, screening by using a plate containing hygromycin resistance, and verifying by PCR.
When the strain needs to reuse the resistance selection marker, the marker is discarded using the following method:
the pSH47 plasmid is transformed by the strain to be lost and a plurality of colonies are picked up and cultured in 5mL YPD medium at 30 ℃ and 220rpm overnight after the colonies grow out, centrifuged at 3,000 rpm for 5min at room temperature, the supernatant is discarded, washed 2 times by 5mL YPDG medium without glucose and with 1% galactose, 5mL of the medium is added and cultured at 30 ℃ and 220rpm overnight, and the SC non-auxotrophic solid medium containing 1 g/L5-fluoro-orotic acid is spread and cultured at 30 ℃. After the fungus grows out, picking the corresponding YPD solid plate with the mark to be lost and coating the single clone for verification.
Example 3 construction of a high lipid-producing synthetic lycopene Strain
The modification strategy used in this example is as follows:
(1) Mainly used for improving triglyceride synthesis, the downstream synthesis flux of triglyceride is improved by over-expressing PAH1 (SEQ ID NO: 8) and DGA1 (SEQ ID NO: 9) genes, and the constructed strain is TM6065, and the construction method is as follows:
the plasmid pTM705 is cut by NotI enzyme, the target fragment is recovered, and is transformed and integrated into the TM606 strain by a lithium acetate method yeast according to the dosage of 200ng, and the strain with resistance is obtained by screening a plate with corresponding resistance and is verified by PCR.
(2) Mainly improves the precursor of triglyceride synthesis, improves the upstream synthesis flux of triglyceride by over-expressing ACC1 (SEQ ID NO: 1) gene with two mutation sites, constructs a strain TM6066, and has the following construction method:
the plasmid pTM706 was digested with NotI, the target fragment was recovered, transformed and integrated into the TM606 strain as described above, and the strain with resistance was obtained by screening using a plate having the corresponding resistance and verified by PCR.
(3) Reducing the degradation of triglyceride on the basis of improving the synthesis of the triglyceride, knocking out TGL3 (SEQ ID NO: 11) gene on the basis of a strain TM6065 to construct a strain TM6068, wherein the construction method comprises the following steps:
the plasmid pTM708 was digested with NotI, the target fragment was recovered, transformed and integrated into the TM606 strain, and the strain with resistance was obtained by screening using a plate containing the corresponding resistance and verified by PCR.
(4) Mainly increasing the size of intracellular lipid droplets, promoting the polymerization of lipid droplets by knocking out FLD1 gene (SEQ ID NO: 10), and constructing a strain TM701 by the following method:
the plasmid pTM701 is digested by NotI, the target fragment is recovered, transformed and integrated into a TM606 strain, a plate containing corresponding resistance is used for screening to obtain a strain with resistance, and PCR verification is carried out.
(5) Mainly increasing intracellular unsaturated fatty acid, and through over-expressing OLE1 (SEQ ID NO: 7) gene, constructing strain TM704, the construction method is as follows:
the plasmid pTM704 was digested with NotI, the target fragment was recovered, transformed and integrated into the TM606 strain, and the strain with resistance was obtained by screening using a plate containing the corresponding resistance and verified by PCR.
(6) The method for upstream and downstream synthesis combination of triglyceride comprises the steps of constructing strains TM60656 or TM60686, digesting plasmids pTM705/pTM708 and pTM706 with NotI respectively, recovering target fragments, integrating the target fragments into a TM606 strain, screening by using a plate containing corresponding resistance to obtain a strain with resistance, and verifying by PCR.
(7) Combining the upstream and downstream synthesis of intracellular triglyceride and the size of intracellular lipid droplets, the strain is constructed as TM70156/TM70186, and the construction method is as follows:
the plasmids pTM705/pTM708 and pTM706 were digested with NotI, the target fragments were recovered, transformed and integrated into TM701, and the resistant strains were obtained by screening using plates containing the corresponding resistance and verified by PCR.
(8) The strain TM707 is constructed by increasing the size of the lipid drop combined with the intracellular unsaturated fatty acid, and the construction method comprises the following steps:
the plasmid pTM707 is digested with NotI, the target fragment is recovered, transformed and integrated into the TM606 strain, and the strain with resistance is obtained by screening with a plate containing corresponding resistance and verified by PCR.
(9) The strain is constructed by combining the upstream and downstream synthesis of intracellular triglyceride and the content of intracellular unsaturated fatty acid, and the construction method is as follows:
the plasmids pTM705/pTM708 and pTM706 were digested with NotI, the target fragments were recovered, transformed and integrated into TM704 strain, and the strain with resistance was selected by using a plate containing the corresponding resistance and verified by PCR.
(10) Combining the method for improving the upstream and downstream synthesis of intracellular triglyceride, the content of intracellular unsaturated fatty acid and the size of lipid droplets, the strain is constructed as TM70756/TM70786, and the construction method is as follows:
the plasmids pTM705/pTM708 and pTM706 were digested with NotI, the target fragments were recovered, transformed and integrated into TM707 strain, and the strain with resistance was obtained by screening using a plate containing the corresponding resistance and verified by PCR.
Example 4 fermentation Process for lycopene engineering bacteria by shake flask culture
In this example, the inventors have described in detail the fermentation culture process of a part of the engineered strain obtained in example 3.
Shake flask fermentation adopts two-stage seed culture, and selects the recombinant strain on the plate to containAfter overnight shake culture (generally 14-18 h) at 30 ℃ in PA flasks with 5mL YPD medium, the bacteria grew to logarithmic growth phase (OD) 600 About 5-8), obtaining a primary seed solution, transferring the strain into a 250mL shaking flask containing 50mL YPD medium by using 1% inoculum size, and carrying out shaking culture for about 14-18h to obtain a secondary seed solution. The final concentration of the cells was OD 600 =0.5 secondary seed liquid was calculated and inoculated into a 500mL shake flask containing 200mL fermentation medium YPD (containing 1% galactose), and shake flask fermentation was carried out at 30 ℃ and 220rpm. Sampling after 96h, storing in a refrigerator at the temperature of 80 ℃ below zero, and measuring the lycopene output accumulation.
EXAMPLE 5 product extraction and detection
In this example, the inventors extracted the product obtained after the fermentation treatment and examined the yield of lycopene.
The sample was thawed by removing the sample from the refrigerator, 500. Mu.L of the fermentation broth was placed in a 15mL centrifuge tube (precooled on ice), centrifuged at 5, 000rpm and 4 ℃ for 2min to collect the cells, and the supernatant was removed. Adding 4mL of acetone (HPLC grade), 0.2g of glass beads, 1% of antioxidant, shaking for 5min, performing ice bath ultrasound for 5-10min, centrifuging for 5, 000rpm at 4 ℃ for 2min, and transferring the supernatant to a 50mL centrifuge tube; repeating the above extraction process and collecting extractive solution until thallus has no obvious yellow color; the collected extracts were mixed well, centrifuged at 2mL,12,000 rpm for 10min, and the supernatant was transferred to a brown sample bottle for HPLC analysis.
The detection method comprises the following steps: lycopene detection was performed using a quaternary HPLC, with the detector being a uv detector, absorption wavelength 474nm, chromatography column being Agilent Zorbax C18 (150mm 4.6mm 5 μm), mobile phase a (acetonitrile: water = 9) and mobile phase B (methanol: isopropanol = 3) analyzed under the following conditions: 0-90% by weight of B (0-15 min), 90% by weight of B (15-30 min), 90% -0B (30-35 min), flow rate of 1mL/min.
As shown in FIG. 1, it can be seen that the yield of lycopene-producing bacteria overexpressing triglyceride is improved to a different extent than that of the original lycopene-producing bacteria TM 606.
Example 6 construction of Natamycin-producing Strain overexpressing triglyceride and fermentation
Natamycin is a odorless, tasteless, low-dose and high-safety food preservative, is prepared by fermenting streptomyces natalensis, is white to milky odorless and tasteless crystalline powder, and has the action mechanism that the natamycin is combined with ergosterol and other sterol groups of fungi to inhibit the biosynthesis of ergosterol, so that cell membranes are distorted, and finally leakage is caused to cause cell death. The natamycin is used for surface treatment of the dough in the baked food, and the natamycin has obvious effect of prolonging the shelf life. Natamycin is slightly soluble in water and poorly soluble in most organic solvents. The solubility in water at room temperature is 30-100 mg/L. At a pH below 3 or above 9, the solubility will increase but the stability of natamycin will decrease.
In this example, the present inventors constructed plasmids shown in Table 2 in Streptomyces natalensis, specifically referring to example 1, corresponding Fragments were obtained by PCR of corresponding genes derived from Streptomyces natalensis, and constructed into each desired plasmid after sequentially linking the corresponding promoters to each other and then linking the resulting Fragments to the ermE promoter in vector pSET152 by the Gibson method (Daniel G.Gibson, enzyme Assembly of overlaying DNA Fragments, methods in Enzymology, volume 498, 2011, pages 349-361.). The constructed plasmid is transformed into Escherichia coli ET12567/pUZ8002 through electricity, the transformed Escherichia coli is cultured in 2ml LB 37 degrees overnight, 200 microliter bacterial liquid is inoculated to 5ml LB 37 degrees culture. Meanwhile, carrying out heat shock and pre-germination treatment on the streptomyces natalensis spores, suspending the streptomyces natalensis spores in 2ml TES buffer solution, carrying out water bath at 50 ℃ for 10 minutes, cooling to room temperature, adding an isovolumetric spore pre-germination culture medium, placing the spore pre-germination culture medium and the prepared escherichia coli together in a shaking table at 37 ℃ for 2.5 hours, washing the escherichia coli for 2 times by using an LB culture medium, meanwhile, collecting the streptomyces spores through centrifugation, mixing the escherichia coli and the streptomyces spores uniformly, and carrying out shaking table culture according to the proportion of 10 8 :10 10 The mixture was mixed with the E.coli cells in equal amounts, spread on plates containing SFM medium, dried and incubated in a 30-degree incubator, after 18 hours the plates were covered with 1ml of sterile water containing 8mg/L of the final concentration of the plates resistant to the adriamycin, and white conjugative transfersomes were visible after 3 days of incubation in a 30-degree incubator. For preliminary verification of the mutant strains, firstPrimers V-apr-F (5 '-GCTCATCGGTCAGCTTCTCA 3') and V-apr-R (5 '-TCGCATTCTTCGCATCCC 3') were used to verify the presence of the Arabic resistance gene, and if the mutant contains the Arabic resistance gene, 726bp of PCR product should be obtained.
Culturing original Streptomyces nataticus J1002 and newly constructed natamycin producing strain for over-expressing triglyceride on SFM plate (soybean cake powder 20g/L, mannitol 20g/L, agar 16g/L, tap water 1L, pH 7.5) at 30 deg.C for 7 days, and picking 4 small blocks of about 3.2cm 2 The spore-containing agar block of (2) was inoculated into a 250mL shake flask containing 30mL COM medium (10 g/L corn starch, 10g/L oat flour, 5g/L malt extract, 2g/L yeast extract, 15g/L agar, pH 7.2) at 30 ℃ and 220rpm for 48 hours, and 3mL of the seed culture was transferred to 25mL NPM medium (50.0 g/L corn starch, 18.0g/L soybean flour, 10.0g/L yeast extract, 1.5g/L CaCO 3 pH 7.2) were incubated at 30 ℃ and 220rpm for 120h in a shaker.
0.5mL of the fermentation broth was centrifuged at 7,000 rpm for 5 minutes, the supernatant was removed, 1.5mL of a methanol glacial acetic acid solution (methanol: glacial acetic acid =95: analytical column Agilent ZORBAX SB-C18 (4.6X 250 nm), flow rate 0.5mL min -1 Detection at UV 303nm the mobile phase is methanol: water: glacial acetic acid = 60.
As a result, as shown in Table 3, the newly constructed strains showed different degrees of improvement in the yield of natamycin as compared to the original production strain J1002.
Table 3: natamycin related strain information
Figure BDA0001433789650000221
Example 7 construction of a Spinosad-producing bacterium overexpressing triglyceride and fermentation
Spinosad, also known as spinosad, is a macrolide nuisanceless high-efficiency biopesticide extracted from fermentation liquor of Saccharopolyspora spinosa. Spinosad is a solid crystal of light gray color with an odor similar to slightly stale soil. Spinosyns have very low solubility in water and are readily soluble in organic solvents, such as: methanol, ethanol, hexanenitrile, acetone, dimethyl sulfoxide, dimethylformamide, and the like.
In this example, the present inventors conjugated the plasmid constructed in example 6 into a spinosyn-producing strain in a similar manner using each of the relevant genes in the spinosyn-producing strain. The newly constructed strains are shown in Table 3.
The original strain of Saccharopolyspora spinosa is purchased from China microorganism strain maintenance center (CGMCC), and the strain number is CGMCC4.1365. Rejuvenation culture medium (yeast extract 1g/L, beef extract 1g/L, casein Acids Hydrolysate 2g/L, glucose 10g/L, agar 15g/L, pH7.3) is adopted for strain rejuvenation. 1mL of the culture after 3 days in the rejuvenation liquid medium was transferred to a 250mL spring flask containing 30mL of the fermentation seed medium. Seed culture medium: TSB30g/L, yeast extract 3g/L, beef extract 3g/L, mgSO 4 ·7H 2 O2 g/L, glucose 10g/L, corn steep liquor 2.5g/L, and pH 7.0. The fermentation seeds were inoculated in the fermentation medium after 50h of cultivation in a shaker at 30 ℃ and a rotational speed of 220rpm. Wherein the fermentation medium is: 40g/L glucose, 10g/L beef extract and MgSO 4 ·7H 2 O2 g/L, naCl 2g/L, soytone 2g/L, soluble starch 30g/L, caCO 3 2.4g/L, yeast extract 0.34g/L, peptone 6.34g/L, pH 7.2. The fermentation medium adopts a 250mL spring shake flask, the liquid loading capacity is 50mL, and the seed inoculation amount is 10%. The fermentation condition was 30 ℃ and the rotation speed was 220rpm.
Spinosyn (CAS No: 168316-95-8) standard, purchased from Sigma, inc. (product code: 33706), contained two components, spinosyn A and spinosyn D, with an HPLC purity of 97%. Chromatographic analysis by HPLC analysis 10% phase a (0.2% aqueous ammonium acetate) and 90% phase B (methanol) were used as mobile phases; the chromatographic column is a C18 column (3 μm,4.6, 150 mm) of DOINEX; the flow rate is 0.8mL/min; the wavelength was 245nm detected using a PDA detector. The MS detector is LCQ FLEET (Thermo scientific). Summary of HPLC standard curves: weighing 5mg pleocidin standard substance, adding 1mL HPLC grade methanol, dissolving and mixing well to obtain 5g/L mother liquor, diluting the standard substance in series, and detecting by HPLC. Taking 0.2mL of fermentation culture, gradually adding acetonitrile with the total volume of 0.8mL, mixing for 2min by vortex oscillation, and standing overnight in a refrigerator at 4 ℃. Centrifuging at 12000rpm for 10min, collecting supernatant, filtering with 0.45 μm nylon membrane, and loading in brown HPLC sample bottle for analysis.
The fermentation results are shown in Table 4, and the yield of the spinosad of J1016-1 is improved to different degrees compared with that of CGMCC4.1365.
Table 4: spinosyn related strain information
Figure BDA0001433789650000231
Example 8 construction of astaxanthin-producing bacteria overexpressing Triglycerides
Natural astaxanthin (also known as ketocarotenoids or astaxanthin) is currently the most powerful natural antioxidant found in nature by humans. Astaxanthin has super-strong antioxidant activity which is more than one hundred times stronger than vitamin E and is called super vitamin E, can effectively remove oxygen free radicals in cells, is the only carotenoid which can pass through a blood brain barrier, and has the effects of enhancing immunity, relieving fatigue, enhancing cell regeneration capacity, preventing cancer, treating cardiovascular diseases, reducing accumulation of aged cells, inhibiting obesity, protecting eyes and central nerves, resisting ultraviolet rays and the like. In recent years, astaxanthin has come to be widely used in pharmaceuticals, health products, cosmetics, food additives, aquaculture, and the like. With the development of national economy of China, the demand for the method is increasing day by day.
In this example, the inventors constructed plasmids as shown In Table 5 on the basis of plasmids pMH1, pFZ81 (Fayin Zhu, in vitro transduction of medium pathway and targeted engineering of farnesene overexpression In Escherichia coli, biotech. Bioeng.2014;111 1396-1405.), and plasmid pFZ153 (TianMa, genome mining of enzyme biochemical genes from Escherichia sp.ATCC 5566for heterologous expression In Escherichia coli, biotech. J.2015 11 (2): 228-237.), and transformed E.coli 1655 competent cells In combination with plasmids as shown In Table 6 to construct an astaxanthin-producing strain of E.coli.
Plasmid construction in this example referring to example 1, the corresponding gene was amplified by PCR, and each desired plasmid was constructed after being sequentially ligated to the corresponding promoter by the Gibson method (Daniel G.Gibson, enzymatic Assembly of overlaying DNA Fragments, methods in Enzymology, volume 498, 2011, pages 349-361.).
TABLE 5 construction of E.coli astaxanthin Synthesis plasmids
Figure BDA0001433789650000241
TABLE 6 construction of E.coli astaxanthin Synthesis strains
Bacterial strains Description of the invention Yield (mg/L)
TM8011 pMH1，pFZ81，pFZ153 72
TM8012 pTM801，pFZ81，pFZ153 85
TM8013 pMH1，pTM802，pFZ153 90
TM8014 pTM801，pTM802，pFZ153 98
TM8015 pTM801，pTM803，pFZ153 110
After the strain is constructed, carrying out shake flask fermentation according to the following method:
after transformation, transformants were picked and cultured overnight at 220rpm at 37 ℃ in LB medium containing 34. Mu.g/mL chloramphenicol, 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin. 200mL of LB medium containing 34. Mu.g/mL chloramphenicol, 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin was inoculated at 1% inoculum size and cultured at 30 ℃ and 200 rpm. OD600 reached 0.7-0.9, inducing with 0.1mM IPTG (isopropyl-. Beta. -D-thiogalactoside) at final concentration, culturing for 15h, sampling for 2mL,12, 000rpm, centrifuging for 3min, removing supernatant, adding 1mL of an extractant (V acetone: V methanol = 4).
Quaternary HPLC (Thermo Fisher Ultimate 3000), column Agilent Zorbax C18 (4.6-mm. Times.150-mm. Times.5-. Mu.m). The chromatographic conditions were as follows: mobile phase a (acetonitrile: water = 9) and mobile phase B (methanol: isopropanol = 3. 0min:0% B,15min:90% B,30min:90% B,35min:0% by weight of B. The flow rate was 1mL/min. Column temperature: at 25 ℃. A detector: and an ultraviolet detector for detecting the wavelength of 474nm.
As a result, the astaxanthin production of the engineered bacteria was higher as shown in Table 6.
Example 9 control of triglyceride overexpression in microorganisms
Based on example 3, we replaced the promoter of PAH1, DGA, ACC1, OLE1 gene with pHXT1 (SEQ ID NO: 28), the promoter is characterized in that the related gene over-expressed in the presence of glucose can be expressed, and the related gene is not expressed in the absence of glucose, so that the lycopene producing strain can accumulate triglyceride in the early stage of fermentation through our fermentation control, and does not accumulate triglyceride after the glucose is consumed in the later stage, thereby controlling the lipid content in the microorganism, and enabling the microorganism to synthesize lycopene by using more substrates and energy in the later stage of fermentation, and further improving the yield of lycopene. The method for constructing the concrete structure is that the promoter is replaced according to the embodiment 1-2. The strains are shown in Table 7 in detail, and the results of fermentation and detection according to examples 4-5 show that the lycopene production is further improved by controlling the expression of triglyceride.
Table 7: lycopene engineering strain transformation information and yield
Newly constructed strains Original strain Gene substituted with pHXT1 promoter Knock-out gene Yield (mg/L)
TM6065H TM6065 PAH1,DGA1 -- 355
TM6066H TM6066 ACC1** --
TM6068H TM6068 PAH1,DGA1 TGL3
TM704H TM704 OLE1 FLD1 340
TM60656H TM60656 PAH1,DGA1,ACC1** -- 460
TM60686H TM60686 PAH1,DGA1,ACC1** TGL3 388
TM70156H TM70156 PAH1,DGA1,ACC1** FLD1 380
TM70186H TM70186 PAH1,DGA1,ACC1** TGL3,FLD1 356
TM707H TM707 OLE1 -- 352
TM70456H TM70456 PAH1,DGA1,ACC1**,OLE1 FLD1 452
TM70486H TM70486 PAH1,DGA1,ACC1**,OLE1 TGL3,FLD1 403
TM70756H TM70756 PAH1,DGA1,ACC1**,OLE1 -- 475
TM70786H TM70786 PAH1,DGA1,ACC1**,OLE1 TGL3 387
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Zhen Zhi Biotechnology GmbH
<120> microorganism and method for increasing fermentation yield of hydrophobic compound of microorganism
<130> PIDC3174947
<160> 28
<170> PatentIn version 3.3
<210> 1
<211> 2233
<212> PRT
<213> Artificial
<220>
<223> ACC1 ×) an amino acid sequence encoding a polypeptide
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Met Ser Glu Glu Ser Leu Phe Glu Ser Ser Pro Gln Lys Met Glu Tyr
1 5 10 15
Glu Ile Thr Asn Tyr Ser Glu Arg His Thr Glu Leu Pro Gly His Phe
20 25 30
Ile Gly Leu Asn Thr Val Asp Lys Leu Glu Glu Ser Pro Leu Arg Asp
35 40 45
Phe Val Lys Ser His Gly Gly His Thr Val Ile Ser Lys Ile Leu Ile
50 55 60
Ala Asn Asn Gly Ile Ala Ala Val Lys Glu Ile Arg Ser Val Arg Lys
65 70 75 80
Trp Ala Tyr Glu Thr Phe Gly Asp Asp Arg Thr Val Gln Phe Val Ala
85 90 95
Met Ala Thr Pro Glu Asp Leu Glu Ala Asn Ala Glu Tyr Ile Arg Met
100 105 110
Ala Asp Gln Tyr Ile Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr
115 120 125
Ala Asn Val Asp Leu Ile Val Asp Ile Ala Glu Arg Ala Asp Val Asp
130 135 140
Ala Val Trp Ala Gly Trp Gly His Ala Ser Glu Asn Pro Leu Leu Pro
145 150 155 160
Glu Lys Leu Ser Gln Ser Lys Arg Lys Val Ile Phe Ile Gly Pro Pro
165 170 175
Gly Asn Ala Met Arg Ser Leu Gly Asp Lys Ile Ser Ser Thr Ile Val
180 185 190
Ala Gln Ser Ala Lys Val Pro Cys Ile Pro Trp Ser Gly Thr Gly Val
195 200 205
Asp Thr Val His Val Asp Glu Lys Thr Gly Leu Val Ser Val Asp Asp
210 215 220
Asp Ile Tyr Gln Lys Gly Cys Cys Thr Ser Pro Glu Asp Gly Leu Gln
225 230 235 240
Lys Ala Lys Arg Ile Gly Phe Pro Val Met Ile Lys Ala Ser Glu Gly
245 250 255
Gly Gly Gly Lys Gly Ile Arg Gln Val Glu Arg Glu Glu Asp Phe Ile
260 265 270
Ala Leu Tyr His Gln Ala Ala Asn Glu Ile Pro Gly Ser Pro Ile Phe
275 280 285
Ile Met Lys Leu Ala Gly Arg Ala Arg His Leu Glu Val Gln Leu Leu
290 295 300
Ala Asp Gln Tyr Gly Thr Asn Ile Ser Leu Phe Gly Arg Asp Cys Ser
305 310 315 320
Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Ala Pro Val Thr Ile
325 330 335
Ala Lys Ala Glu Thr Phe His Glu Met Glu Lys Ala Ala Val Arg Leu
340 345 350
Gly Lys Leu Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr
355 360 365
Ser His Asp Asp Gly Lys Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu
370 375 380
Gln Val Glu His Pro Thr Thr Glu Met Val Ser Gly Val Asn Leu Pro
385 390 395 400
Ala Ala Gln Leu Gln Ile Ala Met Gly Ile Pro Met His Arg Ile Ser
405 410 415
Asp Ile Arg Thr Leu Tyr Gly Met Asn Pro His Ser Ala Ser Glu Ile
420 425 430
Asp Phe Glu Phe Lys Thr Gln Asp Ala Thr Lys Lys Gln Arg Arg Pro
435 440 445
Ile Pro Lys Gly His Cys Thr Ala Cys Arg Ile Thr Ser Glu Asp Pro
450 455 460
Asn Asp Gly Phe Lys Pro Ser Gly Gly Thr Leu His Glu Leu Asn Phe
465 470 475 480
Arg Ser Ser Ser Asn Val Trp Gly Tyr Phe Ser Val Gly Asn Asn Gly
485 490 495
Asn Ile His Ser Phe Ser Asp Ser Gln Phe Gly His Ile Phe Ala Phe
500 505 510
Gly Glu Asn Arg Gln Ala Ser Arg Lys His Met Val Val Ala Leu Lys
515 520 525
Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr Leu Ile
530 535 540
Lys Leu Leu Glu Thr Glu Asp Phe Glu Asp Asn Thr Ile Thr Thr Gly
545 550 555 560
Trp Leu Asp Asp Leu Ile Thr His Lys Met Thr Ala Glu Lys Pro Asp
565 570 575
Pro Thr Leu Ala Val Ile Cys Gly Ala Ala Thr Lys Ala Phe Leu Ala
580 585 590
Ser Glu Glu Ala Arg His Lys Tyr Ile Glu Ser Leu Gln Lys Gly Gln
595 600 605
Val Leu Ser Lys Asp Leu Leu Gln Thr Met Phe Pro Val Asp Phe Ile
610 615 620
His Glu Gly Lys Arg Tyr Lys Phe Thr Val Ala Lys Ser Gly Asn Asp
625 630 635 640
Arg Tyr Thr Leu Phe Ile Asn Gly Ser Lys Cys Asp Ile Ile Leu Arg
645 650 655
Gln Leu Ala Asp Gly Gly Leu Leu Ile Ala Ile Gly Gly Lys Ser His
660 665 670
Thr Ile Tyr Trp Lys Glu Glu Val Ala Ala Thr Arg Leu Ser Val Asp
675 680 685
Ser Met Thr Thr Leu Leu Glu Val Glu Asn Asp Pro Thr Gln Leu Arg
690 695 700
Thr Pro Ser Pro Gly Lys Leu Val Lys Phe Leu Val Glu Asn Gly Glu
705 710 715 720
His Ile Ile Lys Gly Gln Pro Tyr Ala Glu Ile Glu Val Met Lys Met
725 730 735
Gln Met Pro Leu Val Ser Gln Glu Asn Gly Ile Val Gln Leu Leu Lys
740 745 750
Gln Pro Gly Ser Thr Ile Val Ala Gly Asp Ile Met Ala Ile Met Thr
755 760 765
Leu Asp Asp Pro Ser Lys Val Lys His Ala Leu Pro Phe Glu Gly Met
770 775 780
Leu Pro Asp Phe Gly Ser Pro Val Ile Glu Gly Thr Lys Pro Ala Tyr
785 790 795 800
Lys Phe Lys Ser Leu Val Ser Thr Leu Glu Asn Ile Leu Lys Gly Tyr
805 810 815
Asp Asn Gln Val Ile Met Asn Ala Ser Leu Gln Gln Leu Ile Glu Val
820 825 830
Leu Arg Asn Pro Lys Leu Pro Tyr Ser Glu Trp Lys Leu His Ile Ser
835 840 845
Ala Leu His Ser Arg Leu Pro Ala Lys Leu Asp Glu Gln Met Glu Glu
850 855 860
Leu Val Ala Arg Ser Leu Arg Arg Gly Ala Val Phe Pro Ala Arg Gln
865 870 875 880
Leu Ser Lys Leu Ile Asp Met Ala Val Lys Asn Pro Glu Tyr Asn Pro
885 890 895
Asp Lys Leu Leu Gly Ala Val Val Glu Pro Leu Ala Asp Ile Ala His
900 905 910
Lys Tyr Ser Asn Gly Leu Glu Ala His Glu His Ser Ile Phe Val His
915 920 925
Phe Leu Glu Glu Tyr Tyr Glu Val Glu Lys Leu Phe Asn Gly Pro Asn
930 935 940
Val Arg Glu Glu Asn Ile Ile Leu Lys Leu Arg Asp Glu Asn Pro Lys
945 950 955 960
Asp Leu Asp Lys Val Ala Leu Thr Val Leu Ser His Ser Lys Val Ser
965 970 975
Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Lys His Tyr Gln Pro Leu
980 985 990
Cys Lys Leu Ser Ser Lys Val Ser Ala Ile Phe Ser Thr Pro Leu Gln
995 1000 1005
His Ile Val Glu Leu Glu Ser Lys Ala Thr Ala Lys Val Ala Leu
1010 1015 1020
Gln Ala Arg Glu Ile Leu Ile Gln Gly Ala Leu Pro Ser Val Lys
1025 1030 1035
Glu Arg Thr Glu Gln Ile Glu His Ile Leu Lys Ser Ser Val Val
1040 1045 1050
Lys Val Ala Tyr Gly Ser Ser Asn Pro Lys Arg Ser Glu Pro Asp
1055 1060 1065
Leu Asn Ile Leu Lys Asp Leu Ile Asp Ser Asn Tyr Val Val Phe
1070 1075 1080
Asp Val Leu Leu Gln Phe Leu Thr His Gln Asp Pro Val Val Thr
1085 1090 1095
Ala Ala Ala Ala Gln Val Tyr Ile Arg Arg Ala Tyr Arg Ala Tyr
1100 1105 1110
Thr Ile Gly Asp Ile Arg Val His Glu Gly Val Thr Val Pro Ile
1115 1120 1125
Val Glu Trp Lys Phe Gln Leu Pro Ser Ala Ala Phe Ser Thr Phe
1130 1135 1140
Pro Thr Val Lys Ser Lys Met Gly Met Asn Arg Ala Val Ala Val
1145 1150 1155
Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln Ser Ser Pro Leu Arg
1160 1165 1170
Glu Gly Ile Leu Met Ala Val Asp His Leu Asp Asp Val Asp Glu
1175 1180 1185
Ile Leu Ser Gln Ser Leu Glu Val Ile Pro Arg His Gln Ser Ser
1190 1195 1200
Ser Asn Gly Pro Ala Pro Asp Arg Ser Gly Ser Ser Ala Ser Leu
1205 1210 1215
Ser Asn Val Ala Asn Val Cys Val Ala Ser Thr Glu Gly Phe Glu
1220 1225 1230
Ser Glu Glu Glu Ile Leu Val Arg Leu Arg Glu Ile Leu Asp Leu
1235 1240 1245
Asn Lys Gln Glu Leu Ile Asn Ala Ser Ile Arg Arg Ile Thr Phe
1250 1255 1260
Met Phe Gly Phe Lys Asp Gly Ser Tyr Pro Lys Tyr Tyr Thr Phe
1265 1270 1275
Asn Gly Pro Asn Tyr Asn Glu Asn Glu Thr Ile Arg His Ile Glu
1280 1285 1290
Pro Ala Leu Ala Phe Gln Leu Glu Leu Gly Arg Leu Ser Asn Phe
1295 1300 1305
Asn Ile Lys Pro Ile Phe Thr Asp Asn Arg Asn Ile His Val Tyr
1310 1315 1320
Glu Ala Val Ser Lys Thr Ser Pro Leu Asp Lys Arg Phe Phe Thr
1325 1330 1335
Arg Gly Ile Ile Arg Thr Gly His Ile Arg Asp Asp Ile Ser Ile
1340 1345 1350
Gln Glu Tyr Leu Thr Ser Glu Ala Asn Arg Leu Met Ser Asp Ile
1355 1360 1365
Leu Asp Asn Leu Glu Val Thr Asp Thr Ser Asn Ser Asp Leu Asn
1370 1375 1380
His Ile Phe Ile Asn Phe Ile Ala Val Phe Asp Ile Ser Pro Glu
1385 1390 1395
Asp Val Glu Ala Ala Phe Gly Gly Phe Leu Glu Arg Phe Gly Lys
1400 1405 1410
Arg Leu Leu Arg Leu Arg Val Ser Ser Ala Glu Ile Arg Ile Ile
1415 1420 1425
Ile Lys Asp Pro Gln Thr Gly Ala Pro Val Pro Leu Arg Ala Leu
1430 1435 1440
Ile Asn Asn Val Ser Gly Tyr Val Ile Lys Thr Glu Met Tyr Thr
1445 1450 1455
Glu Val Lys Asn Ala Lys Gly Glu Trp Val Phe Lys Ser Leu Gly
1460 1465 1470
Lys Pro Gly Ser Met His Leu Arg Pro Ile Ala Thr Pro Tyr Pro
1475 1480 1485
Val Lys Glu Trp Leu Gln Pro Lys Arg Tyr Lys Ala His Leu Met
1490 1495 1500
Gly Thr Thr Tyr Val Tyr Asp Phe Pro Glu Leu Phe Arg Gln Ala
1505 1510 1515
Ser Ser Ser Gln Trp Lys Asn Phe Ser Ala Asp Val Lys Leu Thr
1520 1525 1530
Asp Asp Phe Phe Ile Ser Asn Glu Leu Ile Glu Asp Glu Asn Gly
1535 1540 1545
Glu Leu Thr Glu Val Glu Arg Glu Pro Gly Ala Asn Ala Ile Gly
1550 1555 1560
Met Val Ala Phe Lys Ile Thr Val Lys Thr Pro Glu Tyr Pro Arg
1565 1570 1575
Gly Arg Gln Phe Val Val Val Ala Asn Asp Ile Thr Phe Lys Ile
1580 1585 1590
Gly Ser Phe Gly Pro Gln Glu Asp Glu Phe Phe Asn Lys Val Thr
1595 1600 1605
Glu Tyr Ala Arg Lys Arg Gly Ile Pro Arg Ile Tyr Leu Ala Ala
1610 1615 1620
Asn Ser Gly Ala Arg Ile Gly Met Ala Glu Glu Ile Val Pro Leu
1625 1630 1635
Phe Gln Val Ala Trp Asn Asp Ala Ala Asn Pro Asp Lys Gly Phe
1640 1645 1650
Gln Tyr Leu Tyr Leu Thr Ser Glu Gly Met Glu Thr Leu Lys Lys
1655 1660 1665
Phe Asp Lys Glu Asn Ser Val Leu Thr Glu Arg Thr Val Ile Asn
1670 1675 1680
Gly Glu Glu Arg Phe Val Ile Lys Thr Ile Ile Gly Ser Glu Asp
1685 1690 1695
Gly Leu Gly Val Glu Cys Leu Arg Gly Ser Gly Leu Ile Ala Gly
1700 1705 1710
Ala Thr Ser Arg Ala Tyr His Asp Ile Phe Thr Ile Thr Leu Val
1715 1720 1725
Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly
1730 1735 1740
Gln Arg Ala Ile Gln Val Glu Gly Gln Pro Ile Ile Leu Thr Gly
1745 1750 1755
Ala Pro Ala Ile Asn Lys Met Leu Gly Arg Glu Val Tyr Thr Ser
1760 1765 1770
Asn Leu Gln Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn Gly Val
1775 1780 1785
Ser His Leu Thr Ala Val Asp Asp Leu Ala Gly Val Glu Lys Ile
1790 1795 1800
Val Glu Trp Met Ser Tyr Val Pro Ala Lys Arg Asn Met Pro Val
1805 1810 1815
Pro Ile Leu Glu Thr Lys Asp Thr Trp Asp Arg Pro Val Asp Phe
1820 1825 1830
Thr Pro Thr Asn Asp Glu Thr Tyr Asp Val Arg Trp Met Ile Glu
1835 1840 1845
Gly Arg Glu Thr Glu Ser Gly Phe Glu Tyr Gly Leu Phe Asp Lys
1850 1855 1860
Gly Ser Phe Phe Glu Thr Leu Ser Gly Trp Ala Lys Gly Val Val
1865 1870 1875
Val Gly Arg Ala Arg Leu Gly Gly Ile Pro Leu Gly Val Ile Gly
1880 1885 1890
Val Glu Thr Arg Thr Val Glu Asn Leu Ile Pro Ala Asp Pro Ala
1895 1900 1905
Asn Pro Asn Ser Ala Glu Thr Leu Ile Gln Glu Pro Gly Gln Val
1910 1915 1920
Trp His Pro Asn Ser Ala Phe Lys Thr Ala Gln Ala Ile Asn Asp
1925 1930 1935
Phe Asn Asn Gly Glu Gln Leu Pro Met Met Ile Leu Ala Asn Trp
1940 1945 1950
Arg Gly Phe Ser Gly Gly Gln Arg Asp Met Phe Asn Glu Val Leu
1955 1960 1965
Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu Val Asp Tyr Lys Gln
1970 1975 1980
Pro Ile Ile Ile Tyr Ile Pro Pro Thr Gly Glu Leu Arg Gly Gly
1985 1990 1995
Ser Trp Val Val Val Asp Pro Thr Ile Asn Ala Asp Gln Met Glu
2000 2005 2010
Met Tyr Ala Asp Val Asn Ala Arg Ala Gly Val Leu Glu Pro Gln
2015 2020 2025
Gly Met Val Gly Ile Lys Phe Arg Arg Glu Lys Leu Leu Asp Thr
2030 2035 2040
Met Asn Arg Leu Asp Asp Lys Tyr Arg Glu Leu Arg Ser Gln Leu
2045 2050 2055
Ser Asn Lys Ser Leu Ala Pro Glu Val His Gln Gln Ile Ser Lys
2060 2065 2070
Gln Leu Ala Asp Arg Glu Arg Glu Leu Leu Pro Ile Tyr Gly Gln
2075 2080 2085
Ile Ser Leu Gln Phe Ala Asp Leu His Asp Arg Ser Ser Arg Met
2090 2095 2100
Val Ala Lys Gly Val Ile Ser Lys Glu Leu Glu Trp Thr Glu Ala
2105 2110 2115
Arg Arg Phe Phe Phe Trp Arg Leu Arg Arg Arg Leu Asn Glu Glu
2120 2125 2130
Tyr Leu Ile Lys Arg Leu Ser His Gln Val Gly Glu Ala Ser Arg
2135 2140 2145
Leu Glu Lys Ile Ala Arg Ile Arg Ser Trp Tyr Pro Ala Ser Val
2150 2155 2160
Asp His Glu Asp Asp Arg Gln Val Ala Thr Trp Ile Glu Glu Asn
2165 2170 2175
Tyr Lys Thr Leu Asp Asp Lys Leu Lys Gly Leu Lys Leu Glu Ser
2180 2185 2190
Phe Ala Gln Asp Leu Ala Lys Lys Ile Arg Ser Asp His Asp Asn
2195 2200 2205
Ala Ile Asp Gly Leu Ser Glu Val Ile Lys Met Leu Ser Thr Asp
2210 2215 2220
Asp Lys Glu Lys Leu Leu Lys Thr Leu Lys
2225 2230
<210> 2
<211> 393
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide coded by TmCrtE of taxus chinensis
<400> 2
Met Ala Tyr Thr Ala Met Ala Ala Gly Thr Gln Ser Leu Gln Leu Arg
1 5 10 15
Thr Val Ala Ser Tyr Gln Glu Cys Asn Ser Met Arg Ser Cys Phe Lys
20 25 30
Leu Thr Pro Phe Lys Ser Phe His Gly Val Asn Phe Asn Val Pro Ser
35 40 45
Leu Gly Ala Ala Asn Cys Glu Ile Met Gly His Leu Lys Leu Gly Ser
50 55 60
Leu Pro Tyr Lys Gln Cys Ser Val Ser Ser Lys Ser Thr Lys Thr Met
65 70 75 80
Ala Gln Leu Val Asp Leu Ala Glu Thr Glu Lys Ala Glu Gly Lys Asp
85 90 95
Ile Glu Phe Asp Phe Asn Glu Tyr Met Lys Ser Lys Ala Val Ala Val
100 105 110
Asp Ala Ala Leu Asp Lys Ala Ile Pro Leu Glu Tyr Pro Glu Lys Ile
115 120 125
His Glu Ser Met Arg Tyr Ser Leu Leu Ala Gly Gly Lys Arg Val Arg
130 135 140
Pro Ala Leu Cys Ile Ala Ala Cys Glu Leu Val Gly Gly Ser Gln Asp
145 150 155 160
Leu Ala Met Pro Thr Ala Cys Ala Met Glu Met Ile His Thr Met Ser
165 170 175
Leu Ile His Asp Asp Leu Pro Cys Met Asp Asn Asp Asp Phe Arg Arg
180 185 190
Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Asp Thr Ala Val Leu
195 200 205
Ala Gly Asp Ala Leu Leu Ser Phe Ala Phe Glu His Ile Ala Val Ala
210 215 220
Thr Ser Lys Thr Val Pro Ser Asp Arg Thr Leu Arg Val Ile Ser Glu
225 230 235 240
Leu Gly Lys Thr Ile Gly Ser Gln Gly Leu Val Gly Gly Gln Val Val
245 250 255
Asp Ile Thr Ser Glu Gly Asp Ala Asn Val Asp Leu Lys Thr Leu Glu
260 265 270
Trp Ile His Ile His Lys Thr Ala Val Leu Leu Glu Cys Ser Val Val
275 280 285
Ser Gly Gly Ile Leu Gly Gly Ala Thr Glu Asp Glu Ile Ala Arg Ile
290 295 300
Arg Arg Tyr Ala Arg Cys Val Gly Leu Leu Phe Gln Val Val Asp Asp
305 310 315 320
Ile Leu Asp Val Thr Lys Ser Ser Glu Glu Leu Gly Lys Thr Ala Gly
325 330 335
Lys Asp Leu Leu Thr Asp Lys Ala Thr Tyr Pro Lys Leu Met Gly Leu
340 345 350
Glu Lys Ala Lys Glu Phe Ala Ala Glu Leu Ala Thr Arg Ala Lys Glu
355 360 365
Glu Leu Ser Ser Phe Asp Gln Ile Lys Ala Ala Pro Leu Leu Gly Leu
370 375 380
Ala Asp Tyr Ile Ala Phe Arg Gln Asn
385 390
<210> 3
<211> 309
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of PaCrtB-encoding polypeptide of pantococcus agglomerans
<400> 3
Met Ser Gln Pro Pro Leu Leu Asp His Ala Thr Gln Thr Met Ala Asn
1 5 10 15
Gly Ser Lys Ser Phe Ala Thr Ala Ala Lys Leu Phe Asp Pro Ala Thr
20 25 30
Arg Arg Ser Val Leu Met Leu Tyr Thr Trp Cys Arg His Cys Asp Asp
35 40 45
Val Ile Asp Asp Gln Thr His Gly Phe Ala Ser Glu Ala Ala Ala Glu
50 55 60
Glu Glu Ala Thr Gln Arg Leu Ala Arg Leu Arg Thr Leu Thr Leu Ala
65 70 75 80
Ala Phe Glu Gly Ala Glu Met Gln Asp Pro Ala Phe Ala Ala Phe Gln
85 90 95
Glu Val Ala Leu Thr His Gly Ile Thr Pro Arg Met Ala Leu Asp His
100 105 110
Leu Asp Gly Phe Ala Met Asp Val Ala Gln Thr Arg Tyr Val Thr Phe
115 120 125
Glu Asp Thr Leu Arg Tyr Cys Tyr His Val Ala Gly Val Val Gly Leu
130 135 140
Met Met Ala Arg Val Met Gly Val Arg Asp Glu Arg Val Leu Asp Arg
145 150 155 160
Ala Cys Asp Leu Gly Leu Ala Phe Gln Leu Thr Asn Ile Ala Arg Asp
165 170 175
Ile Ile Asp Asp Ala Ala Ile Asp Arg Cys Tyr Leu Pro Ala Glu Trp
180 185 190
Leu Gln Asp Ala Gly Leu Thr Pro Glu Asn Tyr Ala Ala Arg Glu Asn
195 200 205
Arg Ala Ala Leu Ala Arg Val Ala Glu Arg Leu Ile Asp Ala Ala Glu
210 215 220
Pro Tyr Tyr Ile Ser Ser Gln Ala Gly Leu His Asp Leu Pro Pro Arg
225 230 235 240
Cys Ala Trp Ala Ile Ala Thr Ala Arg Ser Val Tyr Arg Glu Ile Gly
245 250 255
Ile Lys Val Lys Ala Ala Gly Gly Ser Ala Trp Asp Arg Arg Gln His
260 265 270
Thr Ser Lys Gly Glu Lys Ile Ala Met Leu Met Ala Ala Pro Gly Gln
275 280 285
Val Ile Arg Ala Lys Thr Thr Arg Val Thr Pro Arg Pro Ala Gly Leu
290 295 300
Trp Gln Arg Pro Val
305
<210> 4
<211> 582
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide coded by Blakeslea trispora BtCrtI
<400> 4
Met Ser Asp Gln Lys Lys His Ile Val Val Ile Gly Ala Gly Ile Gly
1 5 10 15
Gly Thr Ala Thr Ala Ala Arg Leu Ala Arg Glu Gly Phe Arg Val Thr
20 25 30
Val Val Glu Lys Asn Asp Phe Ser Gly Gly Arg Cys Ser Phe Ile His
35 40 45
His Asp Gly His Arg Phe Asp Gln Gly Pro Ser Leu Tyr Leu Met Pro
50 55 60
Lys Leu Phe Glu Asp Ala Phe Ala Asp Leu Asp Glu Arg Ile Gly Asp
65 70 75 80
His Leu Asp Leu Leu Arg Cys Asp Asn Asn Tyr Lys Val His Phe Asp
85 90 95
Asp Gly Asp Ala Val Gln Leu Ser Ser Asp Leu Thr Lys Met Lys Gly
100 105 110
Glu Leu Asp Arg Ile Glu Gly Pro Leu Gly Phe Gly Arg Phe Leu Asp
115 120 125
Phe Met Lys Glu Thr His Val His Tyr Glu Gln Gly Thr Phe Ile Ala
130 135 140
Ile Lys Arg Asn Phe Glu Thr Ile Trp Asp Leu Ile Arg Leu Gln Tyr
145 150 155 160
Val Pro Glu Ile Phe Arg Leu His Leu Phe Gly Lys Ile Tyr Asp Arg
165 170 175
Ala Ser Lys Tyr Phe Gln Thr Lys Lys Met Arg Met Ala Phe Thr Phe
180 185 190
Gln Thr Met Tyr Met Gly Met Ser Pro Tyr Asp Ala Pro Ala Val Tyr
195 200 205
Ser Leu Leu Gln Tyr Thr Glu Phe Ala Glu Gly Ile Trp Tyr Pro Arg
210 215 220
Gly Gly Phe Asn Met Val Val Gln Lys Leu Glu Ser Ile Ala Ser Lys
225 230 235 240
Lys Tyr Gly Ala Glu Phe Arg Tyr Gln Ser Pro Val Ala Lys Ile Asn
245 250 255
Thr Val Asp Lys Asp Lys Arg Val Thr Gly Val Thr Leu Glu Ser Gly
260 265 270
Glu Val Ile Glu Ala Asp Ala Val Val Cys Asn Ala Asp Leu Val Tyr
275 280 285
Ala Tyr His His Leu Leu Pro Pro Cys Asn Trp Thr Lys Lys Thr Leu
290 295 300
Ala Ser Lys Lys Leu Thr Ser Ser Ser Ile Ser Phe Tyr Trp Ser Met
305 310 315 320
Ser Thr Lys Val Pro Gln Leu Asp Val His Asn Ile Phe Leu Ala Glu
325 330 335
Ala Tyr Lys Glu Ser Phe Asp Glu Ile Phe Asn Asp Phe Gly Leu Pro
340 345 350
Ser Glu Ala Ser Phe Tyr Val Asn Val Pro Ser Arg Ile Asp Glu Ser
355 360 365
Ala Ala Pro Pro Asn Lys Asp Ser Ile Ile Val Leu Val Pro Ile Gly
370 375 380
His Met Lys Ser Lys Thr Gly Asn Ser Ala Glu Glu Asn Tyr Pro Glu
385 390 395 400
Leu Val Asn Arg Ala Arg Lys Met Val Leu Glu Val Ile Glu Arg Arg
405 410 415
Leu Gly Val Asn Asn Phe Ala Asn Leu Ile Glu His Glu Glu Val Asn
420 425 430
Asp Pro Ser Val Trp Gln Ser Lys Phe Asn Leu Trp Arg Gly Ser Ile
435 440 445
Leu Gly Leu Ser His Asp Val Phe Gln Val Leu Trp Phe Arg Pro Ser
450 455 460
Thr Lys Asp Ser Thr Asn Arg Tyr Asp Asn Leu Phe Phe Val Gly Ala
465 470 475 480
Ser Thr His Pro Gly Thr Gly Val Pro Ile Val Leu Ala Gly Ser Lys
485 490 495
Leu Thr Ser Asp Gln Val Cys Lys Ser Phe Gly Gln Asn Pro Leu Pro
500 505 510
Arg Lys Leu Gln Asp Ser Gln Lys Lys Tyr Ala Pro Glu Gln Thr Arg
515 520 525
Lys Thr Glu Ser His Trp Ile Tyr Tyr Cys Leu Ala Cys Tyr Phe Val
530 535 540
Thr Phe Leu Phe Phe Tyr Phe Phe Pro Arg Asp Asp Thr Thr Thr Pro
545 550 555 560
Ala Ser Phe Ile Asn Gln Leu Leu Pro Asn Val Phe Gln Gly Gln Asn
565 570 575
Ser Asn Asp Ile Arg Ile
580
<210> 5
<211> 652
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of ACS 6-encoding polypeptide
<400> 5
Met Ser Gln Thr His Lys His Ala Ile Pro Ala Asn Ile Ala Asp Arg
1 5 10 15
Cys Leu Ile Asn Pro Glu Gln Tyr Glu Thr Lys Tyr Lys Gln Ser Ile
20 25 30
Asn Asp Pro Asp Thr Phe Trp Gly Glu Gln Gly Lys Ile Leu Asp Trp
35 40 45
Ile Thr Pro Tyr Gln Lys Val Lys Asn Thr Ser Phe Ala Pro Gly Asn
50 55 60
Val Ser Ile Lys Trp Tyr Glu Asp Gly Thr Leu Asn Leu Ala Ala Asn
65 70 75 80
Cys Leu Asp Arg His Leu Gln Glu Asn Gly Asp Arg Thr Ala Ile Ile
85 90 95
Trp Glu Gly Asp Asp Ala Ser Gln Ser Lys His Ile Ser Tyr Arg Glu
100 105 110
Leu His Arg Asp Val Cys Arg Phe Ala Asn Thr Leu Leu Asp Leu Gly
115 120 125
Ile Lys Lys Gly Asp Val Val Ala Ile Tyr Met Pro Met Val Pro Glu
130 135 140
Ala Ala Val Ala Met Leu Ala Cys Ala Arg Ile Gly Ala Val His Ser
145 150 155 160
Val Ile Phe Gly Gly Phe Ser Pro Glu Ala Ile Ala Gly Arg Ile Ile
165 170 175
Asp Ser Ser Ser Arg Leu Val Ile Thr Ala Asp Glu Gly Val Arg Ala
180 185 190
Gly Arg Ser Ile Pro Leu Lys Lys Asn Val Asp Asp Ala Leu Lys Asn
195 200 205
Pro Asn Val Thr Ser Val Glu His Val Ile Val Leu Lys Arg Thr Gly
210 215 220
Asn Asp Ile Asp Trp Gln Glu Gly Arg Asp Leu Trp Trp Arg Asp Leu
225 230 235 240
Ile Glu Lys Ala Ser Pro Glu His Gln Pro Glu Ala Met Asn Ala Glu
245 250 255
Asp Pro Leu Phe Ile Leu Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys
260 265 270
Gly Val Leu His Thr Thr Gly Gly Tyr Leu Val Tyr Ala Ala Thr Thr
275 280 285
Phe Lys Tyr Val Phe Asp Tyr His Pro Gly Asp Ile Tyr Trp Cys Thr
290 295 300
Ala Asp Val Gly Trp Val Thr Gly His Ser Tyr Leu Leu Tyr Gly Pro
305 310 315 320
Leu Ala Cys Gly Ala Thr Thr Leu Met Phe Glu Gly Val Pro Asn Trp
325 330 335
Pro Thr Pro Ala Arg Met Cys Gln Val Val Asp Lys His Gln Val Asn
340 345 350
Ile Leu Tyr Thr Ala Pro Thr Ala Ile Arg Ala Leu Met Ala Glu Gly
355 360 365
Asp Lys Ala Ile Glu Gly Thr Asp Arg Ser Ser Leu Arg Ile Leu Gly
370 375 380
Ser Val Gly Glu Pro Ile Asn Pro Glu Ala Trp Glu Trp Tyr Trp Lys
385 390 395 400
Lys Ile Gly Lys Glu Lys Cys Pro Val Val Asp Thr Trp Trp Gln Thr
405 410 415
Glu Thr Gly Gly Phe Met Ile Thr Pro Leu Pro Gly Ala Ile Glu Leu
420 425 430
Lys Ala Gly Ser Ala Thr Arg Pro Phe Phe Gly Val Gln Pro Ala Leu
435 440 445
Val Asp Asn Glu Gly His Pro Gln Glu Gly Ala Thr Glu Gly Asn Leu
450 455 460
Val Ile Thr Asp Ser Trp Pro Gly Gln Ala Arg Thr Leu Phe Gly Asp
465 470 475 480
His Glu Arg Phe Glu Gln Thr Tyr Phe Ser Thr Phe Lys Asn Met Tyr
485 490 495
Phe Ser Gly Asp Gly Ala Arg Arg Asp Glu Asp Gly Tyr Tyr Trp Ile
500 505 510
Thr Gly Arg Val Asp Asp Val Leu Asn Val Ser Gly His Arg Leu Gly
515 520 525
Thr Ala Glu Ile Glu Ser Ala Leu Val Ala His Pro Lys Ile Ala Glu
530 535 540
Ala Ala Val Val Gly Ile Pro His Ala Ile Lys Gly Gln Ala Ile Tyr
545 550 555 560
Ala Tyr Val Thr Leu Asn His Gly Glu Glu Pro Ser Pro Glu Leu Tyr
565 570 575
Ala Glu Val Arg Asn Trp Val Arg Lys Glu Ile Gly Pro Leu Ala Thr
580 585 590
Pro Asp Val Leu His Trp Thr Asp Ser Leu Pro Lys Thr Arg Ser Gly
595 600 605
Lys Ile Met Arg Arg Ile Leu Arg Lys Ile Ala Ala Gly Asp Thr Ser
610 615 620
Asn Leu Gly Asp Thr Ser Thr Leu Ala Asp Pro Gly Val Val Glu Lys
625 630 635 640
Pro Leu Glu Glu Lys Gln Ala Ile Ala Met Pro Ser
645 650
<210> 6
<211> 172
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of fabA-encoded polypeptide
<400> 6
Met Val Asp Lys Arg Glu Ser Tyr Thr Lys Glu Asp Leu Leu Ala Ser
1 5 10 15
Gly Arg Gly Glu Leu Phe Gly Ala Lys Gly Pro Gln Leu Pro Ala Pro
20 25 30
Asn Met Leu Met Met Asp Arg Val Val Lys Met Thr Glu Thr Gly Gly
35 40 45
Asn Phe Asp Lys Gly Tyr Val Glu Ala Glu Leu Asp Ile Asn Pro Asp
50 55 60
Leu Trp Phe Phe Gly Cys His Phe Ile Gly Asp Pro Val Met Pro Gly
65 70 75 80
Cys Leu Gly Leu Asp Ala Met Trp Gln Leu Val Gly Phe Tyr Leu Gly
85 90 95
Trp Leu Gly Gly Glu Gly Lys Gly Arg Ala Leu Gly Val Gly Glu Val
100 105 110
Lys Phe Thr Gly Gln Val Leu Pro Thr Ala Lys Lys Val Thr Tyr Arg
115 120 125
Ile His Phe Lys Arg Ile Val Asn Arg Arg Leu Ile Met Gly Leu Ala
130 135 140
Asp Gly Glu Val Leu Val Asp Gly Arg Leu Ile Tyr Thr Ala Ser Asp
145 150 155 160
Leu Lys Val Gly Leu Phe Gln Asp Thr Ser Ala Phe
165 170
<210> 7
<211> 510
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide encoded by OLE1
<400> 7
Met Pro Thr Ser Gly Thr Thr Ile Glu Leu Ile Asp Asp Gln Phe Pro
1 5 10 15
Lys Asp Asp Ser Ala Ser Ser Gly Ile Val Asp Glu Val Asp Leu Thr
20 25 30
Glu Ala Asn Ile Leu Ala Thr Gly Leu Asn Lys Lys Ala Pro Arg Ile
35 40 45
Val Asn Gly Phe Gly Ser Leu Met Gly Ser Lys Glu Met Val Ser Val
50 55 60
Glu Phe Asp Lys Lys Gly Asn Glu Lys Lys Ser Asn Leu Asp Arg Leu
65 70 75 80
Leu Glu Lys Asp Asn Gln Glu Lys Glu Glu Ala Lys Thr Lys Ile His
85 90 95
Ile Ser Glu Gln Pro Trp Thr Leu Asn Asn Trp His Gln His Leu Asn
100 105 110
Trp Leu Asn Met Val Leu Val Cys Gly Met Pro Met Ile Gly Trp Tyr
115 120 125
Phe Ala Leu Ser Gly Lys Val Pro Leu His Leu Asn Val Phe Leu Phe
130 135 140
Ser Val Phe Tyr Tyr Ala Val Gly Gly Val Ser Ile Thr Ala Gly Tyr
145 150 155 160
His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala His Trp Pro Leu Arg
165 170 175
Leu Phe Tyr Ala Ile Phe Gly Cys Ala Ser Val Glu Gly Ser Ala Lys
180 185 190
Trp Trp Gly His Ser His Arg Ile His His Arg Tyr Thr Asp Thr Leu
195 200 205
Arg Asp Pro Tyr Asp Ala Arg Arg Gly Leu Trp Tyr Ser His Met Gly
210 215 220
Trp Met Leu Leu Lys Pro Asn Pro Lys Tyr Lys Ala Arg Ala Asp Ile
225 230 235 240
Thr Asp Met Thr Asp Asp Trp Thr Ile Arg Phe Gln His Arg His Tyr
245 250 255
Ile Leu Leu Met Leu Leu Thr Ala Phe Val Ile Pro Thr Leu Ile Cys
260 265 270
Gly Tyr Phe Phe Asn Asp Tyr Met Gly Gly Leu Ile Tyr Ala Gly Phe
275 280 285
Ile Arg Val Phe Val Ile Gln Gln Ala Thr Phe Cys Ile Asn Ser Leu
290 295 300
Ala His Tyr Ile Gly Thr Gln Pro Phe Asp Asp Arg Arg Thr Pro Arg
305 310 315 320
Asp Asn Trp Ile Thr Ala Ile Val Thr Phe Gly Glu Gly Tyr His Asn
325 330 335
Phe His His Glu Phe Pro Thr Asp Tyr Arg Asn Ala Ile Lys Trp Tyr
340 345 350
Gln Tyr Asp Pro Thr Lys Val Ile Ile Tyr Leu Thr Ser Leu Val Gly
355 360 365
Leu Ala Tyr Asp Leu Lys Lys Phe Ser Gln Asn Ala Ile Glu Glu Ala
370 375 380
Leu Ile Gln Gln Glu Gln Lys Lys Ile Asn Lys Lys Lys Ala Lys Ile
385 390 395 400
Asn Trp Gly Pro Val Leu Thr Asp Leu Pro Met Trp Asp Lys Gln Thr
405 410 415
Phe Leu Ala Lys Ser Lys Glu Asn Lys Gly Leu Val Ile Ile Ser Gly
420 425 430
Ile Val His Asp Val Ser Gly Tyr Ile Ser Glu His Pro Gly Gly Glu
435 440 445
Thr Leu Ile Lys Thr Ala Leu Gly Lys Asp Ala Thr Lys Ala Phe Ser
450 455 460
Gly Gly Val Tyr Arg His Ser Asn Ala Ala Gln Asn Val Leu Ala Asp
465 470 475 480
Met Arg Val Ala Val Ile Lys Glu Ser Lys Asn Ser Ala Ile Arg Met
485 490 495
Ala Ser Lys Arg Gly Glu Ile Tyr Glu Thr Gly Lys Phe Phe
500 505 510
<210> 8
<211> 862
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of PAH 1-encoding polypeptide
<400> 8
Met Gln Tyr Val Gly Arg Ala Leu Gly Ser Val Ser Lys Thr Trp Ser
1 5 10 15
Ser Ile Asn Pro Ala Thr Leu Ser Gly Ala Ile Asp Val Ile Val Val
20 25 30
Glu His Pro Asp Gly Arg Leu Ser Cys Ser Pro Phe His Val Arg Phe
35 40 45
Gly Lys Phe Gln Ile Leu Lys Pro Ser Gln Lys Lys Val Gln Val Phe
50 55 60
Ile Asn Glu Lys Leu Ser Asn Met Pro Met Lys Leu Ser Asp Ser Gly
65 70 75 80
Glu Ala Tyr Phe Val Phe Glu Met Gly Asp Gln Val Thr Asp Val Pro
85 90 95
Asp Glu Leu Leu Val Ser Pro Val Met Ser Ala Thr Ser Ser Pro Pro
100 105 110
Gln Ser Pro Glu Thr Ser Ile Leu Glu Gly Gly Thr Glu Gly Glu Gly
115 120 125
Glu Gly Glu Asn Glu Asn Lys Lys Lys Glu Lys Lys Val Leu Glu Glu
130 135 140
Pro Asp Phe Leu Asp Ile Asn Asp Thr Gly Asp Ser Gly Ser Lys Asn
145 150 155 160
Ser Glu Thr Thr Gly Ser Leu Ser Pro Thr Glu Ser Ser Thr Thr Thr
165 170 175
Pro Leu Asp Ser Val Glu Glu Arg Lys Leu Val Glu Gln Arg Thr Lys
180 185 190
Asn Phe Gln Gln Lys Leu Asn Lys Lys Leu Thr Glu Ile His Ile Pro
195 200 205
Ser Lys Leu Asp Asn Asn Gly Asp Leu Leu Leu Asp Thr Glu Gly Tyr
210 215 220
Lys Pro Asn Lys Asn Met Met His Asp Thr Asp Ile Gln Leu Lys Gln
225 230 235 240
Leu Leu Lys Asp Glu Phe Gly Asn Asp Ser Asp Ile Ser Ser Phe Ile
245 250 255
Lys Glu Asp Lys Asn Gly Asn Ile Lys Ile Val Asn Pro Tyr Glu His
260 265 270
Leu Thr Asp Leu Ser Pro Pro Gly Thr Pro Pro Thr Met Ala Thr Ser
275 280 285
Gly Ser Val Leu Gly Leu Asp Ala Met Glu Ser Gly Ser Thr Leu Asn
290 295 300
Ser Leu Ser Ser Ser Pro Ser Gly Ser Asp Thr Glu Asp Glu Thr Ser
305 310 315 320
Phe Ser Lys Glu Gln Ser Ser Lys Ser Glu Lys Thr Ser Lys Lys Gly
325 330 335
Thr Ala Gly Ser Gly Glu Thr Glu Lys Arg Tyr Ile Arg Thr Ile Arg
340 345 350
Leu Thr Asn Asp Gln Leu Lys Cys Leu Asn Leu Thr Tyr Gly Glu Asn
355 360 365
Asp Leu Lys Phe Ser Val Asp His Gly Lys Ala Ile Val Thr Ser Lys
370 375 380
Leu Phe Val Trp Arg Trp Asp Val Pro Ile Val Ile Ser Asp Ile Asp
385 390 395 400
Gly Thr Ile Thr Lys Ser Asp Ala Leu Gly His Val Leu Ala Met Ile
405 410 415
Gly Lys Asp Trp Thr His Leu Gly Val Ala Lys Leu Phe Ser Glu Ile
420 425 430
Ser Arg Asn Gly Tyr Asn Ile Leu Tyr Leu Thr Ala Arg Ser Ala Gly
435 440 445
Gln Ala Asp Ser Thr Arg Ser Tyr Leu Arg Ser Ile Glu Gln Asn Gly
450 455 460
Ser Lys Leu Pro Asn Gly Pro Val Ile Leu Ser Pro Asp Arg Thr Met
465 470 475 480
Ala Ala Leu Arg Arg Glu Val Ile Leu Lys Lys Pro Glu Val Phe Lys
485 490 495
Ile Ala Cys Leu Asn Asp Ile Arg Ser Leu Tyr Phe Glu Asp Ser Asp
500 505 510
Asn Glu Met Asp Thr Glu Glu Lys Ser Thr Pro Phe Phe Ala Gly Phe
515 520 525
Gly Asn Arg Ile Thr Asp Ala Leu Ser Tyr Arg Thr Val Gly Ile Pro
530 535 540
Ser Ser Arg Ile Phe Thr Ile Asn Thr Glu Gly Glu Val His Met Glu
545 550 555 560
Leu Leu Glu Leu Ala Gly Tyr Arg Ser Ser Tyr Ile His Ile Asn Glu
565 570 575
Leu Val Asp His Phe Phe Pro Pro Val Ser Leu Asp Ser Val Asp Leu
580 585 590
Arg Thr Asn Thr Ser Met Val Pro Gly Ser Pro Pro Asn Arg Thr Leu
595 600 605
Asp Asn Phe Asp Ser Glu Ile Thr Ser Gly Arg Lys Thr Leu Phe Arg
610 615 620
Gly Asn Gln Glu Glu Lys Phe Thr Asp Val Asn Phe Trp Arg Asp Pro
625 630 635 640
Leu Val Asp Ile Asp Asn Leu Ser Asp Ile Ser Asn Asp Asp Ser Asp
645 650 655
Asn Ile Asp Glu Asp Thr Asp Val Ser Gln Gln Ser Asn Val Ser Arg
660 665 670
Asn Arg Ala Asn Ser Val Lys Thr Ala Lys Val Thr Lys Ala Pro Gln
675 680 685
Arg Asn Val Ser Gly Ser Thr Asn Asn Asn Glu Val Leu Ala Ala Ser
690 695 700
Ser Asp Val Glu Asn Ala Ser Asp Leu Val Gly Ser His Ser Ser Ser
705 710 715 720
Gly Ser Thr Pro Asn Lys Ser Thr Met Ser Lys Gly Asp Ile Gly Lys
725 730 735
Gln Ile Tyr Leu Glu Leu Gly Ser Pro Leu Ala Ser Pro Lys Leu Arg
740 745 750
Tyr Leu Asp Asp Met Asp Asp Glu Asp Ser Asn Tyr Asn Arg Thr Lys
755 760 765
Ser Arg Arg Ala Ser Ser Ala Ala Ala Thr Ser Ile Asp Lys Glu Phe
770 775 780
Lys Lys Leu Ser Val Ser Lys Ala Gly Ala Pro Thr Arg Ile Val Ser
785 790 795 800
Lys Ile Asp Val Ser Asn Asp Val His Ser Leu Gly Asn Ser Asp Thr
805 810 815
Glu Ser Arg Arg Glu Gln Ser Val Asn Glu Thr Gly Arg Asn Gln Leu
820 825 830
Pro His Asn Ser Met Asp Asp Lys Asp Leu Asp Ser Arg Val Ser Asp
835 840 845
Glu Phe Asp Asp Asp Glu Phe Asp Glu Asp Glu Phe Glu Asp
850 855 860
<210> 9
<211> 418
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of DGA 1-encoding polypeptide
<400> 9
Met Ser Gly Thr Phe Asn Asp Ile Arg Arg Arg Lys Lys Glu Glu Gly
1 5 10 15
Ser Pro Thr Ala Gly Ile Thr Glu Arg His Glu Asn Lys Ser Leu Ser
20 25 30
Ser Ile Asp Lys Arg Glu Gln Thr Leu Lys Pro Gln Leu Glu Ser Cys
35 40 45
Cys Pro Leu Ala Thr Pro Phe Glu Arg Arg Leu Gln Thr Leu Ala Val
50 55 60
Ala Trp His Thr Ser Ser Phe Val Leu Phe Ser Ile Phe Thr Leu Phe
65 70 75 80
Ala Ile Ser Thr Pro Ala Leu Trp Val Leu Ala Ile Pro Tyr Met Ile
85 90 95
Tyr Phe Phe Phe Asp Arg Ser Pro Ala Thr Gly Glu Val Val Asn Arg
100 105 110
Tyr Ser Leu Arg Phe Arg Ser Leu Pro Ile Trp Lys Trp Tyr Cys Asp
115 120 125
Tyr Phe Pro Ile Ser Leu Ile Lys Thr Val Asn Leu Lys Pro Thr Phe
130 135 140
Thr Leu Ser Lys Asn Lys Arg Val Asn Glu Lys Asn Tyr Lys Ile Arg
145 150 155 160
Leu Trp Pro Thr Lys Tyr Ser Ile Asn Leu Lys Ser Asn Ser Thr Ile
165 170 175
Asp Tyr Arg Asn Gln Glu Cys Thr Gly Pro Thr Tyr Leu Phe Gly Tyr
180 185 190
His Pro His Gly Ile Gly Ala Leu Gly Ala Phe Gly Ala Phe Ala Thr
195 200 205
Glu Gly Cys Asn Tyr Ser Lys Ile Phe Pro Gly Ile Pro Ile Ser Leu
210 215 220
Met Thr Leu Val Thr Gln Phe His Ile Pro Leu Tyr Arg Asp Tyr Leu
225 230 235 240
Leu Ala Leu Gly Ile Ser Ser Val Ser Arg Lys Asn Ala Leu Arg Thr
245 250 255
Leu Ser Lys Asn Gln Ser Ile Cys Ile Val Val Gly Gly Ala Arg Glu
260 265 270
Ser Leu Leu Ser Ser Thr Asn Gly Thr Gln Leu Ile Leu Asn Lys Arg
275 280 285
Lys Gly Phe Ile Lys Leu Ala Ile Gln Thr Gly Asn Ile Asn Leu Val
290 295 300
Pro Val Phe Ala Phe Gly Glu Val Asp Cys Tyr Asn Val Leu Ser Thr
305 310 315 320
Lys Lys Asp Ser Val Leu Gly Lys Met Gln Leu Trp Phe Lys Glu Asn
325 330 335
Phe Gly Phe Thr Ile Pro Ile Phe Tyr Ala Arg Gly Leu Phe Asn Tyr
340 345 350
Asp Phe Gly Leu Leu Pro Phe Arg Ala Pro Ile Asn Val Val Val Gly
355 360 365
Arg Pro Ile Tyr Val Glu Lys Lys Ile Thr Asn Pro Pro Asp Asp Val
370 375 380
Val Asn His Phe His Asp Leu Tyr Ile Ala Glu Leu Lys Arg Leu Tyr
385 390 395 400
Tyr Glu Asn Arg Glu Lys Tyr Gly Val Pro Asp Ala Glu Leu Lys Ile
405 410 415
Val Gly
<210> 10
<211> 285
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of FLD 1-encoding polypeptide
<400> 10
Met Lys Ile Asn Val Ser Arg Pro Leu Gln Phe Leu Gln Trp Ser Ser
1 5 10 15
Tyr Ile Val Val Ala Phe Leu Ile Gln Leu Leu Ile Ile Leu Pro Leu
20 25 30
Ser Ile Leu Ile Tyr His Asp Phe Tyr Leu Arg Leu Leu Pro Ala Asp
35 40 45
Ser Ser Asn Val Val Pro Leu Asn Thr Phe Asn Ile Leu Asn Gly Val
50 55 60
Gln Phe Gly Thr Lys Phe Phe Gln Ser Ile Lys Ser Ile Pro Val Gly
65 70 75 80
Thr Asp Leu Pro Gln Thr Ile Asp Asn Gly Leu Ser Gln Leu Ile Pro
85 90 95
Met Arg Asp Asn Met Glu Tyr Lys Leu Asp Leu Asn Leu Gln Leu Tyr
100 105 110
Cys Gln Ser Lys Thr Asp His Leu Asn Leu Asp Asn Leu Leu Ile Asp
115 120 125
Val Tyr Arg Gly Pro Gly Pro Leu Leu Gly Ala Pro Gly Gly Ser Asn
130 135 140
Ser Lys Asp Glu Lys Ile Phe His Thr Ser Arg Pro Ile Val Cys Leu
145 150 155 160
Ala Leu Thr Asp Ser Met Ser Pro Gln Glu Ile Glu Gln Leu Gly Pro
165 170 175
Ser Arg Leu Asp Val Tyr Asp Glu Glu Trp Leu Asn Thr Ile Arg Ile
180 185 190
Glu Asp Lys Ile Ser Leu Glu Ser Ser Tyr Glu Thr Ile Ser Val Phe
195 200 205
Leu Lys Thr Glu Ile Ala Gln Arg Asn Leu Ile Ile His Pro Glu Ser
210 215 220
Gly Ile Lys Phe Arg Met Asn Phe Glu Gln Gly Leu Arg Asn Leu Met
225 230 235 240
Leu Arg Lys Arg Phe Leu Ser Tyr Ile Ile Gly Ile Ser Ile Phe His
245 250 255
Cys Ile Ile Cys Val Leu Phe Phe Ile Thr Gly Cys Thr Ala Phe Ile
260 265 270
Phe Val Arg Lys Gly Gln Glu Lys Ser Lys Lys His Ser
275 280 285
<210> 11
<211> 642
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of TGL 3-encoding polypeptide
<400> 11
Met Lys Glu Thr Ala Gln Glu Tyr Lys Val Ser Ala Val Ile Pro Thr
1 5 10 15
Leu Leu Lys Asn Trp Ile Leu Arg Val Val Tyr Ala Thr Leu Asp His
20 25 30
Ile Pro Pro Phe Val Trp Glu Ile Leu His Val Ile Thr Asp Ile Tyr
35 40 45
Phe Phe Trp Val Gln Lys Leu Ile Asn Tyr Val Arg Pro His Ser Arg
50 55 60
Val Ile Tyr Tyr Asn Ala Ile Lys Lys Leu Asp Glu Cys Asp Thr Tyr
65 70 75 80
Gln Met Trp Cys Gln Gln Ala Ser Val Val Asp Glu Ile Thr Gly Ala
85 90 95
Asn Leu Trp Arg Arg Asn Phe Phe Ser Arg Arg Tyr Asp Phe Asn Ser
100 105 110
Val Ile Glu Gln Tyr Ser Ile Leu Glu Asn Met Leu Arg Glu Glu Lys
115 120 125
Tyr Asp Val Val Lys Glu Lys Phe Ser Thr Thr Gly Pro Cys Met Leu
130 135 140
Arg Asn Phe Ala Gly Ile Gly Asp Lys Lys Leu Phe Thr Lys Ser Leu
145 150 155 160
Met Gly Thr Lys Leu Leu Ile Glu Gln Tyr Leu Thr Arg Ile Leu Glu
165 170 175
Gly Leu Asp Ile Leu Asn Asn Gln Thr Leu Thr Pro Thr Ser Phe Phe
180 185 190
Gln Arg Cys Lys Leu Ser Leu Gly Thr Thr Ala Leu Ile Leu Gln Gly
195 200 205
Gly Ser Leu Phe Gly Leu Phe His Leu Gly Val Ile Arg Gly Leu Leu
210 215 220
Leu Gln Asp Leu Met Pro Asn Ile Ile Ser Gly Ser Ser Met Gly Ala
225 230 235 240
Cys Val Ala Ser Leu Phe Gly Cys Leu Ser Asn Glu Gln Leu Lys Gln
245 250 255
Leu Leu Thr Asp Asp Asn Leu Leu Asn Ile Ile Lys Asn Asp Val Asp
260 265 270
Leu Leu Lys Ser Cys Gly Tyr Gly Asn Leu Glu Gln His Leu Asn Leu
275 280 285
Gly Thr Leu Ile Gln Asn Leu Ile His His Gly Tyr Ser Gln Asp Val
290 295 300
Tyr Leu Phe Ile Arg Phe Val Met Lys Tyr Ile Val Lys Glu Lys Thr
305 310 315 320
Phe Glu Glu Val Tyr Gln Ile Thr Gly Lys Val Phe Asn Ile Val Ile
325 330 335
His Pro Thr Asp Lys Ser Cys Pro Asn Leu Leu Asn Tyr Val Thr Thr
340 345 350
Pro Asn Val Leu Ile Lys Ser Ala Ile Glu Cys Ser Leu Gly Ser Gly
355 360 365
Val Ile Ser Glu Asp Thr Ser Leu Leu Cys Lys Asn Leu Glu Asn Glu
370 375 380
Ile Glu Pro Phe Leu Asn Ile Asn Lys Asn Lys Gln Val Lys Phe Leu
385 390 395 400
Thr Pro Glu Asn Ala Asn Asn Pro Ser Ile Thr Glu Ser Pro Tyr Thr
405 410 415
Arg Leu Thr Glu Leu Phe Asn Val Asn Asn Phe Ile Val Ser Leu Ala
420 425 430
Arg Pro Tyr Leu Ala Pro Leu Val Val Asn Asp Leu Lys His Glu Ile
435 440 445
Lys Thr Ser Lys Tyr Tyr Tyr Tyr Lys His Tyr Pro Asn Met Pro Pro
450 455 460
Ile Asn Ala Asn Thr Val Arg Lys Thr Gln Arg Ser Ser Ser Gln Ser
465 470 475 480
Pro Ile Lys Ala Gly Thr Val Glu Asp Leu Glu Pro Glu Pro Leu Met
485 490 495
Ser Pro Val Pro Pro Ser Ser Ala Val Asn Asp Ser Ala Glu Tyr Ile
500 505 510
Ile Pro Glu Leu Gly Ile Pro Gln Leu Asn Phe Thr Glu Met Glu Pro
515 520 525
Leu Ala Phe Lys Phe Lys Tyr His Leu Glu Arg Lys Leu Lys Asn Ile
530 535 540
Ala Thr Met Glu Phe Arg His Arg Met Glu Val Leu Asp Asn Leu Gly
545 550 555 560
Leu Leu Cys Ser Leu Ile Lys Arg Leu Ile Ile Asp Glu Lys Thr Pro
565 570 575
Arg Ser Ala Thr Glu Ile Ala Val Val Pro Arg Met Lys Ser Leu Ser
580 585 590
Leu Thr Arg Ile Ile Glu Gly Gln Leu Asn Asn Ile Pro Tyr Trp Ile
595 600 605
Lys Ser Gly Glu Arg Ser Thr Trp Pro Ala Leu Ala Leu Ile Lys Thr
610 615 620
Arg Cys Ala Val Glu Phe Lys Leu Asp Asp Ile Ile Arg Ala Arg Arg
625 630 635 640
Ser Arg
<210> 12
<211> 590
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of ACCA 2-encoding polypeptide
<400> 12
Met Arg Lys Val Leu Ile Ala Asn Arg Gly Glu Ile Ala Val Arg Val
1 5 10 15
Ala Arg Ala Cys Arg Asp Ala Gly Ile Ala Ser Val Ala Val Tyr Ala
20 25 30
Asp Pro Asp Arg Asp Ala Leu His Val Arg Ala Ala Asp Glu Ala Phe
35 40 45
Ala Leu Gly Gly Asp Thr Pro Ala Thr Ser Tyr Leu Asp Ile Ala Lys
50 55 60
Val Leu Lys Ala Ala Arg Glu Ser Gly Ala Asp Ala Ile His Pro Gly
65 70 75 80
Tyr Gly Phe Leu Ser Glu Asn Ala Glu Phe Ala Gln Ala Val Leu Asp
85 90 95
Ala Gly Leu Ile Trp Ile Gly Pro Pro Pro His Ala Ile Arg Asp Leu
100 105 110
Gly Asp Lys Val Ala Ala Arg His Ile Ala Gln Arg Ala Gly Ala Pro
115 120 125
Leu Val Ala Gly Thr Pro Asp Pro Val Ser Gly Ala Asp Glu Val Val
130 135 140
Ala Phe Ala Lys Glu His Gly Leu Pro Ile Ala Ile Lys Ala Ala Phe
145 150 155 160
Gly Gly Gly Gly Arg Gly Leu Lys Val Ala Arg Thr Leu Glu Glu Val
165 170 175
Pro Glu Leu Tyr Asp Ser Ala Val Arg Glu Ala Val Ala Ala Phe Gly
180 185 190
Arg Gly Glu Cys Phe Val Glu Arg Tyr Leu Asp Lys Pro Arg His Val
195 200 205
Glu Thr Gln Cys Leu Ala Asp Thr His Gly Asn Val Val Val Val Ser
210 215 220
Thr Arg Asp Cys Ser Leu Gln Arg Arg His Gln Lys Leu Val Glu Glu
225 230 235 240
Ala Pro Ala Pro Phe Leu Ser Glu Ala Gln Thr Glu Gln Leu Tyr Ser
245 250 255
Ser Ser Lys Ala Ile Leu Lys Glu Ala Gly Tyr Val Gly Ala Gly Thr
260 265 270
Val Glu Phe Leu Val Gly Met Asp Gly Thr Ile Ser Phe Leu Glu Val
275 280 285
Asn Thr Arg Leu Gln Val Glu His Pro Val Thr Glu Glu Val Ala Gly
290 295 300
Ile Asp Leu Val Arg Glu Met Phe Arg Ile Ala Asp Gly Glu Glu Leu
305 310 315 320
Gly Tyr Asp Asp Pro Ala Leu Arg Gly His Ser Phe Glu Phe Arg Ile
325 330 335
Asn Gly Glu Asp Pro Gly Arg Gly Phe Leu Pro Ala Pro Gly Thr Val
340 345 350
Thr Leu Phe Asp Ala Pro Thr Gly Pro Gly Val Arg Leu Asp Ala Gly
355 360 365
Val Glu Ser Gly Ser Val Ile Gly Pro Ala Trp Asp Ser Leu Leu Ala
370 375 380
Lys Leu Ile Val Thr Gly Arg Thr Arg Ala Glu Ala Leu Gln Arg Ala
385 390 395 400
Ala Arg Ala Leu Asp Glu Phe Thr Val Glu Gly Met Ala Thr Ala Ile
405 410 415
Pro Phe His Arg Thr Val Val Arg Asp Pro Ala Phe Ala Pro Glu Leu
420 425 430
Thr Gly Ser Thr Asp Pro Phe Thr Val His Thr Arg Trp Ile Glu Thr
435 440 445
Glu Phe Val Asn Glu Ile Lys Pro Phe Thr Thr Pro Ala Asp Thr Glu
450 455 460
Thr Asp Glu Glu Ser Gly Arg Glu Thr Val Val Val Glu Val Gly Gly
465 470 475 480
Lys Arg Leu Glu Val Ser Leu Pro Ser Ser Leu Gly Met Ser Leu Ala
485 490 495
Arg Thr Gly Leu Ala Ala Gly Ala Arg Pro Lys Arg Arg Ala Ala Lys
500 505 510
Lys Ser Gly Pro Ala Ala Ser Gly Asp Thr Leu Ala Ser Pro Met Gln
515 520 525
Gly Thr Ile Val Lys Ile Ala Val Glu Glu Gly Gln Glu Val Gln Glu
530 535 540
Gly Asp Leu Ile Val Val Leu Glu Ala Met Lys Met Glu Gln Pro Leu
545 550 555 560
Asn Ala His Arg Ser Gly Thr Ile Lys Gly Leu Thr Ala Glu Val Gly
565 570 575
Ala Ser Leu Thr Ser Gly Ala Ala Ile Cys Glu Ile Lys Asp
580 585 590
<210> 13
<211> 527
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of ACCB-encoding polypeptide
<400> 13
Met Thr Val Leu Asp Glu Ala Pro Gly Glu Pro Thr Asp Ala Arg Gly
1 5 10 15
Arg Val Ala Glu Leu His Gly Ile Arg Ala Ala Ala Leu Ala Gly Pro
20 25 30
Ser Glu Lys Ala Thr Ala Ala Gln His Ala Lys Gly Lys Leu Thr Ala
35 40 45
Arg Glu Arg Ile Glu Leu Leu Leu Asp Pro Gly Ser Phe Arg Glu Val
50 55 60
Glu Gln Leu Arg Arg His Arg Ala Thr Gly Phe Gly Leu Glu Ala Lys
65 70 75 80
Lys Pro Tyr Thr Asp Gly Val Ile Thr Gly Trp Gly Thr Val Glu Gly
85 90 95
Arg Thr Val Phe Val Tyr Ala His Asp Phe Arg Ile Phe Gly Gly Ala
100 105 110
Leu Gly Glu Ala His Ala Thr Lys Ile His Lys Ile Met Asp Met Ala
115 120 125
Ile Ala Ala Gly Ala Pro Leu Val Ser Leu Asn Asp Gly Ala Gly Ala
130 135 140
Arg Ile Gln Glu Gly Val Ser Ala Leu Ala Gly Tyr Gly Gly Ile Phe
145 150 155 160
Gln Arg Asn Thr Lys Ala Ser Gly Val Ile Pro Gln Ile Ser Val Met
165 170 175
Leu Gly Pro Cys Ala Gly Gly Ala Ala Tyr Ser Pro Ala Leu Thr Asp
180 185 190
Phe Val Phe Met Val Arg Asp Thr Ser Gln Met Phe Ile Thr Gly Pro
195 200 205
Asp Val Val Lys Ala Val Thr Gly Glu Glu Ile Thr Gln Asn Gly Leu
210 215 220
Gly Gly Ala Asp Val His Ala Glu Thr Ser Gly Val Cys His Phe Ala
225 230 235 240
Tyr Asp Asp Glu Glu Thr Cys Leu Ala Glu Val Arg Tyr Leu Leu Ser
245 250 255
Leu Leu Pro Gln Asn Asn Arg Glu Asn Pro Pro Arg Ala Glu Ser Ser
260 265 270
Asp Pro Val Asp Arg Arg Ser Asp Thr Leu Leu Asp Leu Val Pro Ala
275 280 285
Asp Gly Asn Arg Pro Tyr Asp Met Thr Lys Val Ile Glu Glu Leu Val
290 295 300
Asp Glu Gly Glu Tyr Leu Glu Val His Glu Arg Trp Ala Arg Asn Ile
305 310 315 320
Ile Cys Ala Leu Ala Arg Leu Asp Gly Arg Val Val Gly Ile Val Ala
325 330 335
Asn Gln Pro Gln Ala Leu Ala Gly Val Leu Asp Ile Glu Ala Ser Glu
340 345 350
Lys Ala Ala Arg Phe Val Gln Met Cys Asp Ala Phe Asn Ile Pro Ile
355 360 365
Ile Thr Leu Leu Asp Val Pro Gly Phe Leu Pro Gly Val Asp Gln Glu
370 375 380
His Gly Gly Ile Ile Arg His Gly Ala Lys Leu Leu Tyr Ala Tyr Cys
385 390 395 400
Asn Ala Thr Val Pro Arg Ile Ser Leu Ile Leu Arg Lys Ala Tyr Gly
405 410 415
Gly Ala Tyr Ile Val Met Asp Ser Gln Ser Ile Gly Ala Asp Leu Thr
420 425 430
Tyr Ala Trp Pro Thr Asn Glu Ile Ala Val Met Gly Ala Glu Gly Ala
435 440 445
Ala Asn Val Ile Phe Arg Arg Gln Ile Ala Asp Ala Glu Asp Pro Glu
450 455 460
Ala Met Arg Ala Arg Met Val Lys Glu Tyr Lys Ser Glu Leu Met His
465 470 475 480
Pro Tyr Tyr Ala Ala Glu Arg Gly Leu Val Asp Asp Val Ile Asp Pro
485 490 495
Ala Glu Thr Arg Glu Val Leu Ile Thr Ser Leu Ala Met Leu His Thr
500 505 510
Lys His Ala Asp Leu Pro Ser Arg Lys His Gly Asn Pro Pro Gln
515 520 525
<210> 14
<211> 65
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of ACCE-encoding polypeptide
<400> 14
Met Ser Pro Ala Asp Ile Arg Val Glu Lys Gly His Ala Glu Pro Glu
1 5 10 15
Glu Val Ala Ala Ile Thr Ala Leu Leu Leu Ala Arg Ala Ala Ala Arg
20 25 30
Pro Ala Glu Ile Ala Pro Thr His Gly Gly Gly Arg Ala Arg Ala Gly
35 40 45
Trp Arg Arg Leu Glu Arg Glu Pro Gly Phe Arg Ala Pro His Ser Trp
50 55 60
Arg
65
<210> 15
<211> 446
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of DGAT-encoded polypeptide
<400> 15
Met Thr Pro Asp Pro Leu Ala Pro Leu Asp Leu Ala Phe Trp Asn Ile
1 5 10 15
Glu Ser Ala Glu His Pro Met His Leu Gly Ala Leu Gly Val Phe Glu
20 25 30
Ala Asp Ser Pro Thr Ala Gly Ala Leu Ala Ala Asp Leu Leu Ala Ala
35 40 45
Arg Ala Pro Ala Val Pro Gly Leu Arg Met Arg Ile Arg Asp Thr Trp
50 55 60
Gln Pro Pro Met Ala Leu Arg Arg Pro Phe Ala Phe Gly Gly Ala Thr
65 70 75 80
Arg Glu Pro Asp Pro Arg Phe Asp Pro Leu Asp His Val Arg Leu His
85 90 95
Ala Pro Ala Thr Asp Phe His Ala Arg Ala Gly Arg Leu Met Glu Arg
100 105 110
Pro Leu Glu Arg Gly Arg Pro Pro Trp Glu Ala His Val Leu Pro Gly
115 120 125
Ala Asp Gly Gly Ser Phe Ala Val Leu Phe Lys Phe His His Ala Leu
130 135 140
Ala Asp Gly Leu Arg Ala Leu Thr Leu Ala Ala Gly Val Leu Asp Pro
145 150 155 160
Met Asp Leu Pro Ala Pro Arg Pro Arg Pro Glu Gln Pro Pro Arg Gly
165 170 175
Leu Leu Pro Asp Val Arg Ala Leu Pro Asp Arg Leu Arg Gly Ala Leu
180 185 190
Ser Asp Ala Gly Arg Ala Leu Asp Ile Gly Ala Ala Ala Ala Leu Ser
195 200 205
Thr Leu Asp Val Arg Ser Ser Pro Ala Leu Thr Ala Ala Ser Ser Gly
210 215 220
Thr Arg Arg Thr Ala Gly Val Ser Val Asp Leu Asp Asp Val His His
225 230 235 240
Val Arg Lys Thr Thr Gly Gly Thr Val Asn Asp Val Leu Ile Ala Val
245 250 255
Val Ala Gly Ala Leu Arg Arg Trp Leu Asp Glu Arg Gly Asp Gly Ser
260 265 270
Glu Gly Val Ala Pro Arg Ala Leu Ile Pro Val Ser Arg Arg Arg Pro
275 280 285
Arg Ser Ala His Pro Gln Gly Asn Arg Leu Ser Gly Tyr Leu Met Arg
290 295 300
Leu Pro Val Gly Asp Pro Asp Pro Leu Ala Arg Leu Gly Thr Val Arg
305 310 315 320
Ala Ala Met Asp Arg Asn Lys Asp Ala Gly Pro Gly Arg Gly Ala Gly
325 330 335
Ala Val Ala Leu Leu Ala Asp His Val Pro Ala Leu Gly His Arg Leu
340 345 350
Gly Gly Pro Leu Val Ser Gly Ala Ala Arg Leu Trp Phe Asp Leu Leu
355 360 365
Val Thr Ser Val Pro Leu Pro Ser Leu Gly Leu Arg Leu Gly Gly His
370 375 380
Pro Leu Thr Glu Val Tyr Pro Leu Ala Pro Leu Ala Arg Gly His Ser
385 390 395 400
Leu Ala Val Ala Val Ser Thr Tyr Arg Gly Arg Val His Tyr Gly Leu
405 410 415
Leu Ala Asp Ala Lys Ala Val Pro Asp Leu Asp Arg Leu Ala Val Ala
420 425 430
Val Ala Glu Glu Val Glu Thr Leu Leu Thr Ala Cys Arg Pro
435 440 445
<210> 16
<211> 340
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of LPP-encoded polypeptide
<400> 16
Met Arg Thr Glu Arg Lys Pro Thr Arg Leu Asp Arg Val Phe Ala Arg
1 5 10 15
Leu Asp Arg Glu Pro Glu Arg Pro Ala Leu Leu Asp Val Pro Glu Met
20 25 30
Ser Arg His Arg Ile Ala Leu Phe Ala Gly Thr Leu Ala Phe Tyr Ile
35 40 45
Ala Ile Val Trp Ala Val Val Ile Thr Ser Trp Leu Val Arg Leu Asp
50 55 60
Trp Gln Val Met Phe Phe Arg Pro Tyr Gln Gln Trp Pro Glu Ile His
65 70 75 80
Ala Phe Val Asp Tyr Tyr Val Val Leu Gly Gln Arg Gly Pro Thr Ala
85 90 95
Val Met Val Ala Ala Trp Leu Gly Trp Arg Ser Trp Arg Gln His Thr
100 105 110
Leu Arg Pro Leu Leu Ala Leu Gly Val Ser Leu Leu Leu Leu Asn Val
115 120 125
Thr Val Gly Ala Ala Lys Tyr Gly Met Gly Arg Leu Gly Pro His Tyr
130 135 140
Ala Thr Thr Ile Gly Ala Asn Glu Met Trp Leu Gly Gly Asp Ile Phe
145 150 155 160
Pro Ser Gly His Thr Ala Asn Ala Val Val Thr Trp Gly Ile Leu Ala
165 170 175
Tyr Leu Ala Ser Thr His Arg Thr Arg Arg Trp Leu Ser Ala Ile Ser
180 185 190
Ala Val Thr Ser Leu Gly Val Gly Met Ser Thr Val Tyr Leu Gly Thr
195 200 205
His Trp Leu Ser Asp Val Leu Leu Gly Trp Val Ala Gly Leu Leu Ile
210 215 220
Leu Leu Ala Leu Pro Trp Phe Glu Pro Leu Ile Thr Arg Ala Glu Ala
225 230 235 240
Trp Ile Leu Gly Leu Arg Asp Arg Trp Tyr Thr Arg Arg Asp Arg Arg
245 250 255
Ser Thr Thr Arg Pro Pro Leu Gly Pro Pro Val Pro Val Ser Pro Pro
260 265 270
Gly Ser Gly Ser Arg Pro Gln Ala Pro Ala Arg Glu Pro Val Ala Ala
275 280 285
Pro Arg Thr Ala Arg Ala Pro Ala His Leu Ala Pro Gly Pro His Thr
290 295 300
Ala Arg Ser Asp Arg Thr Pro Val Thr Pro Ala Gly Ser Arg Arg Pro
305 310 315 320
Pro His Ser Asp Arg His Ala Arg Asn Thr Ala Pro Thr Ala Arg Pro
325 330 335
Leu Ser Gly Gly
340
<210> 17
<211> 336
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide encoded by OLE1A
<400> 17
Met Thr Thr Ser Ser Asp Val Ile Pro Asp Ala Pro Gln Pro Ala Gly
1 5 10 15
Asp Ala Ala Gly Pro Ser Ala Thr Leu Gly Gly Glu Gln Lys Arg Ser
20 25 30
Ile Glu Gln Ile Thr Leu Leu Leu Phe Ile Thr Leu Pro Phe Leu Ala
35 40 45
Leu Val Ala Ala Val Pro Leu Ala Trp Gly Trp Gly Val Ser Trp Leu
50 55 60
Asp Leu Gly Leu Leu Val Phe Phe Tyr Phe Leu Gly Cys His Gly Ile
65 70 75 80
Thr Ile Gly Phe His Arg His Phe Thr His Gly Ser Phe Lys Ala Lys
85 90 95
Arg Pro Leu Lys Ile Ala Leu Ala Ile Ala Gly Ser Met Ala Val Glu
100 105 110
Gly Pro Leu Val Arg Trp Val Ala Asp His Arg Lys His His Lys Phe
115 120 125
Ser Asp Asp Glu Gly Asp Pro His Ser Pro Trp Arg Tyr Gly Glu Thr
130 135 140
Val Pro Ala Leu Ile Lys Gly Leu Trp Trp Ala His Ile Ala Trp Met
145 150 155 160
Phe Asp Glu Glu Gln Thr Pro Gln Glu Lys Tyr Ala Pro Asp Leu Ile
165 170 175
Lys Asp Pro Ala Leu Arg Ala Val Ser Arg Gln Phe Ile Leu Trp Thr
180 185 190
Val Val Ser Leu Ala Leu Pro Ala Leu Ile Gly Gly Leu Val Thr Met
195 200 205
Ser Trp Trp Gly Ala Phe Thr Gly Phe Phe Trp Gly Ser Leu Val Arg
210 215 220
Val Ala Leu Leu His His Val Thr Trp Ser Ile Asn Ser Ile Cys His
225 230 235 240
Ala Val Gly Lys Arg Pro Phe Lys Ser Arg Asp Arg Ser Gly Asn Val
245 250 255
Trp Trp Leu Ala Ile Leu Ser Cys Gly Glu Ser Trp His Asn Leu His
260 265 270
His Ala Asp Pro Thr Ser Ala Arg His Gly Val Met Arg Gly Gln Leu
275 280 285
Asp Ser Ser Ala Arg Leu Ile Arg Trp Phe Glu Gln Leu Gly Trp Ala
290 295 300
Tyr Asp Val Arg Trp Pro Ser Arg Ser Arg Ile Asp Ser Arg Arg Asn
305 310 315 320
Thr Asp Gln Asp Gly Ala Arg Arg Arg Lys Glu Thr Ala Lys Ala Ala
325 330 335
<210> 18
<211> 345
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide encoded by OLE1B
<400> 18
Met Thr Met Ala Thr Thr Ala Thr Arg Ser Asp Thr Pro Gly Ser Asp
1 5 10 15
Phe Ala Arg Leu Ser Lys Lys Val Ala Asp Ala Gly Leu Leu Gly Arg
20 25 30
Arg Pro Gly Tyr Tyr Thr Leu Arg Ile Thr Ala Val Thr Gly Leu Tyr
35 40 45
Ala Ala Gly Trp Ala Ala Phe Val Leu Val Gly Ala Ser Trp Trp Thr
50 55 60
Leu Ala Ile Ala Ala Phe Leu Ala Val Met Tyr Gly Gln Val Ala Leu
65 70 75 80
Val Ala His Asp Met Ala His Arg Gln Val Phe Arg Arg Arg Arg Ala
85 90 95
Ser Glu Leu Ser Gly Arg Ile Ala Gly Ala Ser Ile Gly Met Ser Tyr
100 105 110
Gly Trp Trp Gln Asp Lys His Thr Arg His His Ala Asn Pro Asn Thr
115 120 125
Glu Asp Leu Asp Pro Asp Ile Gly Pro Asp Leu Leu Val Trp Ser Pro
130 135 140
Asp Gln Ala Arg Ala Ala Thr Gly Leu Pro Arg Leu Leu Gly Arg Trp
145 150 155 160
Gln Ala Phe Leu Phe Phe Pro Leu Leu Thr Leu Glu Gly Phe Asn Leu
165 170 175
His Val Ala Ser Gly Arg Ala Met Ala Asn Arg Arg Leu Lys Arg Arg
180 185 190
Ala Leu Asp Gly Ala Leu Leu Leu Ala His Cys Ala Val Tyr Leu Thr
195 200 205
Ala Leu Phe Trp Val Leu Pro Pro Gly Met Ala Ile Ala Phe Leu Ala
210 215 220
Val His Gln Cys Leu Phe Gly Val Tyr Leu Gly Ser Ala Phe Ala Pro
225 230 235 240
Asn His Lys Gly Met Pro Ile Leu Thr Ala Asp Asp Arg Pro Asp Phe
245 250 255
Leu Arg Arg Gln Val Leu Thr Ser Arg Asn Val Asn Gly Gly Leu Phe
260 265 270
Thr Asp Leu Ala Leu Gly Gly Leu Asn His Gln Ile Glu His His Leu
275 280 285
Phe Pro Ser Met Pro Ser Pro Asn Leu Arg Lys Ala Arg Ala Ile Val
290 295 300
Arg Arg Tyr Cys Arg Asp Leu Gly Val Asp Tyr Ala Glu Thr Gly Leu
305 310 315 320
Val Ala Ser Tyr Arg Leu Ala Leu Thr Ser Leu His Asp Ala Gly Thr
325 330 335
Pro Leu Arg Arg Thr Arg Val Arg Ala
340 345
<210> 19
<211> 350
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide encoded by OLE1C
<400> 19
Met Pro Leu Pro Arg Glu Thr Leu Pro Pro Asp Thr Gly Gly Ser Arg
1 5 10 15
Glu Gly Ser Glu Phe Thr Pro Leu Leu Arg Asp Val Arg Glu Gln Gln
20 25 30
Leu Leu Glu Arg Arg Thr Gly Trp Tyr Ala Arg Thr Ile Ala Val Asn
35 40 45
Ala Leu Gly Leu Ala Ala Val Gly Thr Gly Met Ala Leu Leu Gly Asp
50 55 60
Ser Trp Trp Val Leu Ala Leu Ala Pro Val Leu Ala Val Leu Cys Ala
65 70 75 80
Arg Thr Ala Phe Ile Gly His Asp Ala Gly His Ala Gln Ile Ser Gly
85 90 95
Ser Arg Ala Val Asn Arg Arg Ile Gly Leu Val His Gly Asn Leu Leu
100 105 110
Leu Gly Met Ser Tyr Ala Trp Trp Asn Asp Lys His Asn Arg His His
115 120 125
Ala Asn Pro Asn His Ile Asp Lys Asp Pro Asp Val Ala Ala Asp Val
130 135 140
Leu Val Phe Thr Ser Gly Gln Ala Ala Thr Arg Thr Gly Phe Arg Gly
145 150 155 160
Arg Leu Thr Arg His Gln Ala Trp Leu Phe Phe Pro Leu Thr Leu Leu
165 170 175
Glu Gly Leu Ala Leu Lys Leu His Gly Phe Gln His Leu Arg Arg Gln
180 185 190
Arg Gly Arg Ala Arg Leu Val Glu Gly Ala Leu Leu Val Ala His Val
195 200 205
Ala Gly Tyr Val Thr Leu Leu Leu Ala Thr Met Pro Leu Ala His Ala
210 215 220
Leu Val Phe Ala Ala Leu His Gln Ala Leu Phe Gly Leu His Leu Gly
225 230 235 240
Met Ala Phe Ala Pro Asn His Lys Gly Met Asp Met Pro Asp Pro Asp
245 250 255
Ser Glu Ala Glu Lys Trp Gly His Leu Arg Arg Gln Val Leu Thr Ser
260 265 270
Arg Asn Val Arg Gly Gly Phe Leu Thr Asp Trp Phe Leu Gly Gly Leu
275 280 285
Asn Tyr Gln Ile Glu His His Leu Phe Pro Ser Met Pro Arg Pro His
290 295 300
Leu Gly Leu Ala Gln Ala Ala Val Lys Ala His Cys Arg Asp Leu Gly
305 310 315 320
Ile Pro Tyr Ala Glu Thr Gly Leu Val Asp Ser Tyr Arg Gln Ala Leu
325 330 335
Arg His Met His Glu Val Gly Glu Pro Leu Arg Ala Asp Ile
340 345 350
<210> 20
<211> 347
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide encoded by OLE1D
<400> 20
Met Leu Val Glu Ser Leu Pro Thr Pro Ala Gln Glu Lys Asp Arg Glu
1 5 10 15
Arg Gly Ser Asp Phe Ser Glu Leu Ser Arg Arg Ile Ala Gly Ala Gly
20 25 30
Leu Leu Arg Arg Arg Pro Leu Tyr Tyr Thr Val Arg Phe Gly Ala Val
35 40 45
Ala Leu Ala Leu Ala Gly Gly Val Ala Ala Phe Val Ala Leu Gly Asp
50 55 60
Ser Trp Ser Gln Leu Phe Val Ala Val Ala Leu Ala Val Val Phe Gly
65 70 75 80
Gln Leu Gly Leu Ala Ala His Asp Leu Ala His Arg Gln Val Phe Thr
85 90 95
Arg Arg Arg Pro Ser Glu Ala Gly Gly Leu Leu Thr Ala Asn Leu Leu
100 105 110
Leu Gly Met Ser Tyr Gly Trp Trp Met Asn Lys His Thr Arg His His
115 120 125
Ala Asn Pro Asn His Glu Glu Lys Asp Pro Asp Val Ser Pro Asp Ile
130 135 140
Leu Val Trp Ser Arg Gly Gln Ala Ser Arg Ala Thr Gly Leu Pro Arg
145 150 155 160
Phe Val Gly Arg His Gln Ala Ala Leu Phe Phe Pro Leu Leu Thr Leu
165 170 175
Glu Gly Leu Asn Leu Ser Phe Asn Ser Phe Lys Ala Leu Gly Ser Arg
180 185 190
Ala Val Lys Arg Pro Val Leu Glu Gly Thr Leu Leu Val Ala His Phe
195 200 205
Ala Val Tyr Phe Gly Gly Leu Phe Thr Val Leu Ser Pro Gly Lys Ala
210 215 220
Leu Val Phe Leu Ala Val His Gln Gly Leu Phe Gly Ile Tyr Leu Gly
225 230 235 240
Ser Val Phe Ala Pro Asn His Lys Gly Met Pro Met Ile Glu Glu Gly
245 250 255
Met Arg Leu Asp Phe Leu Arg Arg Gln Val Leu Thr Ser Arg Asn Val
260 265 270
Arg Gly Gly Ala Leu Val Asp Ala Phe Met Gly Gly Leu Asn Tyr Gln
275 280 285
Ile Glu His His Leu Phe Pro Ser Met Pro Thr Pro Ala Leu Gly Arg
290 295 300
Ala Gln Ala Ile Thr Glu Ala Tyr Cys Ala Glu Leu Gly Val Pro Tyr
305 310 315 320
His Gln Thr Gly Leu Leu Ala Ser His Arg Glu Ala Leu Arg His Met
325 330 335
Arg Ser Val Gly Glu Pro Leu Arg Ala Ala Arg
340 345
<210> 21
<211> 319
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of EcACCA-encoding polypeptide
<400> 21
Met Ser Leu Asn Phe Leu Asp Phe Glu Gln Pro Ile Ala Glu Leu Glu
1 5 10 15
Ala Lys Ile Asp Ser Leu Thr Ala Val Ser Arg Gln Asp Glu Lys Leu
20 25 30
Asp Ile Asn Ile Asp Glu Glu Val His Arg Leu Arg Glu Lys Ser Val
35 40 45
Glu Leu Thr Arg Lys Ile Phe Ala Asp Leu Gly Ala Trp Gln Ile Ala
50 55 60
Gln Leu Ala Arg His Pro Gln Arg Pro Tyr Thr Leu Asp Tyr Val Arg
65 70 75 80
Leu Ala Phe Asp Glu Phe Asp Glu Leu Ala Gly Asp Arg Ala Tyr Ala
85 90 95
Asp Asp Lys Ala Ile Val Gly Gly Ile Ala Arg Leu Asp Gly Arg Pro
100 105 110
Val Met Ile Ile Gly His Gln Lys Gly Arg Glu Thr Lys Glu Lys Ile
115 120 125
Arg Arg Asn Phe Gly Met Pro Ala Pro Glu Gly Tyr Arg Lys Ala Leu
130 135 140
Arg Leu Met Gln Met Ala Glu Arg Phe Lys Met Pro Ile Ile Thr Phe
145 150 155 160
Ile Asp Thr Pro Gly Ala Tyr Pro Gly Val Gly Ala Glu Glu Arg Gly
165 170 175
Gln Ser Glu Ala Ile Ala Arg Asn Leu Arg Glu Met Ser Arg Leu Gly
180 185 190
Val Pro Val Val Cys Thr Val Ile Gly Glu Gly Gly Ser Gly Gly Ala
195 200 205
Leu Ala Ile Gly Val Gly Asp Lys Val Asn Met Leu Gln Tyr Ser Thr
210 215 220
Tyr Ser Val Ile Ser Pro Glu Gly Cys Ala Ser Ile Leu Trp Lys Ser
225 230 235 240
Ala Asp Lys Ala Pro Leu Ala Ala Glu Ala Met Gly Ile Ile Ala Pro
245 250 255
Arg Leu Lys Glu Leu Lys Leu Ile Asp Ser Ile Ile Pro Glu Pro Leu
260 265 270
Gly Gly Ala His Arg Asn Pro Glu Ala Met Ala Ala Ser Leu Lys Ala
275 280 285
Gln Leu Leu Ala Asp Leu Ala Asp Leu Asp Val Leu Ser Thr Glu Asp
290 295 300
Leu Lys Asn Arg Arg Tyr Gln Arg Leu Met Ser Tyr Gly Tyr Ala
305 310 315
<210> 22
<211> 156
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of EcACCB-encoding polypeptide
<400> 22
Met Asp Ile Arg Lys Ile Lys Lys Leu Ile Glu Leu Val Glu Glu Ser
1 5 10 15
Gly Ile Ser Glu Leu Glu Ile Ser Glu Gly Glu Glu Ser Val Arg Ile
20 25 30
Ser Arg Ala Ala Pro Ala Ala Ser Phe Pro Val Met Gln Gln Ala Tyr
35 40 45
Ala Ala Pro Met Met Gln Gln Pro Ala Gln Ser Asn Ala Ala Ala Pro
50 55 60
Ala Thr Val Pro Ser Met Glu Ala Pro Ala Ala Ala Glu Ile Ser Gly
65 70 75 80
His Ile Val Arg Ser Pro Met Val Gly Thr Phe Tyr Arg Thr Pro Ser
85 90 95
Pro Asp Ala Lys Ala Phe Ile Glu Val Gly Gln Lys Val Asn Val Gly
100 105 110
Asp Thr Leu Cys Ile Val Glu Ala Met Lys Met Met Asn Gln Ile Glu
115 120 125
Ala Asp Lys Ser Gly Thr Val Lys Ala Ile Leu Val Glu Ser Gly Gln
130 135 140
Pro Val Glu Phe Asp Glu Pro Leu Val Val Ile Glu
145 150 155
<210> 23
<211> 449
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of EcACCC-encoding polypeptide
<400> 23
Met Leu Asp Lys Ile Val Ile Ala Asn Arg Gly Glu Ile Ala Leu Arg
1 5 10 15
Ile Leu Arg Ala Cys Lys Glu Leu Gly Ile Lys Thr Val Ala Val His
20 25 30
Ser Ser Ala Asp Arg Asp Leu Lys His Val Leu Leu Ala Asp Glu Thr
35 40 45
Val Cys Ile Gly Pro Ala Pro Ser Val Lys Ser Tyr Leu Asn Ile Pro
50 55 60
Ala Ile Ile Ser Ala Ala Glu Ile Thr Gly Ala Val Ala Ile His Pro
65 70 75 80
Gly Tyr Gly Phe Leu Ser Glu Asn Ala Asn Phe Ala Glu Gln Val Glu
85 90 95
Arg Ser Gly Phe Ile Phe Ile Gly Pro Lys Ala Glu Thr Ile Arg Leu
100 105 110
Met Gly Asp Lys Val Ser Ala Ile Ala Ala Met Lys Lys Ala Gly Val
115 120 125
Pro Cys Val Pro Gly Ser Asp Gly Pro Leu Gly Asp Asp Met Asp Lys
130 135 140
Asn Arg Ala Ile Ala Lys Arg Ile Gly Tyr Pro Val Ile Ile Lys Ala
145 150 155 160
Ser Gly Gly Gly Gly Gly Arg Gly Met Arg Val Val Arg Gly Asp Ala
165 170 175
Glu Leu Ala Gln Ser Ile Ser Met Thr Arg Ala Glu Ala Lys Ala Ala
180 185 190
Phe Ser Asn Asp Met Val Tyr Met Glu Lys Tyr Leu Glu Asn Pro Arg
195 200 205
His Val Glu Ile Gln Val Leu Ala Asp Gly Gln Gly Asn Ala Ile Tyr
210 215 220
Leu Ala Glu Arg Asp Cys Ser Met Gln Arg Arg His Gln Lys Val Val
225 230 235 240
Glu Glu Ala Pro Ala Pro Gly Ile Thr Pro Glu Leu Arg Arg Tyr Ile
245 250 255
Gly Glu Arg Cys Ala Lys Ala Cys Val Asp Ile Gly Tyr Arg Gly Ala
260 265 270
Gly Thr Phe Glu Phe Leu Phe Glu Asn Gly Glu Phe Tyr Phe Ile Glu
275 280 285
Met Asn Thr Arg Ile Gln Val Glu His Pro Val Thr Glu Met Ile Thr
290 295 300
Gly Val Asp Leu Ile Lys Glu Gln Leu Arg Ile Ala Ala Gly Gln Pro
305 310 315 320
Leu Ser Ile Lys Gln Glu Glu Val His Val Arg Gly His Ala Val Glu
325 330 335
Cys Arg Ile Asn Ala Glu Asp Pro Asn Thr Phe Leu Pro Ser Pro Gly
340 345 350
Lys Ile Thr Arg Phe His Ala Pro Gly Gly Phe Gly Val Arg Trp Glu
355 360 365
Ser His Ile Tyr Ala Gly Tyr Thr Val Pro Pro Tyr Tyr Asp Ser Met
370 375 380
Ile Gly Lys Leu Ile Cys Tyr Gly Glu Asn Arg Asp Val Ala Ile Ala
385 390 395 400
Arg Met Lys Asn Ala Leu Gln Glu Leu Ile Ile Asp Gly Ile Lys Thr
405 410 415
Asn Val Asp Leu Gln Ile Arg Ile Met Asn Asp Glu Asn Phe Gln His
420 425 430
Gly Gly Thr Asn Ile His Tyr Leu Glu Lys Lys Leu Gly Leu Gln Glu
435 440 445
Lys
<210> 24
<211> 304
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of EcACCD-encoding polypeptide
<400> 24
Met Ser Trp Ile Glu Arg Ile Lys Ser Asn Ile Thr Pro Thr Arg Lys
1 5 10 15
Ala Ser Ile Pro Glu Gly Val Trp Thr Lys Cys Asp Ser Cys Gly Gln
20 25 30
Val Leu Tyr Arg Ala Glu Leu Glu Arg Asn Leu Glu Val Cys Pro Lys
35 40 45
Cys Asp His His Met Arg Met Thr Ala Arg Asn Arg Leu His Ser Leu
50 55 60
Leu Asp Glu Gly Ser Leu Val Glu Leu Gly Ser Glu Leu Glu Pro Lys
65 70 75 80
Asp Val Leu Lys Phe Arg Asp Ser Lys Lys Tyr Lys Asp Arg Leu Ala
85 90 95
Ser Ala Gln Lys Glu Thr Gly Glu Lys Asp Ala Leu Val Val Met Lys
100 105 110
Gly Thr Leu Tyr Gly Met Pro Val Val Ala Ala Ala Phe Glu Phe Ala
115 120 125
Phe Met Gly Gly Ser Met Gly Ser Val Val Gly Ala Arg Phe Val Arg
130 135 140
Ala Val Glu Gln Ala Leu Glu Asp Asn Cys Pro Leu Ile Cys Phe Ser
145 150 155 160
Ala Ser Gly Gly Ala Arg Met Gln Glu Ala Leu Met Ser Leu Met Gln
165 170 175
Met Ala Lys Thr Ser Ala Ala Leu Ala Lys Met Gln Glu Arg Gly Leu
180 185 190
Pro Tyr Ile Ser Val Leu Thr Asp Pro Thr Met Gly Gly Val Ser Ala
195 200 205
Ser Phe Ala Met Leu Gly Asp Leu Asn Ile Ala Glu Pro Lys Ala Leu
210 215 220
Ile Gly Phe Ala Gly Pro Arg Val Ile Glu Gln Thr Val Arg Glu Lys
225 230 235 240
Leu Pro Pro Gly Phe Gln Arg Ser Glu Phe Leu Ile Glu Lys Gly Ala
245 250 255
Ile Asp Met Ile Val Arg Arg Pro Glu Met Arg Leu Lys Leu Ala Ser
260 265 270
Ile Leu Ala Lys Leu Met Asn Leu Pro Ala Pro Asn Pro Glu Ala Pro
275 280 285
Arg Glu Gly Val Val Val Pro Pro Val Pro Asp Gln Glu Pro Glu Ala
290 295 300
<210> 25
<211> 254
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of pgpB-encoding polypeptide
<400> 25
Met Arg Ser Ile Ala Arg Arg Thr Ala Val Gly Ala Ala Leu Leu Leu
1 5 10 15
Val Met Pro Val Ala Val Trp Ile Ser Gly Trp Arg Trp Gln Pro Gly
20 25 30
Glu Gln Ser Trp Leu Leu Lys Ala Ala Phe Trp Val Thr Glu Thr Val
35 40 45
Thr Gln Pro Trp Gly Val Ile Thr His Leu Ile Leu Phe Gly Trp Phe
50 55 60
Leu Trp Cys Leu Arg Phe Arg Ile Lys Ala Ala Phe Val Leu Phe Ala
65 70 75 80
Ile Leu Ala Ala Ala Ile Leu Val Gly Gln Gly Val Lys Ser Trp Ile
85 90 95
Lys Asp Lys Val Gln Glu Pro Arg Pro Phe Val Ile Trp Leu Glu Lys
100 105 110
Thr His His Ile Pro Val Asp Glu Phe Tyr Thr Leu Lys Arg Ala Glu
115 120 125
Arg Gly Asn Leu Val Lys Glu Gln Leu Ala Glu Glu Lys Asn Ile Pro
130 135 140
Gln Tyr Leu Arg Ser His Trp Gln Lys Glu Thr Gly Phe Ala Phe Pro
145 150 155 160
Ser Gly His Thr Met Phe Ala Ala Ser Trp Ala Leu Leu Ala Val Gly
165 170 175
Leu Leu Trp Pro Arg Arg Arg Thr Leu Thr Ile Ala Ile Leu Leu Val
180 185 190
Trp Ala Thr Gly Val Met Gly Ser Arg Leu Leu Leu Gly Met His Trp
195 200 205
Pro Arg Asp Leu Val Val Ala Thr Leu Ile Ser Trp Ala Leu Val Ala
210 215 220
Val Ala Thr Trp Leu Ala Gln Arg Ile Cys Gly Pro Leu Thr Pro Pro
225 230 235 240
Ala Glu Glu Asn Arg Glu Ile Ala Gln Arg Glu Gln Glu Ser
245 250
<210> 26
<211> 458
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of atfA-encoding polypeptide
<400> 26
Met Arg Pro Leu His Pro Ile Asp Phe Ile Phe Leu Ser Leu Glu Lys
1 5 10 15
Arg Gln Gln Pro Met His Val Gly Gly Leu Phe Leu Phe Gln Ile Pro
20 25 30
Asp Asn Ala Pro Asp Thr Phe Ile Gln Asp Leu Val Asn Asp Ile Arg
35 40 45
Ile Ser Lys Ser Ile Pro Val Pro Pro Phe Asn Asn Lys Leu Asn Gly
50 55 60
Leu Phe Trp Asp Glu Asp Glu Glu Phe Asp Leu Asp His His Phe Arg
65 70 75 80
His Ile Ala Leu Pro His Pro Gly Arg Ile Arg Glu Leu Leu Ile Tyr
85 90 95
Ile Ser Gln Glu His Ser Thr Leu Leu Asp Arg Ala Lys Pro Leu Trp
100 105 110
Thr Cys Asn Ile Ile Glu Gly Ile Glu Gly Asn Arg Phe Ala Met Tyr
115 120 125
Phe Lys Ile His His Ala Met Val Asp Gly Val Ala Gly Met Arg Leu
130 135 140
Ile Glu Lys Ser Leu Ser His Asp Val Thr Glu Lys Ser Ile Val Pro
145 150 155 160
Pro Trp Cys Val Glu Gly Lys Arg Ala Lys Arg Leu Arg Glu Pro Lys
165 170 175
Thr Gly Lys Ile Lys Lys Ile Met Ser Gly Ile Lys Ser Gln Leu Gln
180 185 190
Ala Thr Pro Thr Val Ile Gln Glu Leu Ser Gln Thr Val Phe Lys Asp
195 200 205
Ile Gly Arg Asn Pro Asp His Val Ser Ser Phe Gln Ala Pro Cys Ser
210 215 220
Ile Leu Asn Gln Arg Val Ser Ser Ser Arg Arg Phe Ala Ala Gln Ser
225 230 235 240
Phe Asp Leu Asp Arg Phe Arg Asn Ile Ala Lys Ser Leu Asn Val Thr
245 250 255
Ile Asn Asp Val Val Leu Ala Val Cys Ser Gly Ala Leu Arg Ala Tyr
260 265 270
Leu Met Ser His Asn Ser Leu Pro Ser Lys Pro Leu Ile Ala Met Val
275 280 285
Pro Ala Ser Ile Arg Asn Asp Asp Ser Asp Val Ser Asn Arg Ile Thr
290 295 300
Met Ile Leu Ala Asn Leu Ala Thr His Lys Asp Asp Pro Leu Gln Arg
305 310 315 320
Leu Glu Ile Ile Arg Arg Ser Val Gln Asn Ser Lys Gln Arg Phe Lys
325 330 335
Arg Met Thr Ser Asp Gln Ile Leu Asn Tyr Ser Ala Val Val Tyr Gly
340 345 350
Pro Ala Gly Leu Asn Ile Ile Ser Gly Met Met Pro Lys Arg Gln Ala
355 360 365
Phe Asn Leu Val Ile Ser Asn Val Pro Gly Pro Arg Glu Pro Leu Tyr
370 375 380
Trp Asn Gly Ala Lys Leu Asp Ala Leu Tyr Pro Ala Ser Ile Val Leu
385 390 395 400
Asp Gly Gln Ala Leu Asn Ile Thr Met Thr Ser Tyr Leu Asp Lys Leu
405 410 415
Glu Val Gly Leu Ile Ala Cys Arg Asn Ala Leu Pro Arg Met Gln Asn
420 425 430
Leu Leu Thr His Leu Glu Glu Glu Ile Gln Leu Phe Glu Gly Val Ile
435 440 445
Ala Lys Gln Glu Asp Ile Lys Thr Ala Asn
450 455
<210> 27
<211> 406
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of polypeptide encoded by fabB
<400> 27
Met Lys Arg Ala Val Ile Thr Gly Leu Gly Ile Val Ser Ser Ile Gly
1 5 10 15
Asn Asn Gln Gln Glu Val Leu Ala Ser Leu Arg Glu Gly Arg Ser Gly
20 25 30
Ile Thr Phe Ser Gln Glu Leu Lys Asp Ser Gly Met Arg Ser His Val
35 40 45
Trp Gly Asn Val Lys Leu Asp Thr Thr Gly Leu Ile Asp Arg Lys Val
50 55 60
Val Arg Phe Met Ser Asp Ala Ser Ile Tyr Ala Phe Leu Ser Met Glu
65 70 75 80
Gln Ala Ile Ala Asp Ala Gly Leu Ser Pro Glu Ala Tyr Gln Asn Asn
85 90 95
Pro Arg Val Gly Leu Ile Ala Gly Ser Gly Gly Gly Ser Pro Arg Phe
100 105 110
Gln Val Phe Gly Ala Asp Ala Met Arg Gly Pro Arg Gly Leu Lys Ala
115 120 125
Val Gly Pro Tyr Val Val Thr Lys Ala Met Ala Ser Gly Val Ser Ala
130 135 140
Cys Leu Ala Thr Pro Phe Lys Ile His Gly Val Asn Tyr Ser Ile Ser
145 150 155 160
Ser Ala Cys Ala Thr Ser Ala His Cys Ile Gly Asn Ala Val Glu Gln
165 170 175
Ile Gln Leu Gly Lys Gln Asp Ile Val Phe Ala Gly Gly Gly Glu Glu
180 185 190
Leu Cys Trp Glu Met Ala Cys Glu Phe Asp Ala Met Gly Ala Leu Ser
195 200 205
Thr Lys Tyr Asn Asp Thr Pro Glu Lys Ala Ser Arg Thr Tyr Asp Ala
210 215 220
His Arg Asp Gly Phe Val Ile Ala Gly Gly Gly Gly Met Val Val Val
225 230 235 240
Glu Glu Leu Glu His Ala Leu Ala Arg Gly Ala His Ile Tyr Ala Glu
245 250 255
Ile Val Gly Tyr Gly Ala Thr Ser Asp Gly Ala Asp Met Val Ala Pro
260 265 270
Ser Gly Glu Gly Ala Val Arg Cys Met Lys Met Ala Met His Gly Val
275 280 285
Asp Thr Pro Ile Asp Tyr Leu Asn Ser His Gly Thr Ser Thr Pro Val
290 295 300
Gly Asp Val Lys Glu Leu Ala Ala Ile Arg Glu Val Phe Gly Asp Lys
305 310 315 320
Ser Pro Ala Ile Ser Ala Thr Lys Ala Met Thr Gly His Ser Leu Gly
325 330 335
Ala Ala Gly Val Gln Glu Ala Ile Tyr Ser Leu Leu Met Leu Glu His
340 345 350
Gly Phe Ile Ala Pro Ser Ile Asn Ile Glu Glu Leu Asp Glu Gln Ala
355 360 365
Ala Gly Leu Asn Ile Val Thr Glu Thr Thr Asp Arg Glu Leu Thr Thr
370 375 380
Val Met Ser Asn Ser Phe Gly Phe Gly Gly Thr Asn Ala Thr Leu Val
385 390 395 400
Met Arg Lys Leu Lys Asp
405
<210> 28
<211> 1123
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence for regulating expression promoter pHXT1
<400> 28
tgcaggtctc atctggaata taattccccc ctcctgaagc aaatttttcc tttgagccgg 60
aatttttgat attccgagtt ctttttttcc attcgcggag gttattccat tcctaaacga 120
gtggccacaa tgaaacttca attcatatcg accgactatt tttctccgaa ccaaaaaaat 180
agcagggcga gattggagct gcggaaaaaa gaggaaaaaa ttttttcgta gttttcttgt 240
gcaaattagg gtgtaaggtt tctagggctt attggttcaa gcagaagaga caacaattgt 300
aggtcctaaa ttcaaggcgg atgtaaggag tattggtttc gaaagttttt ccgaagcggc 360
atggcaggga ctacttgcgc atgcgctcgg attatcttca tttttgcttg caaaaacgta 420
gaatcatggt aaattacatg aagaattctc tttttttttt tttttttttt ttttttacct 480
ctaaagagtg ttgaccaact gaaaaaaccc ttcttcaaga gagttaaact aagactaacc 540
atcataactt ccaaggaatt aatcgatatc ttgcactcct gatttttctt caaagagaca 600
gcgcaaagga ttatgacact gttgcattga gtcaaaagtt tttccgaagt gacccagtgc 660
tctttttttt tttccgtgaa ggactgacaa atatgcgcac aagatccaat acgtaatgga 720
aattcggaaa aactaggaag aaatgctgca gggcattgcc gtgccgatct tttgtctttc 780
agatatatga gaaaaagaat attcatcaag tgctgataga agaataccac tcatatgacg 840
tgggcagaag acagcaaacg taaacatgag ctgctgcgac atttgatggc ttttatccga 900
caagccagga aactccacca ttatctaatg tagcaaaata tttcttaaca cccgaagttg 960
cgtgtccccc tcacgttttt aatcatttga attagtatat tgaaattata tataaaggca 1020
acaatgtccc cataatcaat tccatctggg gtctcatgtt ctttccccac cttaaaatct 1080
ataaagatat cataatcgtc aactagttga tatacgtaaa atc 1123

Claims (8)

1. A microorganism, characterized in that it is saccharomyces cerevisiae having the potential to synthesize lycopene, said microorganism comprising:
overexpressionPAH1、DGA1AndACC1**；
overexpressionPAH1、DGA1AndACC1**silencingTGL3；
Over-expressionPAH1、DGA1AndACC1**silencingFLD1；
Over-expressionPAH1、DGA1、ACC1**AndOLE1；
over-expressionPAH1、DGA1、ACC1**AndOLE1is silentFLD1；
Over-expressionPAH1、DGA1、ACC1**AndOLE1is silentTGL3(ii) a Or
Over-expressionPAH1、DGA1、ACC1**AndOLE1is silentFLD1AndTGL3；
wherein, theACC1* Encoding the polypeptide as set forth in SEQ ID NO: 1;
the describedOLE1Encoding the polypeptide as shown in SEQ ID NO: 7;
the above-mentionedPAH1Encoding the polypeptide as shown in SEQ ID NO: 8;
the above-mentionedDGA1Encoding the polypeptide as shown in SEQ ID NO: 9;
the above-mentionedFLD1Encoding the polypeptide as shown in SEQ ID NO: 10;
the above-mentionedTGL3Encoding the polypeptide as shown in SEQ ID NO: 11.
2. A method for improving the fermentation yield of microbial lycopene, which is characterized by comprising the following steps: increasing the lipid content in a microorganism, wherein the microorganism is saccharomyces cerevisiae having the potential to synthesize lycopene;
the increase of lipid content in a microorganism is achieved by over-expressing and/or silencing in the microorganism at least one of the following genes:
over-expressionPAH1、DGA1AndACC1**；
over-expressionPAH1、DGA1AndACC1**is silentTGL3；
Over-expressionPAH1、DGA1AndACC1**is silentFLD1；
Over-expressionPAH1、DGA1、ACC1**AndOLE1；
over-expressionPAH1、DGA1、ACC1**AndOLE1is silentFLD1；
Over-expressionPAH1、DGA1、ACC1**AndOLE1is silentTGL3(ii) a Or
Over-expressionPAH1、DGA1、ACC1**AndOLE1silencingFLD1AndTGL3；
wherein, theACC1**Encoding the polypeptide as shown in SEQ ID NO:1 of the amino acid sequence shown in the formula (I),
the above-mentionedOLE1Encoding the polypeptide as shown in SEQ ID NO:7, or a polypeptide of an amino acid sequence shown in figure 7,
the describedPAH1Encoding the polypeptide as shown in SEQ ID NO:8, or a polypeptide of an amino acid sequence shown in figure 8,
the describedDGA1Encoding the polypeptide as shown in SEQ ID NO:9, or a polypeptide having the amino acid sequence shown in figure 9,
the above-mentionedFLD1Encoding the polypeptide as shown in SEQ ID NO:10, or a polypeptide having the amino acid sequence shown in figure 10,
the above-mentionedTGL3Encoding the polypeptide as shown in SEQ ID NO: 11.
3. The method of claim 2, wherein the lipid is a triglyceride.
4. The method of claim 2, wherein the increasing the lipid content in the microorganism is achieved by increasing at least one of the amount of triglyceride synthesis, lipid droplet size, and unsaturated fatty acid content.
5. The method of claim 2, wherein the overexpression is achieved by introducing into the microorganism a construct comprising a gene of interest to be overexpressed and a regulated expression promoter operably linked to the gene of interest.
6. The method according to claim 5, wherein the expression-regulating promoter is pHXT1.
7. The method according to claim 6, wherein pHXT1 has a nucleotide sequence as shown in SEQ ID NO 28.
8. Use of a microorganism according to claim 1 for increasing the fermentation yield of lycopene.
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