CN115838644A - Microorganisms and method for improving fermentation yield of microbial hydrophobic compounds - Google Patents

Abstract

The invention provides a microorganism. The microorganisms include: overexpression comprises at least one of the following genes: ACCA2, ACCB, ACCE, DGAT, LPP β, OLE1A, OLE1B, OLE1C, OLE1D, ecaacca, ecACCB, ecACCC, ecACCD, pgpB, atfA, fabA, fabB; 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 increasing the fermentation yield of hydrophobic compounds from microorganisms

This application is a divisional application of a patent application with an application date of October 13, 2017, application number 201710955112.6, and invention name “Microorganisms and methods for increasing the fermentation yield of hydrophobic compounds in microorganisms”.

Technical Field

The invention relates to the field of biotechnology, and in particular to a method for increasing the yield of hydrophobic products through lipid synthesis.

Background Art

Microbial fermentation to produce compounds is already a very mature technology. For example, Saccharomyces cerevisiae is a mature food safety strain that can be fermented on a large scale. It has a clear genetic background and a mature genetic operation system. It is the research and transformation object of many food-grade products and a model strain in industrial production. The mature fermentation platform and surrounding industries have also created convenience for the subsequent promotion of downstream products.

However, how to further improve the products of microbial fermentation remains a key issue that researchers are waiting to solve.

Summary of the invention

This application is made by the inventor based on the discovery of the following problems and facts:

Most hydrophobic compounds cannot accumulate in large quantities due to their low solubility after being synthesized in microorganisms. For example, lycopene is a typical hydrophobic compound. Due to its poor water solubility, the product is located in the cell membrane after synthesis, which not only limits the accumulation of the product, but also produces toxicity to the cells. The accumulation of the product is limited to a certain level, and cell growth is also limited at the same time. This has caused a huge obstacle to the amplification of engineering strains. The inventor unexpectedly found in the experiment that if the lipid content in the microorganism is increased, the output of hydrophobic compounds in the microorganism will be significantly improved. After further research by the inventor, it was found that the increase in the lipid content in the microorganism will provide a carrying environment for the accumulation of hydrophobic products such as lycopene, astaxanthin, natamycin, and spinosad in the body, and will minimize the toxicity of hydrophobic products in the cell. While increasing the accumulation of hydrophobic products, the effect of hydrophobic product accumulation on cell growth can be effectively reduced.

Based on this, in the first aspect of the present invention, the present invention proposes a microorganism. According to an embodiment of the present invention, the microorganism includes: overexpression includes at least one of the following genes: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; and silencing includes at least one of FLD1, TGL3, wherein the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. According to an embodiment of the present invention, the yield of hydrophobic compounds of the microorganism can be increased by more than 10%, such as the yield of lycopene is increased by more than 40%, the yield of natamycin is increased by 14%, the yield of spinosad is increased by 20%, the yield of astaxanthin is increased by 50%, and the product has low toxicity to cell growth.

According to an embodiment of the present invention, the above-mentioned microorganism may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the PAH1, DGA1, OLE1, ACC1** are derived from Saccharomyces cerevisiae, preferably, the ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D are derived from Streptomyces, preferably, the EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB are derived from Escherichia coli.

According to an embodiment of the present invention, the amino acid sequence of the polypeptide encoded by the above gene is shown in SEQ ID NOs: 1, 6-27.

MSEESLFESSPQKMEYEITNYSERHTELPGHFIGLNTVDKLEESPLRDFVKSHGGHTVISKILIANNGIAAVKEIRSVRKWAYETFGDDRTVQFVAMATPEDLEANAEYIRMADQYIEVPGGTNNNNYANVDLIVDIAERADVDAVWAGWGHASENPLLPEKLSQSKRKVIFIGPPGNAMRSLGDKISSTIVAQSAKVPCIPWSGTGVDTVHVDEK TGLVSVDDDIYQKGCCTSPEDGLQKAKRIGFPVMIKASEGGGGKGIRQVEREEDFIALYHQAA NEIPGSPIFIMKLAGRARHLEVQLLADQYGTNISLFGRDCSVQRRHQKIIEEAPVTIAKAETFHEMEKAAVRLGKLVGYVSAGTVEYLYSHDDGKFYFLELNPRLQVEHPTTEMVSGVNLPAAQLQIAMGIPMHRISDIRTLYGMNPHSASEIDFEFKTQDATKKQRRPIPKGHCTACRITSEDPNDGFKPSGGTLHELNFRSSSNVWGY FSVGNNGNIHSFSDSQFGHIFAFGENRQASRKHMVVALKELSIRGDFRTTVEYLIKLLETEDFEDNTITT GWLDDLITHKMTAEKPDPTLAVICGAATKAFLASEEARHKYIESLQKGQVLSKDLLQTMFPVDFIHEGKRYKFTVAKSGNDRYTLFINGSKCDIILRQLADGGLLIAIGGKSHTIYWKEEVAATRLSVDSMTTLLEVENDPTQLRTPSPGKLVKFLVENGEHIIKGQPYAEIEVMKMQMPLVSQENGIVQLLKQPGSTIVAGDIMAIMTL DDPSKVKHALPFEGMLPDFGSPVIEGTKPAYKFKSLVSTLENILKGYDNQVIMNASLQQLIEVLRNPKL PYSEWKLHISALHSRLPAKLDEQMEELVARSLRRGAVFPARQLSKLIDMAVKNPEYNPDKLLGAVVEPLADIAHKYSNGLEAHEHSIFVHFLEEYYEVEKLFNGPNVREENIILKLRDENPKDLDKVALTVLSHSKVSAKNNLILAILKHYQPLCKLSSKVSAIFSTPLQHIVELESKATAKVALQAREILIQGALPSVKERTEQIEHILKS SVVKVAYGSSNPKRSEPDLNILKDLIDSNYVVFDVLLQFLTHQDPVVTAAAAQVYIRRAYRAYTIGDI RVHEGVTVPIVEWKFQLPSAAFSTFPTVKSKMGMNRAVAVSDLSYVANSQSSPLREGILMAVDHLDDVDEILSQSLEVIPRHQSSSNGPAPDRSGSSASLSNVANVCVASTEGFESEEEILVRLREILDLNKQELINASIRRITFMFGFKDGSYPKYYTFNGPNYNENETIRHIEPALAFQLELGRLSNFNIKPIFTDNRNIHVYEAVSKTSPLDKRFFTRGIIRTGHI RDDISIQEYLTSEANRLMSDILDNLEVTDTSNSDLNHIFINFIAVFDISP EDVEAAFGGFLERFGKRLLRLRVSSAEIRIIIKDPQTGAPVPLRALINNVSGYVIKTEMYTEVKNAKGEWVFKSLGKPGSMHLRPIATPYPVKEWLQPKRYKAHLMGTTYVYDFPELFRQASSSQWKNFSADVKLTDDFFISNELIEDENGELTEVEREPGANAIGMVAFKITVKTPEYPRGRQFVVVANDITFKIGSFGPQEDEFFNKVTEY ARKRGIPRIYLAANSGARIGMAEEIVPLFQVAWNDAANPDKGFQYLYLTSEGMETLKKFDKENSVLT ERTVINGEERFVIKTIIGSEDGLGVECLRGSGLIAGATSRAYHDIFTITLVTCRSVGIGAYLVRLGQRAIQVEGQPIILTGAPAINKMLGREVYTSNLQLGGTQIMYNNGVSHLTAVDDLAGVEKIVEWMSYVPAKRNMPVPILETKDTWDRPVDFTPTNDETYDVRWMIEGRETESGFEYGLFDKGSFFETLSGWAKGVVVGRARLGGIPLGVIG VETRTVENLIPADPANPNSAETLIQEPGQVWHPNSAFKTAQAINDFNNGEQLPMMILANWRGFS GGQRDMFNEVLKYGSFIVDALVDYKQPIIIYIPPTGELRGGSWVVVDPTINADQMEMYADVNARAGVLEPQGMVGIKFRREKLLDTMNRLDDKYRELRSQLSNKSLAPEVHQQISKQLADRERELLPIYGQISLQFADLHDRSRMVAKGVISKELEWTEARRFFFWRLRRRLNEEYLIKRLSHQVGEASRLEKIARIRSWYPASV DHEDDRQVATWEENYKTLDDKLKGLKLESFAQDLAKKIRSDHDNAIDGLSEVIKMLSTDDKEKLLKTLK (SEQ ID NO: 1).

MVDKRESYTKEDLLASGRGELFGAKGPQLPAPNMLMMDRVVKMTETGGNFDKGYVEAELDINPDLWFFGCHFIGDPVMPGGLDAMWQLVGFYLGWLGGEGKGRALGVGEVKFTGQVLPTAKKVTYRIHFKRIVNRRLIMGLADGEVLVDGRLIYTASDLKVGLFQDTSAF (SEQ ID NO: 6).

MPTSGTTIELIDDQFPKDDSASSGIVDEVDLTEANILATGLNKKAPRIVNGFGSLMGSKEMVSVEFDKKGNEKKSNLDRLLEKDNQEKEEAKTKIHISEQPWTLNNWHQHLNWLNMVLVCGMPMIGWYFALSGKVPLHLNVFLFSVFYYAVGGVSITAGYHRLWSHRSYSAHWPLRLFYAIFGCASVEGSAKWWGHSHRIHHRYTDTLRDPY DARRGLWYSHMGWMLLKPNPKYKARADITDMTDDWTIRFQHRHYI LLMLLTAFVIPTLICGYFFNDYMGGLIYAGFIRVFVIQQATFCINSLAHYIGTQPFDDRRTPRDNWITAIVTFGEGYHNFHHEFPTDYRNAIKWYQYDPTKVIIYLTSLVGLAYDLKKFSQNAIEEALIQQEQKKINKKKAKINWGPVLTDLPMWDKQTFLAKSKENKGLVIISGIVHDVSGYISEHPGGETLIKTALGKDAT KAFSGGVYRHSNAAQNVLADMRVAVIKESKNSAIRMASKRGEIYETGKFF (SEQ ID NO: 7).

MQYVGRALGSVSKTWSSINPATLSGAIDVIVVEHPDGRLSCSPFHVRFGKFQILKPSQKKVQVFINEKLSNMPMKLSDSGEAYFVFEMGDQVTDVPDELLVSPVMSATSSPPQSPETSILEGGTEGEGEGENENKKKEKKVLEEPDFLDINDTGDSGSKNSETTGSLSPTESSTTTPLDSVEERKLVEQRTKNFQQKLNKKLTEIHIPSKLDNNG D LLLDTEGYKPNKNMMHDTDIQLKQLLKDEFGNDSDISSFIKEDKNGNIKIVNPYEHLTDLSPPGTPPTMATSGSVLGLDAMESGSTLNSSSPSGSDTEDETSFSKEQSSKSEKTSKKGTAGSGETEKRYIRTIRLTNDQLKCLNLTYGENDLKFSVDHGKAIVTSKLFVWRWDVPIVISDIDGTITKSDALGHVLAMIGKDWTHLGVAKLFSEIS RNGYNILYLTARSAGQADSTRSYLRSIEQNGSKLPNGPVILSPDRTMAALRREVILKKPEVFKIACLNDIRSLYFEDSDNEMDTEEKSTPFFAGFGNRITDALSYRTVGIPSSRIFTINTEGEVHMELLELAGYRSSYIHINELVDHFFPPVSLDSVDLRTNTSMVPGSPPNRTLDNFDSEITSGRKTLFRGNQEEKFTDVNFWRDPLVDIDNLSDISNDDSDNIDEDTDTD VSQQSNVSRNRANSVKTAKVTKAPQRNVSGSTNNNEVLAASSDVENASDLVGSHSSSGSTPNKSTMSKGDIGKQIYLELGSPLASPKLRYLDDMDDEDSNYNRTKSRRASSAAATSIDKEFKKLSVSKAGAPTRIVSKIDVSNDVHSLGNSDTESRREQSVNETGRNQLPHNSMDDKDLDSRVSDEFDDDEFDEDEFED (SEQ ID NO: 8).

MSGTFNDIRRRKEEGSPTAGITERHENKSLSSIDKREQTLKPQLESCCPLATPFERRLQTLAVAWHTSSFVLFSIFTLFAISTPALWVLAIPYMIYFFFDRSPATGEVVNRYSLRFRSLPIWKWYCDYFPISLIKTVNLKPTFTLSKNKRVNEKNYKIRLWPTKYSINLKSNSTIDYRNQECTGPTYLFGYHPHGIGALGAFGAFATEGC NEW YORK (SEQ ID NO: 9).

MKINVSRPLQFLQWSSYIVVAFLIQLLIILPLSILIYHDFYLRLLPADSSNVVPLNTFNILNGVQFGTKFFQSIKSIPVGTDLPQTIDNGLSQLIPMRDNMEYKLDLNLQLYCQSKTDHLNLDNLLIDVYRGPGPLLGAPGGSNSKDEKIFHTSRPIVCLALTDSMSPQEIEQLGPSRLDVYDEEWLNTIRIEDKISLESSYETIS VFLKTEIAQRNLIIHPESGIKFRMNFEQGLRNLMLRKRFLSYIIGISIFHCIICVLFFITGCTAFIFVRKGQEKSKKHS (SEQ ID NO: 10).

MKETAQEYKVSAVIPTLLKNWILRVVYATLDHIPPFVWEILHVITDIYFFWVQKLINYVRPHSRVIYYNAIKKLDECDTYQMWCQQASVVDEITGANLWRRNFFSRRYDFNSVIEQYSILENMLREEKYDVVKEKFSTTGPCMLRNFAGIGDKKLFTKSLMGTKLLIEQYLTRILEGLDILNNQTLTPTSFFQRCKLSLGTTALILQGGSL FGLFHLGVIRGLLLQDLMPNIISGSSMGACVASLFGCLSNEQLKQLLTDDNLLNIIKNDVDLLKSCGYGNLEQHLNLGTLIQNLIHHGYSQDVYLFIRFVMKYIVKEKTFEE VYQITGKVFNIVIHPTDKSCPNLLNYVTTPNVLIKSAIECSLGSGVISEDTSLLCKNLENEIEPFLNINKNKQVKFLTPENANNPSITESPYTRLTELFNVNNFIVSLARPYLAPLVVNDLKHEIKTSKYYYYKHYPNMPPINANTVRKTQRSSSQSPIKAGTVEDLEPEPLMSPVPPSSAVNDSAEYIIPELGIPQLNFTEMEPLAFKFKYHLER KLKNIATMEFRHRMEVLDNLGLLCSLIKRLIIDEKTPRSATEIAVVPRMKSLSLTRIIEGQLNNIPYWIKSGERSTWPALALIKTRCAVEFKLDDIIRARRSR (SEQ ID NO: 11).

MRKVLIANRGEIAVRVARACRDAGIASVAVYADPDRDALHVRAADEAFALGGDTPATSYLDIAKVLKAARESGADAIHPGYGFLSENAEFAQAVLDAGLIWIGPPPHAIRDLGDKVAARHIAQRAGAPLVAGTPDPVSGADEVVAFAKEHGLPIAIKAAFGGGGRGLKVARTLEEVPELYDSAVREAVAAFGRGECFVERYLDKPRHVETQCLADTHGNVVVV STRDCSLQRRHQKLVEEAPAPFLSEAQTEQLYSSSKAILKEAGYVGAGTVEFLVGMDGTISFLEVNTRLQVEHP VTEEVAGIDLVREMFRIADGEELGYDDPALRGHSFEFRINGEDPGRGFLPAPGTVTLFDAPTGPGVRLDAGVESGSVIGPAWDSLLAKLIVTGRTRAEALQRAARALDEFTVEGMATAIPFHRTVVRDPAFAPELTGSTDPFTVHTRWIETEFVNEIKPFTTPADTETDEESGRETVVVEVGGKRLEVSLPSSLGMSLARTGLAAGARPKRRAAKKSGPAASGDTLASPMQ GTIVKIAVEEGQEVQEGDLIVVLEAMKMEQPLNAHRSGTIKGLTAEVGASLTSGAAICEIKD (SEQ ID NO: 12).

MTVLDEAPGEPTDARGRVAELHGIRAAALAGPSEKATAAQHAKGKLTARERIELLLDPGSFREVEQLRRHRATGFGLEAKKPYTDGVITGWGTVEGRTVFVYAHDFRIFGGALGEAHATKIHKIMDMAIAAGAPLVSLNDGAGARIQEGVSALAGYGGIFQRNTKASGVIPQISVMLGPCAGGAAYSPALTDFVFMVRDTSQMFITGPDVVKAVTGEEITQNG LGGADVHAETSGVCHFAYDDEETCLAEVRYLLSLLPQNNREN PPRAESSDPVDRRSDTLLDLVPADGNRPYDMTKVIEELVDEGEYLEVHERWARNIICALARLDGRVVGIVANQPQALAGVLDIEASEKAARFVQMCDAFNIPIITLLDVPGFLPGVDQEHGGIIRHGAKLLYAYCNATVPRISLILRKAYGGAYIVMDSQSIGADLTYAWPTNEIAVMGAEGAANVIFRRQIADAEDPEAMRARMVKEYKSELMH PYYAAERGLVDDVIDPAETREVLITSLAMLHTKHADLPSRKHGNPPQ (SEQ ID NO: 13).

MSPADIRVEKGHAEPEEVAAITALLLARAAARPAEIAPTHGGGRARAGWRRLEREPGFRAPHSWR (SEQ ID NO: 14).

MTPDPLAPLDLAFWNIESAEHPMHLGALGVFEADSPTAGALAADLLAARAPAVPGLRMRIRDTWQPPMALRRPFAFGGATREPDPRFDPLDHVRLHAPATDFHARAGRLMERPLERGRPPWEAHVLPGADGGSFAVLFKFHHALADGLRALTLAAGVLDPMDLPAPRPRPEQPPRGLLPDVRALPDRLRGALSDAGRALDIGAAAALSTLDVRSSPALTAASSGT RRTAGVSVDLDDVHHVRKTTGGTVNDVLIAVVAGALRRWLDERGDGSEGVAPRALIPVSRRRPRSAHPQGNRLSGYLMRLPVGDPDPLARLGTVRAAMDRNKDAGPGRGAGAVALLADHVPALGHRLGGPLVSGAARLWFDLLVTSVPLPSLGLRLGGHPLTEVYPLAPLARGHSLAVAVSTYRGRVHYGLLADAKAVPDLDRLAVAVAEEVETLLTACRP(SEQ ID NO :15).

MRTERKPTRLDRVFARLDREPERPALLDVPEMSRHRIALFAGTLAFYIAIVWAVVITSWLVRLDWQVMFFRPYQQWPEIHAFVDYYVVLGQRGPTAVMVAAWLGWRSWRQHTLRPLLALGVSLLLLNVTVGAAKYGMGRLGPHYATTIGANEMWLGGDIFPSGHTANAVVTWGILAYLASTHRTRRWLSAISAVTSLGVGMSTVYLGTHWLSDV LLGWVAGLLILLALPWFEPLITRAEAWILGLRDRWYTRRDRRSTTRPPLGPPVPVSPPGSGSRPQAPAREPVAAPRTARAPAHLAPGPHTARSDRTPVTPAGSRRPPHSDRHARNTAPTARPLSGG (SEQ ID NO: 16).

MTTSSDVIPDAPQPAGDAAGPSATLGGEQKRSIEQITLLLFITLPFLALVAAVPLAWGWGVSWLDLGLLVFFYFLGCHGITIGFHRHFTHGSFKAKRPLKIALAIAGSMAVEGPLVRWVADHRKHHKFSDDEGDPHSPWRYGETVPALIKGLWWAHIAWMFDEEQTPQEKYAPDLIKDPALRAVSRQFILWTVVSLALPALIGGLVTMSWWGAFT GFFWGSLVRVALLHHVTWSINSICHAVGKRPFKSRDRSGNVWWLAILSCGESWHNLHHADPTSARHGVMRGQLDSSARLIRWFEQLGWAYDVRWPSRSRIDSRRNTDQDGARRRKETAKAA (SEQ ID NO: 17).

MTMATTATRSDTPGSDFARLSKKVADAGLLGRRPGYYTLRITAVTGLYAAGWAAFVLVGASWWTLAIAAFLAVMYGQVALVAHDMAHRQVFRRRRASELSGRIAGASIGMSYGWWQDKHTRHHANPNTEDLDPDIGPDLLVWSPDQARAATGLPRLLGRWQAFLFFPLLTLEGFNLHVASGRAMANRRLKRRALDGALLLAHCAVYLTALFWVLPPGMAIAFLAVHQCL FGVYLGSAFAPNHKGMPILTADDRPDFLRRQVLTSRNVNGGLFTDLALLGGLNHQIEHHLFPSMPSPNLRKARAIVRRYCRDLGVDYAETGLVASYRLALTSLHDAGTPLRRTRVRA (SEQ ID NO: 18).

MPLPRETLPPDTGGSREGSEFTPLLRDVREQQLLERRTGWYARTIAVNALGLAAVGTGMALLGDSWWVLALAPVLAVLCARTAFIGHDAGHAQISGSRAVNRRIGLVHGNLLLGMSYAWWNDKHNRHHANPNHIDKDPDVAADVLVFTSGQAATRTGFRGRLTRHQAWLFFPLTLLEGLALKLHGFQHLRRQRGRARLVEGALLVAHVAGYVTLLLATMPLAHA LVFAALHQALFGLHLGMAFAPNHKGMDMPDPDSEAEKWGHLRRQVLTSRNVRGGFLTDWFLGGLNYQIEHHLFPSMPRPHLGLAQAAVKAHCRDLGIPYAETGLVDSYRQALRHMHEVGEPLRADI (SEQ ID NO: 19).

MLVESLPTPAQEKDRERGSDFSELSRRIAGAGLLRRRPLYYTVRFGAVALALAGGVAAFVALGDSWSQLFVAVALAVVFGQLGLAAHDLAHRQVFTRRRPSEAGGLLTANLLLGMSYGWWMNKHTRHHANPNHEEKDPDVSPDILVWSRGQASRATGLPRFVGRHQAALFFPLLTLEGLNLSFNSFKALGSRAVKRPVLEGTLLVAHFAVYFGGLFTVLSPG KALVFLAVHQGLFGIYLGSVFAPNHKGMPMIEEGMRLDFLRRQVLTSRNVRGGALVDAFMGGLNYQIEHHLFPSMPTPALGRAQAITEAYCAELGVPYHQTGLLASHREALRHMRSVGEPLRAAR (SEQ ID NO: 20).

MSLNFLDFEQPIAELEAKIDSLTAVSRQDEKLDINIDEEVHRLREKSVELTRKIFADLGAWQIAQLARHPQRPYTLDYVRLAFDEFDELAGDRAYADDKAIVGGIARLDGRPVMIIGHQKGRETKEKIRRNFGMPAPEGYRKALRLMQMAERFKMPIITFIDTPGAYPGVGAEERGQSEAIARNLREMSRLGVPVVCTVIGEGGSGGALAIGVG DKVNMLQYSTYSVISPEGCASILWKSADKAPLAAEAMGIIAPRLKELKLIDSIIPEPLGGAHRNPEAMAASLKAQLLADLADLDVLSTEDLKNRRYQRLMSYGYA (SEQ ID NO: 21).

MDIRKIKKLIELVEESGISELEISEGEESVRISRAAPAASFPVMQQAYAAPMMQQPAQSNAAAPATVPSMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAFIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPLVVIE (SEQ ID NO: 22).

MLDKIVIANRGEIALRILRACKELGIKTVAVHSSADRDLKHVLLADETVCIGPAPSVKSYLNIPAIISAAEITGAVAIHPGYGFLSENANFAEQVERSGFIFIGPKAETIRLMGDKVSAIAAMKKAGVPCVPGSDGPLGDDMDKNRAIAKRIGYPVIIKASGGGGGRGMRVVRGDAELAQSISMTRAEAKAAFSNDMVYMEKYLENPRHVEIQVLADGQGNAIY LA ERDCSMQRRHQKVVEEAPAPGITPELRRYIGERCAKACVDIGYRGAGTFEFLFENGEFYFIEMNTRIQVEHPVTEMITGVDLIKEQLRIAAGQPLSIKQEEVHVRGHAVECRINAEDPNTFLPSPGKITRFHAPGGFGVRWESHIYAGYTVPPYYDSMIGKLICYGENRDVAIARMKNALQELIIDGIKTNVDLQIRIMNDENFQHGGTNI HYLEKKLGLQEK (SEQ ID NO: 23).

MSWIERIKSNITPTRKASIPEGVWTKCDSCGQVLYRAELERNLEVCPKCDHHMRMTARNRLHSLLDEGSLVELGSELEPKDVLKFRDSKKYKDRLASAQKETGEKDALVVMKGTLYGMPVVAAAFEFAFMMGGSMGSVVGARFVRAVEQALEDNCPLICFSASGGARMQEALMSLMQMAKTSAALAKMQERGLPYISVLTDPTMGGVSASFAMLGDLNIAEP KALIGFAGPRVIEQTVREKLPPGFQRSEFLIEKGAIDMIVRRPEMRLKLASILAKLMNLPAPNPEAPREGVVVPPVPDQEPEA (SEQ ID NO: 24).

MRSIARRTAVGAALLLVMPVAVWISGWRWQPGEQSWLLKAAFWVTETVTQPWGVITHLILFGWFLWCLRFRIKAAFVLFAILAAAILVGQGVKSWIKDKVQEPRPFVIWLEKTHHIPVDEFYTLKRAERGNLVKEQLAEEKNIPQYLRSHWQKETGFAFPSGHTMFAASWALLAVGLLWPRRRTLTIAILLVWATGVMGSRLLLGMHWP RDLVVATLISWALVAVATWLAQRICGPLTPPAEENREIAQREQES (SEQ ID NO: 25).

MRPLHPIDFIFLSLEKRQQPMHVGGLFLFQIPDNAPDTFIQDLVNDIRISKSIPVPPFNNKLNGLFWDEDEEFDLDHFRHIALPHPGRIRELLIYISQEHSTLLDRAKPLWTCNIIEGIEGNRFAMYFKIHHAMVDGVAGMRLIEKSLSHDVTEKSIVPPWCVEGKRAKRLREPKTGKIKKIMSGIKSQLQATPTVIQELSQTVFKDIGRNPDH VSSFQAPCSILNQRVS SSRRFAAQSFDLDRFRNIAKSLNVTINDVVLAVCSGALRAYLMSHNSLPSKPLIAMVPASIRNDDSDVSNRITMILANLATHKDDPLQRLEIIRRSVQNSKQRFKRMTSDQILNYSAVVYGPAGLNIISGMMPKRQAFNLVISNVPGPREPLYWNGAKLDALYPASIVLDGQALNITMTSYLDKLEVGLIACRNALPRMQNLLTHLEEEIQLFEGVIA KQEDIKTAN (SEQ ID NO: 26).

MKRAVITLGIVSSIGNNQQEVLASLREGRSGITFSQELKDSGMRSHVWGNVKLDTTGLIDRKVVRFMSDASIYAFLSMEQAIADAGLSPEAYQNNPRVGLIAGSGGGSPRFQVFGADAMRGPRGLKAVGPYVVTKAMASGVSACLATPFKIHGVNYSISSACATSAHCIGNAVEQIQLGKQDIVFAGGGEELCWEMACEFDAMGALSTKYNDTPEKA SRTYDAHRDGFVIAGGGGMVVEELEHALARGAHIYAEIVGYGATSDGADMVAPSGEGAVRCMKMAMHGVDTPIDYLNSHGTSTPVGDVKELAAIREVFGDKSPAISATKAMTGHSLGAAGVQEAIYSLLMLEHGFIAPSINIEELDEQAAGLNIVTETTDRELTTVMSNSFGFGGTNATLVMRKLKD (SEQ ID NO: 7).

The inventors have found that overexpression of the genes derived from the above-mentioned microorganisms in microorganisms having the potential to synthesize hydrophobic compounds can significantly promote the accumulation of hydrophobic compounds in the microorganisms.

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 and Trichoderma reesei. The inventors found that by overexpressing at least one of the above genes and silencing at least one selected from FLD1 and TGL3 in the above microorganism, the accumulation of hydrophobic compounds in the microorganism is further significantly improved.

According to an embodiment of the present invention, the hydrophobic compound comprises at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosad polyketide, diterpenes, triterpenes and tetraterpenes. The yield of the hydrophobic compound in the microorganism according to the embodiment of the present invention is higher.

In the second aspect of the present invention, the present invention proposes a method for increasing the fermentation yield of microbial hydrophobic compounds. According to an embodiment of the present invention, the compound includes: increasing the lipid content in the microorganism, wherein the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. The inventor unexpectedly found in the experiment that increasing the lipid content in the microorganism with the potential to synthesize hydrophobic compounds can significantly increase the fermentation yield of microbial hydrophobic compounds, and the fermentation yield of microbial hydrophobic compounds can be increased by more than 10%, such as the yield of lycopene increased by more than 40%, the yield of natamycin increased by 14%, the yield of spinosad increased by 20%, and the yield of astaxanthin increased by 50%, and the product has low toxicity to cell growth.

According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the lipid is triglyceride. The inventors have found that increasing the content of triglyceride in microorganisms has a more significant effect on promoting the fermentation yield of hydrophobic compounds.

According to an embodiment of the present invention, the increase in lipid content in the microorganism is achieved by increasing at least one of the synthesis of triglycerides, the size of lipid droplets, and the content of unsaturated fatty acids, thereby further increasing the fermentation yield of hydrophobic compounds.

According to an embodiment of the present invention, the lipid content in the microorganism is increased by overexpressing at least one of the following genes in the microorganism: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; and silencing at least one of FLD1 and TGL3. By the above method, the lipid content in the microorganism can be effectively increased, thereby providing a carrying environment for the accumulation of hydrophobic products in the body, and reducing the toxicity of hydrophobic products in the cells as much as possible. While further increasing the accumulation of hydrophobic products, the effect of the accumulation of hydrophobic products on cell growth can be further effectively reduced.

According to an embodiment of the present invention, the overexpression is achieved by introducing a construct into the microorganism, the construct comprising a target gene to be overexpressed and a regulated expression promoter, the regulated expression promoter being operably connected to the target gene. Thus, the target gene can be overexpressed in a controllable manner, the target gene is overexpressed during the cell growth period, and expression stops during the product accumulation period.

According to an embodiment of the present invention, the regulated expression promoter is pHXT1. pHXT1 is a glucose-controlled promoter, which can achieve more convenient and efficient controllable expression of the target gene.

According to an embodiment of the present invention, the pHXT1 has the nucleotide sequence shown in SEQ ID NO:28.

TGCAGGTCTCATCTGGAATATAATTCCCCCCTCCTGAAGCAAATTTTTCCTTTGAGCCGGAATTTTTGATATTCCGAGTTCTTTTTTTCCATTCGCGGAGGTTATTCCATTCCTAAACGAGTGGCCACAATGAAACTTCAATTCATATCGACCGACTATTTTTCTCCGAACCAAAAAAATAGCAGGGCGAGATTGGAGCTGCGGAAAAAAGAGGAAAAAAATTTTTTCGTAGTTTTCTTGTGCAAATTAGGGTGTAAGGTTTCTAGG GCTTATTGGTTCAAG CAGAAGAGACAACAATTGTAGGTCCTAAATTCAAGGCGGATGTAAGGAGTATTGGTTTCGAAAGTTTTTCCGAAGCGGCATGGCAGGGACTACTTGCGCATGCGCTCGGATTATCTTCATTTTTGCTTGCAAAAACGTAGAATCATGGTAAATTACATGAAGAATTCTCTTTTTTTTTTTTTTTTTTTTTTTACCTCTAAAGAGTGTTGACCAACTGAAAAAACCCTTCTTCAAGAGAGTTAAACTAAGACTAACCATC ATAACTTCCAAGGAATTAAT CGATATCTTGCACTCCTGATTTTTCTTCAAAGAGACAGCGCAAAGGATTATGACACTGTTGCATTGAGTCAAAAGTTTTTCCGAAGTGACCCAGTGCTCTTTTTTTTTCCGTGAAGGACTGACAAATATGCGCACAAGATCCAATACGTAATGGAAATTCGGAAAAACTAGGAAGAAATGCTGCAGGGCATTGCCGTGCCGATCTTTTGTCTTTCAGATATATGAGAAAAAGAATATTCATCAAGTGCTGATAGAAGAATAACCACT CATATGACGTGGGGC AGAAGACAGCAAACGTAAACATGAGCTGCTGCGACATTTGATGGCTTTTATCCGACAAGCCAGGAAACTCCACCATTATCTAATGTAGCAAAATATTTCTTAACACCCGAAGTTGCGTGTCCCCCTCACGTTTTTAATCATTTGAATTAGTATATTGAAATTATATATAAAGGCAACAATGTCCCCATAATCAATTCCATCTGGGGTCTCATGTTCTTTCCCCACCTTAAAATCTATAAAGATATCATAATCGTCAACTAGTTGATA TACGTAAAATC (SEQ ID NO: 28).

According to an embodiment of the present invention, the microorganism comprises at least one selected from yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride and Trichoderma reesei, etc. By increasing the lipid content in the above microorganisms, the accumulation of hydrophobic compounds in the microorganisms is further significantly improved.

According to an embodiment of the present invention, the hydrophobic compound comprises at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosad, polyketide, diterpenoid, triterpenoid and tetraterpenoid compounds. Using the method according to an embodiment of the present invention, the yield of the hydrophobic compound is higher.

In the third aspect of the present invention, the present invention proposes the use of the aforementioned microorganism in increasing the yield of hydrophobic compound fermentation. As mentioned above, the microorganism according to the embodiment of the present invention has the performance of high yield of hydrophobic compounds. The microorganism according to the embodiment of the present invention can be effectively used in the fermentation production of hydrophobic compounds, and the yield of hydrophobic compounds is high and the toxicity to the microorganism is low.

It should be noted that the amino acid sequence of the polypeptide encoded by the gene disclosed in the present application means that any nucleotide sequence encoding a polypeptide having this amino acid sequence is within the protection scope of the present application.

It should be 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 with 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 all within the scope of protection of the present application.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG. 1 is a result diagram showing that the yield of lycopene-producing bacteria overexpressing triglyceride according to an embodiment of the present invention is improved to varying degrees compared with the original lycopene-producing bacteria TM606.

DETAILED DESCRIPTION

The embodiments of the present invention are described in detail below, with Saccharomyces cerevisiae with high lycopene production, Escherichia coli with high astaxanthin production, Streptomyces with high natamycin production, and Saccharopolyspora with high spinosad production as representatives. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limitations of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. If the specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in the art or in accordance with the product instructions shall be followed. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

In the following examples, the amino acid sequences of the polypeptides encoded by the PAH1, DGA1, OLE1, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, and fabB genes are shown in the sequence listing.

Example 1 Construction of plasmids required for lycopene production

The primers used in the examples are shown in Table 1.

Table 1: Primers used to construct lycopene engineering strains

The plasmids constructed in the examples and the fragments used for plasmid construction were amplified by PCR using the corresponding primers and templates, as shown in Table 2 for details.

Table 2: Plasmids constructed in this example

PCR reaction system: 30.5μL H ₂ O, 10μL 5×reaction buffer, 4μL 2.5mM dNTPs, 2μL 10mM forward primer, 2μL 10mM reverse primer, 1μL template DNA (1-100ng), 0.5μL Phusion High-Fidelity DNA Polymerase. PCR reaction program: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 56℃ annealing for 30s, 72℃ extension (30s/K), 30 cycles; finally extension at 72℃ for 10min.

The plasmids constructed in the examples were all constructed using the yeast assembly method: different plasmids were assembled according to the fragments listed in Table 2.

The PCR fragments were cut from the gel and recovered, and the volume used was calculated based on 300 ng of each fragment.

According to the needs of each plasmid, take the corresponding fragment, mix well, calculate the volume, add 10% volume of 3M NaAc, 2% volume of 10mg/mL glycogen, mix well, add 2 times volume of anhydrous ethanol, place at -80℃ for 2h, centrifuge at 13,000rpm, 4℃ for 20min, discard the supernatant. Add 500μL 70% ethanol to wash once, centrifuge at 13,200rpm at room temperature for 3min, discard the supernatant, dry, add 4μL ddH ₂ O to re-dissolve, and set aside.

The processed mixed fragments were transformed into Saccharomyces cerevisiae by the lithium acetate method, coated with the corresponding YPD resistance plate, and cultured at 30°C. (The colony growth takes about 3 days). When the colony grows to an appropriate size, a single clone is picked and transferred to the liquid medium with the corresponding YPD resistance, cultured at 30°C, 220rpm for 20h, plasmids are extracted, and transformed into E.coli DH10B, coated with LB solid plate (Ampicillin resistance), cultured at 37°C for about 2 days, and a single clone is picked and transferred to LB-Amp liquid medium, cultured at 37°C, 220rpm for 16h to extract plasmids, and enzyme digestion verification is performed.

Example 2 Construction of Saccharomyces cerevisiae Lycopene Strain

The lycopene-producing yeast strain TM606 was constructed as follows:

The expression of tHMG1 improves the conversion rate of the MVA pathway; the silencing of GAL1, 7, 10 utilizes galactose to induce the production of lycopene; the construction method is to digest the plasmid pZY141 with NotI to recover the target fragment, integrate it into the Saccharomyces cerevisiae (CEN.PK2-1D) strain by lithium acetate yeast transformation at a dosage of 200 ng, screen the target strain using a plate containing the corresponding nutritional deficiency, and verify it by PCR.

PaCrtB (SEQ ID NO: 3) from Panococcus agglomerans, TmCrtE (SEQ ID NO: 2) from Taxus chinensis, and BtCrtI (SEQ ID NO: 4) from Trispora pyrenoidosa were expressed to heterologously synthesize lycopene, and the copy number was adjusted.

MAYTAMAAGTQSLQLRTVASYQECNSMRSCFKLTPFKSFHGVNFNVPSLGAANCEIMGHLKLGSLPYKQCSVSSKSTKTMAQLVDLAETEKAEGKDIEFDFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPALCIAACELVGGSQDLAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKPTNHKVFGEDTAVLAGDALLSFAFE HIAVATSKTVPSDRTLRVISELGKTIGSQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILGGATEDEIARIRRYARCVGLLFQVVDDILDVTKSSEELGKTAGKDLLTDKATYPKLMGLEKAKEFAAELATRAKEELSSFDQIKAAPLLGLADYIAFRQN (SEQ ID NO: 2).

MSQPPLLDHATQTMANGSKSFATAAKLFDPATRRSVLMLYTWCRHCDDVIDDQTHGFASEAAAEEEATQRLARLRTLTLAAFEGAEMQDPAFAAFQEVALTHGITPRMALDHLDGFAMDVAQTRYVTFEDTLRYCYHVAGVVGLMMARVMGVRDERVLDRACDLGLAFQLTNIARDIIDDAAIDRCYLPAEWLQDAGLTPENYAARENRAALARVAERLIDAAEPYY ISSQAGLHDLPPRCAWAIATARSVYREIGIKVKAAGGSAWDRRQHTSKGEKIAMLMAAPGQVIRAKTTRVTPRPAGLWQRPV (SEQ ID NO: 3).

MSDQKKHIVVIGAGIGGTATAARLAREGFRVTVVEKNDFSGGRCSFIHHDGHRFDQGPSLYLMPKLFEDAFADLDERIGDHLDLLRCDNNYKVHFDDGDAVQLSSDLTKMKGELDRIEGPLGFGRFLDFMKETHVHYEQGTFIAIKRNFETIWDLIRLQYVPEIFRLHLFGKIYDRASKYFQTKKMRMAFTFQTMYMGMSPYDAPAVY SLLQYTEFAEGIWYPRGGFNMVVQKLESIASKKYGAEFRYQSPVAKINTVDKDKRVTGVTLESGEVIEADAVVCNADLVYAYHHL LPPCNWTKKTLASKKLTSSSISFYWSMSTKVPQLDVHNIFLAEAYKESFDEIFNDFGLPSEASFYVNVPSRIDESAAPPNKDSIIVLVPIGHMKSKTGNSAEENYPELVNRARKMVLEVIERRLGVNNFANLIEHEEVNDPSVWQSKFNLWRGSILGLSHDVFQVLWFRPSTKDSTNRYDNLFFVGASTHPGTGVPIVLAGSKLTSDQVCKSFGQ NPLPRKLQDSQKKYAPEQTRKTESHWIYYCLACYFVTFLFFYFFPRDDTTTPASFINQLLPNVFQGQNSNDIRI (SEQ ID NO: 4).

The construction method is to digest plasmids pZY151, pZY184, and pZY196 with NotI, recover the target fragment, continuously integrate it into the above strains through lithium acetate yeast transformation, screen with plates containing corresponding nutritional deficiencies to obtain the target strains, and verify by PCR.

The POS5 gene was expressed to balance the reducing power in yeast. The construction method was to digest the plasmid pTM206 with NotI, recover the target fragment, transform and integrate it into the above strain, screen it with a plate containing the corresponding resistance to obtain the target strain, and verify it with PCR.

The expression of ADH2, ACS6 (SEQ ID NO: 5), and ALD6 genes increases the supply of precursor substances for the synthesis of lycopene.

MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTFWGEQGKILDWITPYQKVKNTSFAPGNVSIKWYEDGTLNLAANCLDRHLQENGDRTAIIWEGDDASQSKHISYRELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAVAMLACARIGAVHSVIFGGFSPEAIAGRIIDSSSRLVITADEGVRAGRSIPLKKNVDDALKNPNVTS VEHVIVLKRTGNDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPLFILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYHPGDIYWCTADVGWVTGHSYLLYGPLACGATTL MFEGVPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEGDKAIEEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKEKCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFFGVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLFGDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITGRVDDVLNVSGHRLGTAEIESALVAHPKIA EAAVVGIPHAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIGPLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNLGDTSTLADPGVVEKPLEEKQAIAMPS (SEQ ID NO: 5).

The construction method is to digest the plasmid pTM303 with NotI to recover the target fragment, transform and integrate it into the above-mentioned strain, screen it with a plate containing corresponding resistance to obtain the target strain, and verify it with PCR.

Silencing Ypl062W and Exg1 genes regulates the yeast lycopene synthesis system as a whole. Construction method: In the Ypl062W gene to be inactivated, the corresponding knockout cassette fragment is constructed with the hygromycin resistance gene as a marker, and the Exg1 gene is constructed with the G418 resistance gene as a marker. The fragments are respectively integrated into the above-mentioned engineering bacteria through lithium acetate yeast transformation, screened with a plate containing hygromycin resistance, and verified by PCR.

When the strain needs to reuse the resistance selection marker, use the following method to discard the marker:

Transform the strain to be marked with pSH47 plasmid, and after colonies grow, pick multiple colonies and culture them in 5 mL YPD medium at 30°C, 220 rpm overnight, centrifuge at 3,000 rpm for 5 min at room temperature, discard the supernatant, wash twice with 5 mL YPDG medium without glucose and containing 1% galactose, add 5 mL of the medium and culture at 30°C, 220 rpm overnight, and spread on SC non-nutrition-deficient solid medium containing 5-fluoro-orotic acid with a final concentration of 1 g/L and culture at 30°C. After colonies grow, pick a single clone and spread on the corresponding YPD solid plate of the marker to be marked for verification.

Example 3 Construction of a high-yield lycopene-producing strain

The transformation strategy used in this embodiment is as follows:

(1) To improve triglyceride synthesis, the downstream synthesis flux of triglyceride was increased by overexpressing PAH1 (SEQ ID NO: 8) and DGA1 (SEQ ID NO: 9). The strain TM6065 was constructed. The construction method was as follows:

The plasmid pTM705 was digested with NotI to recover the target fragment, and 200 ng of the fragment was integrated into the TM606 strain by the lithium acetate yeast transformation method. The strain with resistance was screened using a plate containing the corresponding resistance and verified by PCR.

(2) To improve the precursor of triglyceride synthesis, the upstream synthesis flux of triglyceride was increased by overexpressing the ACC1** (SEQ ID NO: 1) gene with two mutation sites, and the strain TM6066 was constructed. The construction method is as follows:

The plasmid pTM706 was digested with NotI to recover the target fragment, which was transformed and integrated into the TM606 strain as above. The strain with resistance was screened with a plate containing the corresponding resistance and verified by PCR.

(3) On the basis of improving triglyceride synthesis and reducing triglyceride degradation, strain TM6068 was constructed by knocking out the TGL3 (SEQ ID NO: 11) gene based on strain TM6065. The construction method is as follows:

The plasmid pTM708 was digested with NotI to recover the target fragment, which was then transformed and integrated into the TM606 strain. The strain with resistance was screened using a plate containing the corresponding resistance and verified by PCR.

(4) In order to increase the size of intracellular lipid droplets, lipid droplet aggregation was promoted by knocking out the FLD1 gene (SEQ ID NO: 10). The strain TM701 was constructed. The construction method was as follows:

The plasmid pTM701 was digested with NotI to recover the target fragment, which was then transformed and integrated into the TM606 strain. The strain with resistance was screened using a plate containing the corresponding resistance and verified by PCR.

(5) To increase intracellular unsaturated fatty acids, strain TM704 was constructed by overexpressing the OLE1 (SEQ ID NO: 7) gene. The construction method is as follows:

The plasmid pTM704 was digested with NotI to recover the target fragment, which was then transformed and integrated into the TM606 strain. The strain with resistance was screened using a plate containing the corresponding resistance and verified by PCR.

(6) A method for combining upstream and downstream synthesis of triglycerides was used to construct strain TM60656 or TM60686. The plasmids pTM705/pTM708 and pTM706 were respectively digested with NotI to recover the target fragments and then integrated into the TM606 strain. The strains with resistance were screened using plates containing corresponding resistance and verified by PCR.

(7) Combining the upstream and downstream synthesis of intracellular triglycerides and the size of intracellular lipid droplets, the strain TM70156/TM70186 was constructed. The construction method is as follows:

Plasmids pTM705/pTM708 and pTM706 were digested with NotI to recover the target fragments, which were transformed and integrated into TM701. The resistant strains were screened using plates containing corresponding resistance and verified by PCR.

(8) In order to increase the size of intracellular unsaturated fatty acids bound to lipid droplets, the strain TM707 was constructed, and the construction method was as follows:

The plasmid pTM707 was digested with NotI to recover the target fragment, which was then transformed and integrated into the TM606 strain. The strain with resistance was screened using a plate containing the corresponding resistance and verified by PCR.

(9) In combination with improving the upstream and downstream synthesis of intracellular triglycerides and the content of intracellular unsaturated fatty acids, the strain TM70456/TM70486 was constructed, and the construction method was as follows:

Plasmids pTM705/pTM708 and pTM706 were digested with NotI to recover the target fragments, which were transformed and integrated into the TM704 strain. The strains with resistance were screened using plates containing corresponding resistance and verified by PCR.

(10) Combined with the method of increasing the upstream and downstream synthesis of intracellular triglycerides, the content of intracellular unsaturated fatty acids, and the size of lipid droplets, the strain TM70756/TM70786 was constructed. The construction method is as follows:

Plasmids pTM705/pTM708 and pTM706 were digested with NotI to recover the target fragments, which were transformed and integrated into the TM707 strain. The strains with resistance were screened using plates containing corresponding resistance and verified by PCR.

Example 4 Lycopene engineering bacteria shake flask culture fermentation process

In this example, the inventors introduce in detail the fermentation culture process of some of the engineered strains obtained in Example 3.

The shake flask fermentation adopts two-stage seed culture. The recombinant strain on the plate is picked into a PA bottle containing 5mL YPD medium. After overnight culture at 30°C (generally 14-18h), the bacteria grow to the logarithmic growth phase ( _OD600 is about 5-8), and the first-stage seed liquid is obtained. The strain is transferred to a 250mL shake flask containing 50mL YPD medium with a 1% inoculation amount. After about 14-18h of shake flask culture, the second-stage seed liquid is obtained. The final concentration of the bacteria is calculated as _OD600 = 0.5 and inoculated into a 500mL shake flask containing 200mL fermentation medium YPD (containing 1% galactose), and the shake flask fermentation is carried out at 30°C 220rpm. After 96h, the sample is stored in a -80°C refrigerator to measure the accumulation of lycopene production.

Example 5 Product extraction and detection

In this example, the inventors extracted the product obtained after the fermentation treatment and detected the yield of lycopene.

Take out the sample from the refrigerator and thaw, take 500μL of fermentation liquid in a 15mL centrifuge tube (precooled on ice), centrifuge at 5,000rpm, 4℃ for 2min to collect the bacteria, and remove the supernatant. Add 4mL acetone (HPLC grade), 0.2g glass beads, 1% antioxidant, shake for 5min, ultrasonicate in an ice bath for 5-10min, centrifuge at 5,000rpm, 4℃ for 2min, transfer the supernatant to a 50mL centrifuge tube; repeat the above extraction process and collect the extract until the bacteria have no obvious yellow color; mix the collected extract, take 2mL, centrifuge at 12,000rpm for 10min, take the supernatant to a brown injection bottle, and perform HPLC analysis.

Detection method: Lycopene detection was carried out using quaternary HPLC, the detector was a UV detector, the absorption wavelength was 474nm, the chromatographic column was Agilent Zorbax C18 (150mm*4.6mm*5μm), mobile phase A (acetonitrile: water = 9:1) and mobile phase B (methanol: isopropanol = 3:2) were analyzed under the following conditions: 0-90% B (0-15min), 90% B (15-30min), 90% -0B (30-35min), flow rate 1mL/min.

The test results are shown in FIG1 , and it can be seen that the lycopene production of the triglyceride-overexpressing bacteria is improved to varying degrees compared with the original lycopene production bacteria TM606.

Example 6 Construction of Natamycin Producing Bacteria Overexpressing Triglyceride and Fermentation

Natamycin is an odorless, tasteless, low-dose and highly safe food preservative made by fermentation of Streptomyces natamycin. It is a white to milky white odorless and tasteless crystalline powder. Its mechanism of action is to combine with ergosterol and other sterol groups of fungi to inhibit ergosterol biosynthesis, thereby deforming the cell membrane, eventually leading to leakage and cell death. In baked foods, using natamycin to treat the dough surface has a significant effect of extending the shelf life. Natamycin is slightly soluble in water and difficult to dissolve in most organic solvents. The solubility in water at room temperature is 30-100 mg/L. When the pH is lower than 3 or higher than 9, its solubility will increase, but the stability of natamycin will be reduced.

In this example, the inventors constructed plasmids as shown in Table 2 in Streptomyces nata. The specific method is to obtain the corresponding fragments by PCR from the corresponding genes derived from Streptomyces in Example 1, and then connect them to the corresponding promoters and the ermE* promoter in the vector pSET152 in sequence using the Gibson method (Daniel G. Gibson, Enzymatic Assembly of Overlapping DNA Fragments, Methods in Enzymology, Volume 498, 2011, Pages 349-361.) to construct the required plasmids. The constructed plasmids were transformed into Escherichia coli ET12567/pUZ8002 by electroporation, and the transformed Escherichia coli was cultured in 2 ml LB at 37 degrees overnight, and 200 microliters of bacterial solution was transferred to 5 ml LB and cultured at 37 degrees. At the same time, the natalidomyces spores were subjected to heat shock and pre-germination treatment. The natalidomyces spores were suspended in 2 ml of TES buffer, in a 50-degree water bath for 10 minutes, and after cooling to room temperature, an equal volume of spore pre-germination medium was added, and the spores were cultured in a 37-degree shaker together with the previously prepared Escherichia coli for 2.5 hours. The Escherichia coli was washed twice with LB medium. At the same time, Streptomyces spores were collected by centrifugation, and the Escherichia coli and Streptomyces spores were mixed evenly. The mixture was mixed with an equal amount of Escherichia coli cells at a ratio of 10 ⁸ :10 ¹⁰ , and spread on a plate containing SFM medium, blown dry and placed in a 30-degree incubator for culture. After 18 hours, the plate was covered with 1 ml of sterile water containing a final concentration of 8 mg/L apramycin resistance on the plate, and cultured in a 30-degree incubator. White conjugative transfer products can be seen after 3 days of culture. For the initial verification of the mutant strain, first primers V-apr-F (5'-GCTCATCGGTCAGCTTCTCA 3') and V-apr-R (5'-TCGCATTCTTCGCATCCC 3') were used to verify the presence of the Abra resistance gene. If the mutant strain contains the Abra resistance gene, a PCR product of 726 bp should be obtained.

The original Streptomyces natatomica J1002 and the newly constructed natamycin-producing strain overexpressing triglycerides were cultured on SFM plates (20 g/L soybean cake powder, 20 g/L mannitol, 16 g/L agar, 1 L tap water, pH 7.5) at 30 degrees for 7 days, and then 4 small pieces of spore-containing agar blocks with a total size ^of about 3.2 cm2 were picked and inoculated into 30 mL COM medium (10 g/L corn starch, 10 g/L oatmeal, 5 g/L malt extract, 2 g/L yeast extract, 15 g/L agar, pH 7.2) in a 250 mL shake flask. After culturing for 48 h at 30 degrees Celsius and 220 rpm, 3 mL of seed culture was transferred to 25 mL NPM medium (50.0 g/L corn starch, 18.0 g/L soybean powder, 10.0 g/L yeast extract, 1.5 g/L CaCO3, pH _7.2 ). 7.2) Incubate in a shaker at 30°C, 220 rpm for 120 h.

Take 0.5 mL of fermentation broth and centrifuge at 7,000 rpm for 5 minutes, remove the supernatant, add 1.5 mL of methanol glacial acetic acid solution (methanol: glacial acetic acid = 95:5, v/v) to mix and precipitate. After ultrasonication for 20 minutes, centrifuge at 7,000 rpm for 5 minutes, remove 50 μl of supernatant and add 950 μl of methanol glacial acetic acid solution (methanol: glacial acetic acid = 95:5, v/v) for HPLC detection. HPLC detection conditions are: analytical column Agilent ZORBAX SB-C18 (4.6×250 nm), flow rate 0.5 mL min ^-1 , UV 303 nm detection. The mobile phase is methanol: water: glacial acetic acid = 60:40:5 (V/V/V).

The results are shown in Table 3. The natamycin production of the newly constructed strains was improved to varying degrees compared with the original production strain J1002.

Table 3: Natamycin related strain information

Example 7 Construction of a spinosad-producing bacterium overexpressing triglycerides and fermentation

Spinosad, also known as spinosad, is a macrolide non-toxic and highly effective biopesticide extracted from the fermentation broth of Saccharopolyspora spinosa. Spinosad is a light grey solid crystal with a slightly stale earthy smell. Spinosad has a very low solubility in water and is easily soluble in organic solvents, such as methanol, ethanol, cyanonitrile, acetone, dimethyl sulfoxide, dimethylformamide, etc.

In this example, the inventors expressed relevant genes in the spinosyn-producing bacteria, and transferred the plasmid constructed in Example 6 into the spinosyn-producing bacteria by conjugation in a similar manner. The newly constructed strains are shown in Table 3.

The original strain of Saccharopolyspora spinosa was purchased from the China Microbial Strain Maintenance Center (CGMCC), and the strain number is CGMCC4.1365. The strain was rejuvenated using rejuvenation medium (yeast extract 1g/L, beef extract 1g/L, Casein Acids Hydrolysate 2g/L, glucose 10g/L, agar 15g/L, pH7.3). After 3 days of culture in the rejuvenation liquid medium, 1mL of the culture was transferred to a 250mL spring shake flask containing 30mL of fermentation seed medium. Seed medium: TSB 30g/L, yeast extract 3g/L, beef extract 3g/L, MgSO ₄ ·7H ₂ O 2g/L, glucose 10g/L, corn steep liquor 2.5g/L, pH 7.0. The fermentation seeds were cultured in a shaker at 30°C and a speed of 220rpm for 50h and then inoculated into the fermentation medium. The fermentation medium is: glucose 40g/L, beef extract 10g/L, MgSO ₄ ·7H ₂ O 2g/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 uses a 250mL spring shake flask with a liquid volume of 50mL and a seed inoculation amount of 10%. The fermentation conditions are 30°C and the rotation speed is 220rpm.

Spinosad (CAS No: 168316-95-8) standard was purchased from Sigma (product number: 33706). The standard contains two components, spinosyn A and spinosyn D, with an HPLC purity of 97%. The mobile phase used for HPLC analysis was 10% phase A (0.2% ammonium acetate aqueous solution) and 90% phase B (methanol); the chromatographic column was a DOINEX C18 column (3μm, 4.6, 150mm); the flow rate was 0.8mL/min; a PDA detector was used, and the detection wavelength was 245nm. The MS detector was LCQ FLEET (Thermo scientific). Summary of HPLC standard curve: Weigh 5mg of spinosyn standard, add 1mL of HPLC grade methanol, dissolve and mix to obtain a 5g/L mother solution, make a series of dilutions of the standard, and detect it by HPLC. Take 0.2 mL of the fermentation culture, add 0.8 mL of acetonitrile gradually, vortex and mix for 2 minutes, and place in a refrigerator at 4°C overnight. Centrifuge at 12000 rpm for 10 minutes, take the supernatant, filter it with a 0.45 μm nylon membrane, and put it in a brown HPLC sample bottle for analysis.

The fermentation results are shown in Table 4. The spinosad production of J1016-1 was higher than that of CGMCC4.1365 to varying degrees.

Table 4: Information on spinosad-related strains

Example 8 Construction of astaxanthin-producing bacteria overexpressing triglycerides

Natural astaxanthin (also known as ketocarotenoid or astaxanthin) is the strongest natural antioxidant found in nature. Astaxanthin has super antioxidant activity, more than 100 times stronger than vitamin E, and is called "super vitamin E". It can effectively remove oxygen free radicals in cells and is the only carotenoid that can pass through the blood-brain barrier. It has the effects of enhancing immunity, relieving fatigue, enhancing cell regeneration, preventing cancer, treating cardiovascular diseases, reducing the accumulation of aging cells, inhibiting obesity, protecting eyes and central nervous system, and resisting ultraviolet rays. In recent years, astaxanthin has begun to be widely used in medicines, health products, cosmetics, food additives, aquaculture, etc. With the development of my country's national economy, the demand for it is also increasing.

In this embodiment, the inventors constructed plasmids as shown in Table 5 based on plasmids pMH1, pFZ81 (Fayin Zhu, In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli, Biotechnol. Bioeng. 2014; 111: 1396-1405.), and plasmid pFZ153 (Tian Ma, Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC55669 for heterologous overproduction in Escherichia coli, Biotechnol. J. 2015; 11 (2): 228-237.), and transformed E. coli MG1655 competent cells according to the plasmid combination shown in Table 6 to construct an Escherichia coli astaxanthin production strain.

The specific method of constructing the plasmid in this example is as described in Example 1. The corresponding gene is amplified by PCR and sequentially connected to the corresponding promoter using the Gibson method (Daniel G. Gibson, Enzymatic Assembly of Overlapping DNA Fragments, Methods in Enzymology, Volume 498, 2011, Pages 349-361.) to construct the required plasmids.

Table 5: E. coli astaxanthin synthesis plasmid construction

Table 6: Construction of Escherichia coli astaxanthin synthesis strain

Strains describe 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 construction is completed, shake flask fermentation is carried out as follows:

After transformation, the transformants were selected and cultured overnight in LB medium containing 34 μg/mL chloramphenicol, 50 μg/mL kanamycin, and 100 μg/mL ampicillin at 37°C and 220 rpm. 200 mL of LB medium containing 34 μg/mL chloramphenicol, 50 μg/mL kanamycin, and 100 μg/mL ampicillin was transferred with a 1% inoculum and cultured at 30°C and 200 rpm. When OD600 reached 0.7-0.9, 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) was added for induction. After 15 h of culture, 2 mL of the sample was taken, centrifuged at 12,000 rpm for 3 min, the supernatant was removed, 1 mL of the extractant (V acetone: V methanol = 4:1) was added, the cells were shaken and dispersed, ultrasonicated for 10 min, centrifuged at 13,000 rpm and 4°C for 10 min, and the supernatant was taken for high performance liquid chromatography (HPLC) detection according to the following method. The operation process was protected from light.

Quaternary HPLC (Thermo Fisher Ultimate 3000), chromatographic column Agilent Zorbax C18 (4.6-mm×150-mm×5-μm). Chromatographic conditions are as follows: mobile phase A (acetonitrile: water = 9:1, V/V) and mobile phase B (methanol: isopropanol = 3:2, V/V). 0 min: 0% B, 15 min: 90% B, 30 min: 90% B, 35 min: 0% B. Flow rate 1 mL/min. Column temperature: 25°C. Detector: UV detector, detection wavelength 474 nm.

The results are shown in Table 6, and the astaxanthin production of the modified engineered bacteria is higher.

Example 9 Controlling triglyceride overexpression in microorganisms

On the basis of Example 3, we replaced the promoters of the PAH1, DGA, ACC1, and OLE1 genes with pHXT1 (SEQID NO: 28), which is characterized by the overexpression of related genes in the presence of glucose, and the non-expression of related genes in the absence of glucose. Therefore, through our fermentation control, the lycopene production strain can accumulate triglycerides in the early stage of fermentation, and no longer accumulate triglycerides after glucose is consumed in the later stage, thereby controlling the lipid content in the microorganism, so that the microorganism can use more substrates and energy to synthesize lycopene in the later stage of fermentation, thereby further increasing the yield of lycopene. The specific construction method refers to Example 1-2, and the promoter is replaced. The strain details are shown in Table 7, and the fermentation and detection methods of Examples 4-5 are used for detection. The results show that the yield of lycopene is further increased by controlling the expression of triglycerides.

Table 7: Lycopene engineering strain transformation information and yield

Newly constructed strains Original strain Gene replaced with pHXT1 promoter Knockout 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 of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (11)

1. A microorganism, characterized in that it comprises: Overexpression includes at least one selected from the following genes: ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACC A, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; Wherein, the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. 2. The microorganism according to claim 1, characterized in that ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, and OLE1D are derived from Streptomyces, Optionally, the EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, and fabB are derived from Escherichia coli, Optionally, the fabA encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, Optionally, the ACCA2 encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 12, Optionally, the ACCB encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 13, Optionally, the ACCE encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 14, Optionally, the DGAT encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 15, Optionally, the LPPb encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 16, Optionally, the OLE1A encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 17, Optionally, the OLE1B encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 18, Optionally, the OLE1C encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 19, Optionally, the OLE1D encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 20, Optionally, the EcACCA encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 21, Optionally, the EcACCB encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 22, Optionally, the EcACCC encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 23, Optionally, the EcACCD encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 24, Optionally, the pgpB encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 25, Optionally, the atfA encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 26, Optionally, the fabB encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:27. 3. The microorganism according to claim 1, characterized in that the microorganism comprises at least one selected from yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride and Trichoderma reesei. 4. The microorganism according to claim 1, characterized in that the hydrophobic compound comprises at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosad, polyketide, diterpenoid, triterpenoid and tetraterpenoid compounds. 5. A method for increasing the fermentation yield of hydrophobic compounds in microorganisms, characterized in that it includes: increasing the lipid content in the microorganism, wherein the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. The method according to claim 5 , wherein the lipid is triglyceride. 7. The method according to claim 5, characterized in that the increasing of lipid content in the microorganism is achieved by increasing at least one of the synthesis amount of triglycerides, the size of lipid droplets and the content of unsaturated fatty acids. 8. The method according to claim 5, characterized in that the increase in lipid content in the microorganism is achieved by overexpressing at least one of the following genes in the microorganism: ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB, Wherein, the fabA encodes a polypeptide having an amino acid sequence shown in SEQ ID NO: 6, The ACCA2 encodes a polypeptide having an amino acid sequence shown in SEQ ID NO: 12, The ACCB encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 13, The ACCE encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 14, The DGAT encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 15, The LPPb encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 16, The OLE1A encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 17, The OLE1C encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 19, The OLE1D encodes a polypeptide having an amino acid sequence shown in SEQ ID NO: 20, The EcACCA encodes a polypeptide having an amino acid sequence shown in SEQ ID NO: 21, The EcACCB encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 22, The EcACCC encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 23, The EcACCD encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 24, The pgpB encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 25, The atfA encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 26, The fabB encodes a polypeptide having an amino acid sequence shown in SEQ ID NO: 27. 9. The method according to claim 8, characterized in that the overexpression is achieved by introducing a construct into the microorganism, wherein the construct comprises a target gene to be overexpressed and a regulated expression promoter, wherein the regulated expression promoter is operably connected to the target gene, Preferably, the inducible promoter is pHXT1, Optionally, the PHXT1 has the nucleotide sequence shown in SEQ ID NO:28. 10. The method according to claim 5, characterized in that the microorganism comprises at least one selected from yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride and Trichoderma reesei, etc. Optionally, the hydrophobic compound comprises at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosad, polyketide, diterpenoid, triterpenoid, tetraterpenoid compound. 11. Use of the microorganism according to claims 1 to 4 in increasing the fermentation yield of hydrophobic compounds.

Images