Microorganisms and methods to increase the fermentation yield of microbial hydrophobic compounds

Abstract

This invention proposes a microorganism. The microorganism comprises: overexpressing at least one of the following genes: ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; wherein the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. According to embodiments of the invention, the yield of hydrophobic compounds from the microorganism can be increased by more than 10%, such as increasing the yield of lycopene by more than 40%, natamycin by 14%, spinosad by 20%, and astaxanthin by 50%, and the products have low toxicity to cell growth.

Description

[0001] This application is a divisional application of the patent application filed on October 13, 2017, with application number 201710955112.6 and entitled "Microorganisms and a method for increasing the fermentation yield of hydrophobic compounds of microorganisms". Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to a method for increasing the yield of hydrophobic products through lipid synthesis. Background Technology

[0003] Microbial fermentation for compound production is a well-established technology. For example, *Saccharomyces cerevisiae*, a mature, large-scale fermentable food-grade yeast, possesses a clear genetic background and a mature genetic manipulation system. It is a research and modification target for many food-grade products and a model strain in industrial production. Mature fermentation platforms and related industries also facilitate the promotion of downstream products.

[0004] However, how to further improve the quality of microbial fermentation products remains a key issue that researchers are eager to address. Summary of the Invention

[0005] This application was made by the inventor based on the discovery of the following problems and facts:

[0006] Most hydrophobic compounds, after synthesis in microorganisms, suffer from low solubility, preventing large-scale accumulation. For example, lycopene, a typical hydrophobic compound, is poorly water-soluble and, after synthesis, is localized to the cell membrane. This not only limits product accumulation but also exerts toxicity on cells, restricting accumulation and consequently limiting cell growth. This poses a significant obstacle to the scale-up production of engineered strains. The inventors unexpectedly discovered in their experiments that increasing the lipid content within microorganisms significantly increases the yield of hydrophobic compounds. Further research revealed that increased lipid content provides a supportive environment for the accumulation of hydrophobic products such as lycopene, astaxanthin, natamycin, and spinosad, while minimizing their intracellular toxicity. This effectively reduces the impact of hydrophobic product accumulation on cell growth while simultaneously increasing accumulation.

[0007] Based on this, in a first aspect, the present invention provides a microorganism. According to embodiments of the present invention, the microorganism comprises: overexpression of at least one of the genes selected from: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; and silencing of at least one of the genes selected from FLD1 and TGL3, wherein the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. According to embodiments of the present invention, the yield of hydrophobic compounds from the microorganism can be increased by more than 10%, such as an increase of more than 40% in lycopene yield, a 14% increase in natamycin yield, a 20% increase in spinosad yield, and a 50% increase in astaxanthin yield, and the products have low toxicity to cell growth.

[0008] According to embodiments of the present invention, the above-mentioned microorganisms may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, PAH1, DGA1, OLE1, and ACC1** are derived from Saccharomyces cerevisiae; preferably, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, and OLE1D are derived from Streptomyces; and preferably, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, and fabB are derived from Escherichia coli.

[0010] According to an embodiment of the present invention, the amino acid sequence of the polypeptide encoded by the above-mentioned gene is shown in SEQ ID NO: 1, 6 to 27.

[0011]

[0012] MVDKRESYTKEDLLASGRGELFGAKGPQLPAPNMLMMDRVVKMTETGGNFDKGYVEAELDINPDLWFFGCHFIGDPVMPGCLGLDAMWQLVGFYLGWLGGEGKGRALGVGEVKFTGQVLPTAKKVTYRIHFKRIVNRRLIMGLADGEVLVDGRLIYTASDLKVGLFQDTSAF(SEQ ID NO:6)。

[0013] MPTSGTTIELIDDQFPKDDSASSGIVDEVDLTEANILATGLNKKAPRIVNGFGSLMGSKEMVSVEFDKKGNEKKSNLDRLLEKDNQEKEEAKTKIHISEQPWTLNNWHQHLNWLNMVLVCGMPMIGWYFALSGKVPLHLNVFLFSVFYYAVGGVSITAGYHRLWSHRSYSAHWPLRLFYAIFGCASVEGSAKWWGHSHRIHHRYTDTLRDPYDARRGLWYSHMGWMLLKPNPKYKARADITDMTDDWTIRFQHRHYILLMLLTAFVIPTLICGYFFNDYMGGLIYAGFIRVFVIQQATFCINSLAHYIGTQPFDDRRTPRDNWITAIVTFGEGYHNFHHEFPTDYRNAIKWYQYDPTKVIIYLTSLVGLAYDLKKFSQNAIEEALIQQEQKKINKKKAKINWGPVLTDLPMWDKQTFLAKSKENKGLVIISGIVHDVSGYISEHPGGETLIKTALGKDATKAFSGGVYRHSNAAQNVLADMRVAVIKESKNSAIRMASKRGEIYETGKFF(SEQ ID NO:7)。

[0014] (SEQ ID NO: 8).

[0015] MSGTFNDIRRRKKEEGSPTAGITERHENKSLSSIDKREQTLKPQLESCCPLATPFFERRLQTLAVAWHTSSFVLFSIFTLFAISTPALWVLAIPYMIYFFFDRSPATGEVVNRYSLRFRSLPIWKWYCDYFPISLIKTVNLKPTFTLSKNKRVNEKNYKIRLWPTKYSINLKSNSTIDYRNQECTGPTYLFGYHPHGIGALGAFGAFATEGCNYSKIFPGIPISLMTLVTQFHIPLYRDYLLALGISSVSRKNALRTLSKNQSICIVVGGARESLSSTNGTQLILNKRKGFIKLAIQTGNINLPVFAFGEVDCYNVLSTKKDSVLGKMQLWFKENFGFTIPIFYARGLFNYDFGLLPFRAPINVVGRPIYVEKKITNPPDDVVNHFHDLYIAELKRLYYENREKYGVPDAELKIVG (SEQ ID NO: 9).

[0016] MKINVSRPLQFLQWSSYIVVAFLIQLLIILPLSILIYHDFYLRLLPADSSNVVPLNTFNILNGVQFGTKFFQSIKSIPVGTDLPQTIDNGLSQLIPMRDNMEYKLDLNLQLYCQSKTDHNLDNLLIDVYRGPGPLLAGAPGGSN SKDEKIFHTSRPIVCLALTDSMSPQEIEQLGPSRLDVYDEEWLNTIRIEDKISLESSYETISVFLKTEIAQRNLIIHPESGIKFRMNFEQGLRNMLRKRFLYIIGISIFHCIICVLFFITGCTAFIFVRKGQEKSKKHS(SEQ ID NO:10)。

[0017] (SEQ ID NO: 11).

[0018] (SEQ ID NO: 12).

[0019] MTVLDEAPGEPTDARGRVAELHGIRAAALAGPSEKATAAQHAKGKLTARERIELLLDPGSFREVEQLRRHRATGFGLEAKKPYTDGVITGWGTVEGRTVFVYAHDFRIFGGALGEAHATKIHKIMDMAIAAGAPLVSLNDGAGARIQEGVSALAGYGGIFQRNTKASGVIPQISVMLGPCAGGAAYSPALTDFVFMVRDTSQMFITGPDVVKAVTGEEITQNGLGGADVHAETSGVCHFAYDDEETCLAEVRYLLSLLPQNNRENPPRAESSDPVDRRSDTLLDLVPADGNRPYDMTKVIEELVDEGEYLEVHERWARNIICALARLDGRVVGIVANQPQALAGVLDIEASEKAARFVQMCDAFNIPIITLLDVPGFLPGVDQEHGGIIRHGAKLLYAYCNATVPRISLILRKAYGGAYIVMDSQSIGADLTYAWPTNEIAVMGAEGAANVIFRRQIADAEDPEAMRARMVKEYKSELMHPYYAAERGLVDDVIDPAETREVLITSLAMLHTKHADLPSRKHGNPPQ(SEQ ID NO:13)。

[0020] MSPADIRVEKGHAEPEEVAAITALLLARAAARPAEIAPTHGGGRARAGWRRLEREPGFRAPHSWR(SEQID NO:14)。

[0021] MTPDPLAPLDLAFWNIESAEHPMHLGALGVFEADSPTAGALAADLLAARAPAVPGLRMRIRDTWQPPMALRRPFAFGGATREPDPRFDPLDHVRLHAPATDFHARAGRLMERPLERGRPPWEAHVLPGADGGSFAVLFKFHHALADGLRALTLAAGVLDPMDLPAPRPRPEQPPRGLLPDVRALPDRLRGALSDAGRALDIGAAAALSTLDVRSSPALTAASSGTRRTAGVSVDLDDVHHVRKTTGGTVNDVLIAVVAGALRRWLDERGDGSEGVAPRALIPVSRRRPRSAHPQGNRLSGYLMRLPVGDPDPLARLGTVRAAMDRNKDAGPGRGAGAVALLADHVPALGHRLGGPLVSGAARLWFDLLVTSVPLPSLGLRLGGHPLTEVYPLAPLARGHSLAVAVSTYRGRVHYGLLADAKAVPDLDRLAVAVAEEVETLLTACRP(SEQ ID NO:15)。

[0022] MRTERKPTRLDRVFARLDREPERPALLDVPEMSRHRIALFAGTLAFYIAIVWAVVITSWLVRLDWQVMFFRPYQQWPEIHAFVDYYVVLGQRGPTAVMVAAWLGWRSWRQHTLRPLLALGVSLLLLNVTVGAAKYGMGRLGPHYATTIGANEMWLGGDIFPSGHTANAVVTWGILAYLASTHRTRRWLSAISAVTSLGVGMSTVYLGTHWLSDVLLGWVAGLLILLALPWFEPLITRAEAWILGLRDRWYTRRDRRSTTRPPLGPPVPVSPPGSGSRPQAPAREPVAAPRTARAPAHLAPGPHTARSDRTPVTPAGSRRPPHSDRHARNTAPTARPLSGG(SEQ ID NO:16)。

[0023] MTTSSDVIPDAPQPAGDAAGPSATLGGEQKRSIEQITLLLFITLPFLALVAAVPLAWGWGVSWLDLGLLVFFYFLGCHGITIGFHRHFTHGSFKAKRPLKIALAIAGSMAVEGPLVRWVADHRKHHKFSDDEGDPHSPWRYGETVPALIKGLWWAHIAWMFDEEQTPQEKYAPDLIKDPALRAVSRQFILWTVVSLALPALIGGLVTMSWWGAFTGFFWGSLVRVALLHHVTWSINSICHAVGKRPFKSRDRSGNVWWLAILSCGESWHNLHHADPTSARHGVMRGQLDSSARLIRWFEQLGWAYDVRWPSRSRIDSRRNTDQDGARRRKETAKAA(SEQ ID NO:17)。

[0024] MTMATTATRSDTPGSDFARLSKKVADAGLLGRRPGYYTLRITAVTGLYAAGWAAFVLVGASWWTLAIAAFLAVMYGQVALVAHDMAHRQVFRRRRASELSGRIAGASIGMSYGWWQDKHTRHHANPNTEDLDPDIGPDLLVWSPDQARAATGLPRLLGRWQAFLFFPLLTLEGFNLHVASGRAMANRRLKRRALDGALLLAHCAVYLTALFWVLPPGMAIAFLAVHQCLFGVYLGSAFAPNHKGMPILTADDRPDFLRRQVLTSRNVNGGLFTDLALGGLNHQIEHHLFPSMPSPNLRKARAIVRRYCRDLGVDYAETGLVASYRLALTSLHDAGTPLRRTRVRA(SEQ ID NO:18)。

[0025] MPLPRETLPPDTGGSREGSEFTPLLRDVREQQLLERRTGWYARTIAVNALGLAAVGTGMALLGDSWWVLALAPVLAVLCARTAFIGHDAGHAQISGSRAVNRRIGLVHGNLLLGMSYAWWNDKHNRHHANPNHIDKDPDVAADVLVFTSGQAATRTGFRGRLTRHQAWLFFPLTLLEGLALKLHGFQHLRRQRGRARLVEGALLVAHVAGYVTLLLATMPLAHALVFAALHQALFGLHLGMAFAPNHKGMDMPDPDSEAEKWGHLRRQVLTSRNVRGGFLTDWFLGGLNYQIEHHLFPSMPRPHLGLAQAAVKAHCRDLGIPYAETGLVDSYRQALRHMHEVGEPLRADI(SEQ ID NO:19)。

[0026] MLVESLPTPAQEKDRERGSDFSELSRRIAGAGLLRRRPLYYTVRFGAVALALAGGVAAFVALGDSWSQLFVAVALAVVFGQLGLAAHDLAHRQVFTRRRPSEAGGLLTANLLLGMSYGWWMNKHTRHHANPNHEEKDPDVSPDILVWSRGQASRATGLPRFVGRHQAALFFPLLTLEGLNLSFNSFKALGSRAVKRPVLEGTLLVAHFAVYFGGLFTVLSPGKALVFLAVHQGLFGIYLGSVFAPNHKGMPMIEEGMRLDFLRRQVLTSRNVRGGALVDAFMGGLNYQIEHHLFPSMPTPALGRAQAITEAYCAELGVPYHQTGLLASHREALRHMRSVGEPLRAAR(SEQ ID NO:20)。

[0027] MSLNFLDFEQPIAELEAKIDSLTAVSRQDEKLDINIDEEVHRLREKSVELTRKIFADLGAWQIAQLARHPQRPYTLDYVRLAFDEFDELAGDRAYADDKAIVGGIARLDGRPVMIIGHQKGRETKEKIRRNFGMPAPEGYRKALRLMQMAERFKMPIITFIDTPGAYPGVGAEERGQSEAIARNLREMSRLGVPVVCTVIGEGGSGGALAIGVGDKVNMLQYSTYSVISPEGCASILWKSADKAPLAAEAMGIIAPRLKELKLIDSIIPEPLGGAHRNPEAMAASLKAQLLADLADLDVLSTEDLKNRRYQRLMSYGYA(SEQ ID NO:21)。

[0028] MDIRKIKKLIELVEESGISELEISEGEESVRISRAAPAASFPVMQQAYAAPMMQQPAQSNAAAPATVPSMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAFIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPLVVIE(SEQ ID NO:22)。

[0029] MLDKIVIANRGEIALRILRACKELGIKTVAVHSSADRDLKHVLLADETVCIGPAPSVKSYLNIPAIISAAEITGAVAIHPGYGFLSENANFAEQVERSGFIFIGPKAETIRLMGDKVSAIAAMKKAGVPCVPGSDGDPLGDDMDKNRAIAKRIGYPVIIKASGGGGRGMRVVRGDAELAQSISMTRAEAKAAFSNDMVYMEKYLENPRHVEIQVLADGQGNAIYLAERDCSMQRRHQKVVEEAPAPGITPELRRYIGERCAKACVDIGYRGAGTFEFLFENGEFYFIEMNTRIQVEHPVTEMITGVDLIKEQLRIAAGQPLSIKQEEVHVRGHAVECRINAEDPNTFLPSPGKITRFHAPGGFGVRWESHIYAGYTVPPYYDSMIGKLICYGENRDVAIARMKNALQELIIDGIKTNVDLQIRIMNDENFQHGGTNIHYLEKKLGLQEK (SEQ ID NO: 23).

[0030] MSWIERIKSNITPTRKASIPEGVWTKCDSCGQVLYRAELERNLEVCPKCDHHMRMTARNRLHSLLDEGSLVELGSELEPKDVLKFRDSKKYKDRLASAQKETGEKDALVVMKGTLYGMPVVAAAFEFAFMGGSMGSVVGARFVRAVEQALEDNC PLICFSASGGARMQEALMSLMQMAKTSAALAKMQERGLPYISVLTDPTMGGVSASFAMLGDLNIAEPKALIGFAGPRVIEQTVREKLPPGFQRSEFLIEKGAIDMIVRRPEMRLKLASILAKLMNLPANPEAPREGVVVPPVDQEPEA(SEQ ID NO:24).

[0031] MRSIARRTAVGAALLLVMPVAVWISGWRWQPGEQSWLLKAAFWVTETVTQPWGVITHLILFGWFLWCLRFRIKAAFVLFAILAAAILVGQGVKSWIKDKVQEPRPFVIWLEKTHHIPVDEFYTLKRAERGNLVKEQLAEEKNIPQYLRSHWQKETGFAFPSGHTMFAASWALLAVGLLWPRRRTLTIAILLVWATGVMGSRLLLGMHWPRDLVVATLISWALVAVATWLAQRICGPLTPPAEENREIAQREQES(SEQ ID NO:25)。

[0032] MRPLHPIDFIFLSLEKRQQPMHVGGLFLFQIPDNAPDTFIQDLVNDIRISKSIPVPPFNNKLNGLFWDEDEEFDLDHHFRHIALPHPGRIRELLIYISQEHSTLLDRAKPLWTCNIIEGIEGNRFAMYFKIHHAMVDGVAGMRLIEKSLSHDVTEKSIVPPWCVEGKRAKRLREPKTGKIKKIMSGIKSQLQATPTVIQELSQTVFKDIGRNPDHVSSFQAPCSILNQRVSSSRRFAAQSFDLDRFRNIAKSLNVTINDVVLAVCSGALRAYLMSHNSLPSKPLIAMVPASIRNDDSDVSNRITMILANLATHKDDPLQRLEIIRRSVQNSKQRFKRMTSDQILNYSAVVYGPAGLNIISGMMPKRQAFNLVISNVPGPREPLYWNGAKLDALYPASIVLDGQALNITMTSYLDKLEVGLIACRNALPRMQNLLTHLEEEIQLFEGVIAKQEDIKTAN(SEQ ID NO:26)。

[0033] MKRAVITLGIVSSIGNNQQEVLASLREGRSGITFSQELKDSGMRSHVWGNVKLDTTGLIDRKVVRFMSDASIYAFLSMEQAIADAGLSPEAYQNNPRVGLIAGSGGGSPRFQVFGADAMRGPRGLKAVGPYVVTKAMASGVSACLATPFKIHGVNYSISSACATSAHCIGNAVEQIQLGKQDIVFAGGGEELCWEMACEFDAMG ALSTKYNDTPEKASRTYDAHRDGFVIAGGGGMVVEELEHALARGAHIYAEIVGYGATSDGADMVAPSGEGAVRCMKMAMHGVDTPIDYLNSHGTSTPVGDVKELAAIREVFGDKSPAISATKAMTGHSLGAAGVQEAIYSLLMLEHGFIAPSINIEELDEQAAGLNIVTETTDRELTTVMSNSFGFGGTNATLVMRKLKD(SEQ ID NO: 7).

[0034] The inventors discovered that overexpression of genes derived from the aforementioned microorganisms in microorganisms with the potential to synthesize hydrophobic compounds can significantly promote the accumulation of hydrophobic compounds in microorganisms.

[0035] According to embodiments 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 have found that overexpression of at least one of the aforementioned genes and silencing of at least one selected from FLD1 and TGL3 in the above-mentioned microorganisms further significantly enhances the accumulation of hydrophobic compounds in the microorganisms.

[0036] According to embodiments of the present invention, the hydrophobic compound includes at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosad polyketide, disesquiterpenes, triterpenes, and tetraterpenoids. The microorganisms according to embodiments of the present invention produce higher yields of the above-mentioned hydrophobic compounds.

[0037] In a second aspect, the present invention provides a method for increasing the fermentation yield of hydrophobic compounds from microorganisms. According to embodiments of the invention, the compound comprises: increasing the lipid content within the microorganism, wherein the microorganism is a microorganism with the potential to synthesize hydrophobic compounds. The inventors unexpectedly discovered in experiments that increasing the lipid content within microorganisms with the potential to synthesize hydrophobic compounds can significantly increase the fermentation yield of microbial hydrophobic compounds, increasing the yield by more than 10%, such as increasing the yield of lycopene by more than 40%, natamycin by 14%, spinosad by 20%, and astaxanthin by 50%, with the products exhibiting low toxicity to cell growth.

[0038] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0039] According to an embodiment of the present invention, the lipid is a triglyceride. The inventors have found that increasing the triglyceride content in microorganisms has a more significant effect on promoting the fermentation yield of hydrophobic compounds.

[0040] According to embodiments of the present invention, the increase in lipid content within microorganisms is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids. This further increases the fermentation yield of hydrophobic compounds.

[0041] According to embodiments of the present invention, the increase in lipid content within microorganisms is achieved by overexpressing at least one of the following genes in the microorganisms: 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 the genes selected from FLD1 and TGL3. This method effectively increases lipid content within microorganisms, thereby providing a suitable environment for the accumulation of hydrophobic products in vivo, minimizing the toxicity of hydrophobic products within cells, and further effectively reducing the impact of hydrophobic product accumulation on cell growth while simultaneously increasing the accumulation of hydrophobic products.

[0042] According to an embodiment of the present invention, the overexpression is achieved by introducing a construct into the microorganism. The construct includes a target gene to be overexpressed and a regulatory promoter, wherein the regulatory promoter is operatively linked to the target gene. This allows for controlled overexpression of the target gene, with overexpression occurring during cell growth and ceasing during product accumulation.

[0043] According to an embodiment of the present invention, the regulatory expression promoter is pHXT1. pHXT1 is a glucose-controlled promoter, which enables more convenient and efficient controllable expression of the target gene.

[0044] According to an embodiment of the present invention, the pHXT1 has the nucleotide sequence shown in SEQ ID NO:28.

[0045]

[0046] According to embodiments of the present invention, the microorganisms include at least one selected from yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, and Trichoderma reesei. Increasing the lipid content in the above-mentioned microorganisms further significantly enhances the accumulation of hydrophobic compounds within them.

[0047] According to embodiments of the present invention, the hydrophobic compound comprises at least one selected from lycopene, carotene, astaxanthin, natamycin, spinosad, polyketides, disesquiterpenes, triterpenes, and tetraterpenoids. Using the method according to embodiments of the present invention, the yield of the above-mentioned hydrophobic compound is higher.

[0048] In a third aspect, the present invention proposes the use of the aforementioned microorganisms in increasing the fermentation yield of hydrophobic compounds. As previously stated, the microorganisms according to embodiments of the present invention possess the ability to produce high yields of hydrophobic compounds. These microorganisms can be effectively used in the fermentation production of hydrophobic compounds, resulting in high yields and low toxicity to the microorganisms.

[0049] It should be noted that the amino acid sequence of the polypeptide encoded by the gene disclosed in this application means that any nucleotide sequence encoding a polypeptide having this amino acid sequence is within the protection scope of this application.

[0050] It should be noted that the amino acid sequence of the polypeptide encoded by the gene disclosed in this application means a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the disclosed amino acid sequence; or

[0051] Peptides with one or more amino acid substitutions, deletions, and / or additions to the disclosed amino acid sequence are all within the scope of protection of this application.

[0052] 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. Attached Figure Description

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0054] Figure 1 The graph shows the results of the lycopene-producing bacteria that overexpress triglycerides according to embodiments of the present invention, which showed varying degrees of yield improvement compared to the original lycopene-producing bacteria TM606. Detailed Implementation

[0055] The embodiments of the present invention are described in detail below, using *Saccharomyces cerevisiae* (high lycopene producer), *Escherichia coli* (high astaxanthin producer), *Streptomyces* (high natamycin producer), and *Saccharopolysporum* (high spinosad producer) as examples. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are all within the scope of the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0056] 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.

[0057] Example 1: Construction of plasmids required for lycopene production bacteria

[0058] The primers used in the examples are shown in Table 1.

[0059] Table 1: Primers used to construct lycopene-producing engineered strains

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] The plasmids constructed in the examples and the fragments used in plasmid construction were amplified by PCR using the corresponding primers and templates, as detailed in Table 2.

[0067] Table 2: Plasmids constructed in this embodiment

[0068]

[0069]

[0070]

[0071]

[0072] PCR reaction system: 30.5 μL H2O, 10 μL 5×reaction buffer, 4 μL 2.5 mM dNTPs, 2 μL 10 mM forward primer, 2 μL 10 mM reverse primer, 1 μL template DNA (1-100 ng), 0.5 μL Phusion High-Fidelity DNA Polymerase. PCR reaction program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 56℃ annealing for 30 s, 72℃ extension (30 s / K), 30 cycles; final extension at 72℃ for 10 min.

[0073] All plasmids constructed in the examples were constructed using the yeast assembly method: different plasmids were assembled according to the fragments listed in Table 2.

[0074] The PCR fragments were excised and recovered using a gel, and the volume used was calculated based on 300 ng per fragment.

[0075] Take the required fragment for each plasmid, mix well, calculate the volume, add 10% (v / v) of 3M NaAc and 2% (v / v) of 10 mg / mL glycogen, mix well, then add 2 times the volume of anhydrous ethanol and mix again. Incubate at -80℃ for 2 h, centrifuge at 13,000 rpm at 4℃ for 20 min, and discard the supernatant. Wash once with 500 μL of 70% ethanol, centrifuge at 13,200 rpm at room temperature for 3 min, discard the supernatant, air dry, and redissolve in 4 μL of ddH2O for later use.

[0076] The processed mixed fragments were transformed into *Saccharomyces cerevisiae* using the lithium acetate method, plated on YPD-resistant plates, and incubated at 30°C (colony growth takes approximately 3 days). Once colonies reached a suitable size, single colonies were picked and cultured on YPD-resistant liquid medium at 30°C and 220 rpm for 20 hours. Plasmids were extracted, transformed into *E. coli* DH10B, plated on LB solid plates (Ampicillin resistant), and incubated at 37°C for approximately 2 days. Single colonies were then picked and cultured on LB-Amp liquid medium at 37°C and 220 rpm for 16 hours to extract plasmids, which were then verified by enzyme digestion.

[0077] Example 2 Construction of Saccharomyces cerevisiae Lycopene Strain

[0078] The Saccharomyces cerevisiae lycopene strain TM606 was constructed using the following method:

[0079] Expression of tHMG1 improves the conversion rate of the MVA pathway; silencing GAL1, 7, and 10 induces lycopene production using galactose; the construction method involves digesting plasmid pZY141 with NotI enzyme, recovering the target fragment, and integrating it into Saccharomyces cerevisiae (CEN.PK2-1D) strain via lithium acetate transformation at a dosage of 200 ng. The target strain was obtained by screening on plates containing the corresponding auxotrophic factors and verified by PCR.

[0080] Lycopene was expressed heterologously from PaCrtB (SEQ ID NO: 3) derived from Pancoccus clumps, TmCrtE (SEQ ID NO: 2) derived from the plant Taxus chinensis, and BtCrtI (SEQ ID NO: 4) derived from Trispora trispora, with copy number adjustment.

[0081] MAYTAMAAGTQSLQLRTVASYQECNSMRSCFKLTPFKSFHGVNFNVPSLGAANCEIMGHLKLGSLPYKQCSVSSKSTKTMAQLVDLAETEKAEGKDIEFDFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPALCIAACELVGGSQDLAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKPTNH KVFGEDTAVLAGDALLSFAFEHIAVATSKTVPSDRTLRVISELGKTIGSQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILGGATEDEIARIRRYARCVGLLFQVVDDILDVTKSSEELGKTAGKDLLTDKATYPKLMGLEKAKEFAAELATRAKEELSSFDQIKAAPLLGLADYIAFRQN(SEQ ID NO: 2).

[0082] MSQPPLLDHATQTMANGSKSFATAAKLFDPATRRSVLMLYTWCRHCDDVIDDQTHGFASEAAAEEEATQRLARLRTLTLAAFEGAEMQDPAFAAFQEVALTHGITPRMALDHLDGFAMDVAQTRYVTFEDTLRYCYHVAGVVGLMMARVMGVRDERVLDRACDLGLAFQLTNIARDIIDDAAIDRCYLPAEWLQDAGLTPENYAARENRAALARVAERLIDAAEPYYISSQAGLHDLPPRCAWAIATARSVYREIGIKVKAAGGSAWDRRQHTSKGEKIAMLMAAPGQVIRAKTTRVTPRPAGLWQRPV(SEQ ID NO:3)。

[0083] MSDQKKHIVVIGAGIGGTATAARLAREGFRVTVVEKNDFSGGRCSFIHHDGHRFDQGPSLYLMPKLFEDAFADLDERIGDHLDLLRCDNNYKVHFDDGDAVQLSSDLTKMKGELDRIEGPLGFGRFLDFMKETHVHYEQGTFIAIKRNFETIWDLIRLQYVPEIFRLHLFGKIYDRASKYFQTKKMRMAFTFQTMYMGMSPYDAPAVYSLLQYTEFAEGIWYPRGGFNMVVQKLESIASKKYGAEFRYQSPVAKINTVDKDKRVTGVTLESGEVIEADAVVCNADLVYAYHHLLPPCNWTKKTLASKKLTSSSISFYWSMSTKVPQLDVHNIFLAEAYKESFDEIFNDFGLPSEASFYVNVPSRIDESAAPPNKDSIIVLVPIGHMKSKTGNSAEENYPELVNRARKMVLEVIERRLGVNNFANLIEHEEVNDPSVWQSKFNLWRGSILGLSHDVFQVLWFRPSTKDSTNRYDNLFFVGASTHPGTGVPIVLAGSKLTSDQVCKSFGQNPLPRKLQDSQKKYAPEQTRKTESHWIYYCLACYFVTFLFFYFFPRDDTTTPASFINQLLPNVFQGQNSNDIRI(SEQ ID NO:4)。

[0084] The construction method involved digesting plasmids pZY151, pZY184, and pZY196 with NotI enzyme, recovering the target fragments, and continuously integrating them into the above strains via yeast transformation using the lithium acetate method. The target strains were obtained by screening on plates containing the corresponding nutrient deficiencies and then verified by PCR.

[0085] The expression of the POS5 gene balances the reducing power in yeast. The construction method is as follows: plasmid pTM206 is digested with NotI enzyme, the target fragment is recovered, transformed and integrated into the above strain, and the target strain is obtained by screening with plates containing the corresponding resistance. PCR verification is then performed.

[0086] Expression of the ADH2, ACS6 (SEQ ID NO: 5), and ALD6 genes increases the supply of precursors for lycopene synthesis.

[0087] (SEQ ID NO: 5).

[0088] The construction method involves digesting plasmid pTM303 with NotI enzyme, recovering the target fragment, transforming and integrating it into the above-mentioned strain, screening with plates containing the corresponding resistance to obtain the target strain, and verifying it by PCR.

[0089] Silencing the Ypl062W and Exg1 genes regulates the yeast lycopene synthesis system holistically. Construction method: A knockout cassette fragment was constructed in the Ypl062W gene (which required inactivation) using a hygromycin resistance gene as a marker, and a knockout cassette fragment was constructed in the Exg1 gene using a G418 resistance gene as a marker. These fragments were integrated into the engineered strains via lithium acetate transformation. Screening was performed using plates containing hygromycin resistance, and verification was conducted by PCR.

[0090] When a strain needs to be reused with an resistance selection marker, discard the marker using the following method:

[0091] The strain to be labeled was transformed into the pSH47 plasmid. After colonies grew, several colonies were picked and cultured overnight at 30°C and 220 rpm in 5 mL YPD medium. The culture was then centrifuged at 3,000 rpm for 5 min at room temperature, the supernatant was discarded, and the colonies were washed twice with 5 mL of YPDG medium (containing 1% galactose and no glucose). 5 mL of this medium was added and cultured overnight at 30°C and 220 rpm. The colonies were then plated on SC non-auxiliary auxotrophic solid medium containing 1 g / L 5-fluoro-orotic acid and incubated at 30°C. After colonies grew, single colonies were picked and plated on the corresponding YPD solid plates for verification.

[0092] Example 3: Construction of a lycopene-producing strain with high lipid synthesis

[0093] The modification strategy used in this embodiment is as follows:

[0094] (1) To enhance triglyceride synthesis, the downstream synthesis throughput of triglycerides was increased by overexpressing PAH1 (SEQ ID NO: 8) and DGA1 (SEQ ID NO: 9) genes. The strain TM6065 was constructed using the following method:

[0095] The target fragment of plasmid pTM705 was recovered after digestion with NotI enzyme and integrated into yeast strain TM606 via lithium acetate transformation at a dosage of 200 ng. The strain was screened using plates containing the corresponding resistance to obtain resistant strains, and then verified by PCR.

[0096] (2) To enhance the precursors for triglyceride synthesis, the upstream synthesis throughput of triglycerides was increased by overexpressing the ACC1** (SEQ ID NO: 1) gene with two mutation sites. The strain was named TM6066 and the construction method is as follows:

[0097] The target fragment was recovered after digestion of plasmid pTM706 with NotI enzyme and transformed and integrated into strain TM606 as described above. The strain was screened using plates containing the corresponding resistance to obtain resistant strains, and then verified by PCR.

[0098] (3) To reduce triglyceride degradation while improving triglyceride synthesis, strain TM6068 was constructed by knocking out the TGL3 (SEQ ID NO: 11) gene from strain TM6065. The construction method is as follows:

[0099] The target fragment was recovered after digestion of plasmid pTM708 with NotI enzyme, transformed and integrated into strain TM606, and strains with corresponding resistance were obtained by screening using plates containing the corresponding resistance, and verified by PCR.

[0100] (4) 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 was named TM701 and the construction method is as follows:

[0101] The target fragment was recovered after digestion of plasmid pTM701 with NotI enzyme, transformed and integrated into strain TM606, and strains with corresponding resistance were obtained by screening using plates containing the corresponding resistance, and verified by PCR.

[0102] (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:

[0103] The target fragment was recovered after digestion of plasmid pTM704 with NotI enzyme, transformed and integrated into strain TM606, and strains with corresponding resistance were obtained by screening using plates containing the corresponding resistance, and verified by PCR.

[0104] (6) Method for upstream and downstream synthesis of triglycerides: construct strains TM60656 or TM60686, digest plasmids pTM705 / pTM708 and pTM706 with NotI enzyme, recover the target fragment and integrate it into strain TM606. Use plates containing the corresponding resistance to screen for strains with resistance and verify by PCR.

[0105] (7) Based on the upstream and downstream synthesis of intracellular triglycerides and the size of intracellular lipid droplets, strains TM70156 / TM70186 were constructed using the following method:

[0106] Plasmids pTM705 / pTM708 and pTM706 were digested with NotI enzyme, and the target fragments were recovered and transformed into TM701. The strains with the corresponding resistance were screened using plates containing the corresponding resistance and verified by PCR.

[0107] (8) To increase the size of intracellular unsaturated fatty acid-binding lipid droplets, strain TM707 was constructed using the following method:

[0108] The target fragment was recovered after digestion of plasmid pTM707 with NotI enzyme, transformed and integrated into strain TM606, and strains with corresponding resistance were obtained by screening using plates containing the corresponding resistance, and verified by PCR.

[0109] (9) To enhance the upstream and downstream synthesis of intracellular triglycerides and the content of intracellular unsaturated fatty acids, strains TM70456 / TM70486 were constructed using the following method:

[0110] Plasmids pTM705 / pTM708 and pTM706 were digested with NotI and the target fragments were recovered and transformed into TM704 strain. The strains with the corresponding resistance were screened using plates and verified by PCR.

[0111] (10) Combining methods to enhance the upstream and downstream synthesis of intracellular triglycerides, increase the content of intracellular unsaturated fatty acids, and improve lipid droplet size, strains TM70756 / TM70786 were constructed, and the construction method is as follows:

[0112] Plasmids pTM705 / pTM708 and pTM706 were digested with NotI and the target fragments were recovered and transformed into TM707 strain. The strains with the corresponding resistance were screened using plates and verified by PCR.

[0113] Example 4: Shake-flask culture and fermentation process of engineered lycopene bacteria

[0114] In this embodiment, the inventors describe in detail the fermentation and culture process of some of the engineered strains obtained in Example 3.

[0115] Shake-flask fermentation employs a two-stage seed culture. The recombinant strain from the plate is transferred to a PA flask containing 5 mL of YPD medium and incubated overnight at 30°C in a shaker (generally 14-18 hours). The cells then grow to the logarithmic growth phase (OD). 600 At approximately 5-8 hours, the primary seed culture is obtained. The strain is then transferred at a 1% inoculum to a 250 mL shake flask containing 50 mL of YPD medium. After shaking and incubating for about 14-18 hours, the secondary seed culture is obtained. The final bacterial concentration is calculated as OD0.05. 600 =0.5 The secondary seed culture was calculated and inoculated into a 500 mL shake flask containing 200 mL of YPD fermentation medium (containing 1% galactose), and shake-flask fermentation was carried out at 30℃ and 220 rpm. After 96 h, samples were taken and stored in a -80℃ freezer to measure lycopene yield accumulation.

[0116] Example 5 Product Extraction and Detection

[0117] In this embodiment, the inventors extracted the product obtained after fermentation and tested the lycopene yield.

[0118] Thaw the sample after removing it from the freezer. Transfer 500 μL of fermentation broth to a 15 mL centrifuge tube (pre-cooled on ice), centrifuge at 5,000 rpm and 4°C for 2 min to collect the cells, and discard the supernatant. Add 4 mL of acetone (HPLC grade), 0.2 g of glass beads, and 1% antioxidant, shake for 5 min, sonicate on ice for 5-10 min, centrifuge at 5,000 rpm and 4°C for 2 min, and transfer the supernatant to a 50 mL centrifuge tube. Repeat the above extraction process and collect the extract until the cells no longer show obvious yellow color. Mix the collected extract, take 2 mL, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to a brown sample vial for HPLC analysis.

[0119] Detection method: Lycopene was detected by quaternary HPLC with a UV detector at an absorption wavelength of 474 nm. The chromatographic column was an Agilent Zorbax C18 (150 mm * 4.6 mm * 5 μm). Mobile phase A (acetonitrile:water = 9:1) and mobile phase B (methanol:isopropanol = 3:2) were analyzed under the following conditions: 0-90% B (0-15 min), 90% B (15-30 min), 90%-0 B (30-35 min), and the flow rate was 1 mL / min.

[0120] Test results as follows Figure 1 As shown, the lycopene-producing bacteria that overexpress triglycerides have increased the yield to varying degrees compared to the original lycopene-producing bacteria TM606.

[0121] Example 6: Construction of Natamycin-producing strain overexpressing triglycerides and fermentation

[0122] Natamycin is an odorless, tasteless, low-dose, and highly safe food preservative produced by the fermentation of *Streptomyces natamycin*. It is a white to off-white, odorless, and tasteless crystalline powder. Its mechanism of action involves binding to ergosterol and other sterol groups in the fungus, inhibiting ergosterol biosynthesis, thereby causing cell membrane distortion, ultimately leading to leakage and cell death. In baked goods, surface treatment of dough with natamycin significantly extends shelf life. Natamycin is slightly soluble in water and poorly soluble in most organic solvents. Its solubility in water at room temperature is 30–100 mg / L. Solubility increases at pH below 3 or above 9, but this decreases the stability of natamycin.

[0123] In this embodiment, the inventors constructed plasmids as shown in Table 2 in *Streptomyces natalata*. Specifically, following Example 1, the corresponding genes from *Streptomyces* were obtained via PCR to obtain corresponding fragments. Using the Gibson method (Daniel G. Gibson, *Enzymatic Assembly of Overlapping DNA Fragments*, *Methods in Enzymology*, Volume 498, 2011, Pages 349-361), these fragments were sequentially ligated after the corresponding promoters and then after the ermE* promoter in the vector pSET152, thus constructing the required plasmids. The constructed plasmids were electroporated into *Escherichia coli* ET12567 / pUZ8002. The transformed *E. coli* were cultured overnight at 37°C in 2 mL LB broth. 200 μL of the bacterial culture was then transferred to 5 mL LB broth and cultured at 37°C. Meanwhile, *Streptomyces nata* spores were subjected to heat shock and pre-germination treatment. *Streptomyces nata* spores were suspended in 2 mL of TES buffer and incubated in a 50°C water bath for 10 minutes. After cooling to room temperature, an equal volume of spore pre-germination medium was added, and the mixture was incubated with the previously prepared *E. coli* at 37°C in a shaker for 2.5 hours. The *E. coli* were washed twice with LB medium. Simultaneously, *Streptomyces* spores were collected by centrifugation. The *E. coli* and *Streptomyces* spores were mixed thoroughly at a ratio of 10:1. 8 :10 10 The mixture was mixed in equal volumes with E. coli cells and spread onto plates containing SFM medium. The plates were dried and incubated at 30°C. After 18 hours, the plates were covered with 1 mL of sterile water containing 8 mg / L abeprazole resistance. After incubation at 30°C for 3 days, white conjugation transferons were observed. For preliminary verification of the mutant strain, primers V-apr-F (5'-GCTCATCGGTCAGCTTCTCA 3') and V-apr-R (5'-TCGCATTCTTCGCATCCC 3') were used to verify the presence of the abeprazole resistance gene. If the mutant strain contained the abeprazole resistance gene, a 726 bp PCR product should be obtained.

[0124] The original *Streptomyces natamycin* J1002 and the newly constructed natamycin-producing strain overexpressing triglycerides were cultured on SFM plates (20 g / L soybean meal, 20 g / L mannitol, 16 g / L agar, 1 L tap water, pH 7.5) at 30°C for 7 days. Four small pieces, totaling approximately 3.2 cm in size, were then collected. 2The spore-containing agar blocks were inoculated into 250 mL shake flasks containing 30 mL of COM medium (10 g / L corn starch, 10 g / L oat flour, 5 g / L malt extract, 2 g / L yeast extract, 15 g / L agar, pH 7.2) and cultured at 30 °C and 220 rpm for 48 h. Then, 3 mL of the seed culture was transferred to 25 mL of NPM medium (50.0 g / L corn starch, 18.0 g / L soybean flour, 10.0 g / L yeast extract, 1.5 g / L CaCO3, pH 7.2) and cultured in a shaker at 30 °C and 220 rpm for 120 h.

[0125] Centrifuge 0.5 mL of fermentation broth at 7,000 rpm for 5 minutes, discard the supernatant, and add 1.5 mL of methanol-glacial acetic acid solution (methanol:glacial acetic acid = 95:5, v / v) to mix and precipitate. After sonication for 20 minutes, centrifuge at 7,000 rpm for 5 minutes, discard 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: Agilent ZORBAX SB-C18 column (4.6 × 250 nm), flow rate 0.5 mL / min. -1 Detected by UV 303nm. The mobile phase was methanol:water:glacial acetic acid = 60:40:5 (V / V / V).

[0126] The results are shown in Table 3. The newly constructed strains showed varying degrees of increase in natamycin production compared to the original production strain J1002.

[0127] Table 3: Information on Natamycin-Related Strains

[0128]

[0129] Example 7: Construction of a spinosad-producing strain overexpressing triglycerides and fermentation.

[0130] Spinosad, also known as spinosad, is a macrolide-based, environmentally friendly, and highly effective biological insecticide extracted from the fermentation broth of *Saccharopolyspora spinosa*. Spinosad is a light gray solid crystalline powder with a slightly stale, earthy odor. It has very low solubility in water but is readily soluble in organic solvents such as methanol, ethanol, acetonitrile, acetone, dimethyl sulfoxide, and dimethylformamide.

[0131] In this embodiment, the inventors extracted relevant genes into the spinosad-producing bacteria and then transferred the plasmid constructed in Example 6 into the spinosad-producing bacteria using a similar method. The newly constructed strains are shown in Table 3.

[0132] The original strain of *Polysporium sacchariformis* was purchased from the Chinese Center for Microbial Culture Collection (CGMCC), strain number CGMCC4.1365. The strain was rejuvenated using a rejuvenation medium (1 g / L yeast extract, 1 g / L beef extract, 2 g / L casein acid hydrolysate, 10 g / L glucose, 15 g / L agar, pH 7.3). After 3 days of incubation in the rejuvenation liquid medium, 1 mL of the culture was transferred to a 250 mL spring-loaded shake flask containing 30 mL of fermentation seed medium. The seed medium consisted of 30 g / L TSB, 3 g / L yeast extract, 3 g / L beef extract, 2 g / L MgSO4·7H2O, 10 g / L glucose, 2.5 g / L corn steep liquor, pH 7.0. The fermentation seed was cultured at 30℃ and 220 rpm for 50 h before being inoculated into the fermentation medium. The fermentation medium consisted of: glucose 40 g / L, beef extract 10 g / L, MgSO4·7H2O 2 g / L, NaCl 2 g / L, soybean peptone 2 g / L, soluble starch 30 g / L, CaCO3 2.4 g / L, yeast extract 0.34 g / L, peptone 6.34 g / L, and pH 7.2. The fermentation medium was prepared in 250 mL spring-loaded shake flasks with a 50 mL liquid volume and a seed inoculum size of 10%. Fermentation conditions were 30 °C and a rotation speed of 220 rpm.

[0133] Spinosad (CAS No: 168316-95-8) standard was purchased from Sigma (product code: 33706). This standard contains two components, spinosad A and spinosad D, with an HPLC purity of 97%. The HPLC analysis used a mobile phase of 10% phase A (0.2% ammonium acetate aqueous solution) and 90% phase B (methanol); a DOINEX C18 column (3 μm, 4.6, 150 mm); a flow rate of 0.8 mL / min; and a PDA detector at a detection wavelength of 245 nm. The MS detector was an LCQ FLEET (Thermo Scientific). The HPLC standard curve was prepared by dissolving 5 mg of spinosad standard in 1 mL of HPLC-grade methanol to obtain a 5 g / L stock solution. The standard was then serially diluted and analyzed by HPLC. Take 0.2 mL of fermentation culture and add 0.8 mL of acetonitrile in batches. Vortex to mix for 2 min and then place in a refrigerator at 4°C overnight. Centrifuge at 12000 rpm for 10 min, then take the supernatant, filter it through a 0.45 μm nylon membrane, and store it in a brown HPLC sample vial for analysis.

[0134] The fermentation results are shown in Table 4. J1016-1 showed a greater yield of spinosad than CGMCC4.1365.

[0135] Table 4: Information on spinosad-related bacterial strains

[0136]

[0137] Example 8: Construction of an astaxanthin-producing bacterium overexpressing triglycerides

[0138] Astaxanthin (also known as ketocarotenoids or astaxanthin) is currently the strongest natural antioxidant discovered by humans in nature. Astaxanthin possesses extremely strong antioxidant activity, more than 100 times stronger than vitamin E, earning it the title of "super vitamin E." It effectively scavenge intracellular oxygen free radicals and is the only carotenoid that can cross the blood-brain barrier. It has numerous benefits, including enhancing immunity, relieving fatigue, improving cell regeneration, preventing cancer, treating cardiovascular diseases, reducing the accumulation of senescent cells, inhibiting obesity, protecting the eyes and central nervous system, and providing UV protection. In recent years, astaxanthin has begun to be widely used in pharmaceuticals, health products, cosmetics, food additives, and aquaculture. With the development of my country's national economy, the demand for it is also increasing.

[0139] In this embodiment, the inventors constructed 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 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.) as shown in Table 5, and transformed E. coli MG1655 competent cells with the plasmid combinations shown in Table 6 to construct an Escherichia coli astaxanthin-producing strain.

[0140] The specific method for plasmid construction in this embodiment is the same as in Example 1. The corresponding genes are amplified by PCR and sequentially ligated to the corresponding promoters 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.

[0141] Table 5: Construction of plasmids for astaxanthin synthesis in Escherichia coli

[0142]

[0143] Table 6: Construction of Escherichia coli astaxanthin synthesizing strains

[0144] 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

[0145] After the strain is constructed, shake-flask fermentation is performed as follows:

[0146] After transformation, transformants were selected and cultured overnight at 37°C and 220 rpm in LB medium containing 34 μg / mL chloramphenicol, 50 μg / mL kanamycin, and 100 μg / mL ampicillin. 200 mL of LB medium containing the same three compounds was then inoculated at a 1% inoculum and cultured at 30°C and 200 rpm. When the OD600 reached 0.7-0.9, 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added for induction. After 15 h of culture, 2 mL of the culture was collected, centrifuged at 12,000 rpm for 3 min, the supernatant was discarded, and 1 mL of extraction solvent (V:V:Methanol = 4:1) was added. The cells were shaken to disperse the bacteria, sonicated for 10 min, and centrifuged at 13,000 rpm and 4°C for 10 min. The supernatant was then analyzed by high-performance liquid chromatography (HPLC) as described below, with the operation performed in the dark.

[0147] Quaternary HPLC (Thermo Fisher Ultimate 3000), Agilent Zorbax C18 column (4.6 mm × 150 mm × 5 μm). Chromatographic conditions were 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.

[0148] The results are shown in Table 6. The modified engineered bacteria produced higher astaxanthin yields.

[0149] Example 9: Controlling Triglyceride Overexpression in Microorganisms

[0150] Based on Example 3, we replaced the promoters of the PAH1, DGA, ACC1, and OLE1 genes with pHXT1 (SEQ ID NO: 28). This promoter is characterized by the ability to express the relevant genes overexpressed under glucose conditions, but not under glucose-free conditions. Therefore, through our fermentation control, the lycopene-producing strain can accumulate triglycerides in the early stages of fermentation, and stop accumulating triglycerides after glucose is consumed in the later stages. This controls the lipid content within the microorganism, allowing the microorganism to utilize more substrate and energy to synthesize lycopene in the later stages of fermentation, thereby further increasing lycopene yield. The specific construction method is the same as in Examples 1-2, with the promoters replaced. Details of the strain are shown in Table 7. Fermentation and detection methods according to Examples 4-5 were used for testing. The results showed that by controlling triglyceride expression, lycopene yield was further increased.

[0151] Table 7: Information on the modification and yield of lycopene-producing engineered strains

[0152] 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

[0153] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0154] 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. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: OLE1A The OLE1A The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 17, wherein the *Streptomyces natamycin* has the potential to synthesize natamycin.

2. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: OLE1B The OLE1B The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 18, wherein the *Streptomyces natamycin* has the potential to synthesize natamycin.

3. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: OLE1C The OLE1C The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 19, wherein the *Streptomyces natamycin* has the potential to synthesize natamycin.

4. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: OLE1D, The OLE1D The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 20, wherein the *Streptomyces natamycin* has the potential to synthesize natamycin.

5. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: LPPβ and DGAT, The LPPβ encodes a polypeptide with the amino acid sequence shown in SEQ ID NO: 16, the DGAT encodes a polypeptide with the amino acid sequence shown in SEQ ID NO: 15, and the *Streptomyces natamycin* has the potential to synthesize natamycin.

6. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: ACCA2, ACCE and ACCB The ACCA2 A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 12, The ACCB A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 13, The ACCE The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 14, wherein the *Streptomyces natamycin* has the potential to synthesize natamycin.

7. A type of *Streptomyces natalata*, characterized in that, include: Overexpression: ACCA2, ACCE, ACCB, LPPβ and DGAT , The ACCA2 A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 12, The ACCB A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 13, The ACCE A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 14, The LPPβ A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 16, The DGAT The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 15, wherein the *Streptomyces natamycin* has the potential to synthesize natamycin.

8. A method for increasing the yield of natamycin fermentation by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: OLE1A; The OLE1A A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 17, Increase the lipid content in Streptomyces natamata, wherein Streptomyces natamata has the potential to synthesize natamycin.

9. The method according to claim 8, characterized in that, The lipid is a triglyceride.

10. The method according to claim 8, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

11. The method according to claim 8, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

12. The method according to claim 11, characterized in that, The expression promoter is pHXT1.

13. The method according to claim 12, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

14. A method for increasing the fermentation yield of natamycin by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: OLE1B The OLE1B A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 18 increases the lipid content in Streptomyces natamalia, wherein the Streptomyces natamalia has the potential to synthesize natamycin.

15. The method according to claim 14, characterized in that, The lipid is a triglyceride.

16. The method according to claim 14, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

17. The method according to claim 14, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

18. The method according to claim 17, characterized in that, The expression promoter is pHXT1.

19. The method according to claim 18, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

20. A method for increasing the yield of natamycin fermentation by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: OLE1C The OLE1C A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 19, Increase the lipid content in Streptomyces natamata, wherein Streptomyces natamata has the potential to synthesize natamycin.

21. The method according to claim 20, characterized in that, The lipid is a triglyceride.

22. The method according to claim 20, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

23. The method according to claim 20, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

24. The method according to claim 23, characterized in that, The expression promoter is pHXT1.

25. The method according to claim 24, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

26. A method for increasing the yield of natamycin fermentation by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: OLE1D The OLE1D A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 20, Increase the lipid content in Streptomyces natamata, wherein Streptomyces natamata has the potential to synthesize natamycin.

27. The method according to claim 26, characterized in that, The lipid is a triglyceride.

28. The method according to claim 26, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

29. The method according to claim 26, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

30. The method according to claim 29, characterized in that, The expression promoter is pHXT1.

31. The method according to claim 30, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

32. A method for increasing the fermentation yield of natamycin by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: LPPβ and DGAT The LPPβ The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 16, wherein DGAT A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 15; Increase the lipid content in Streptomyces natamata, wherein Streptomyces natamata has the potential to synthesize natamycin.

33. The method according to claim 32, characterized in that, The lipid is a triglyceride.

34. The method according to claim 32, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

35. The method according to claim 32, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

36. The method according to claim 35, characterized in that, The expression promoter is pHXT1.

37. The method according to claim 36, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

38. A method for increasing the yield of natamycin fermentation by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: ACCA2, ACCE and ACCB The ACCA2 The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 12, wherein ACCB The polypeptide encoding the amino acid sequence shown in SEQ ID NO: 13, wherein ACCE A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 14, Increase the lipid content in Streptomyces natamata, wherein Streptomyces natamata has the potential to synthesize natamycin.

39. The method according to claim 38, characterized in that, The lipid is a triglyceride.

40. The method according to claim 38, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

41. The method according to claim 38, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

42. The method according to claim 41, characterized in that, The expression promoter is pHXT1.

43. The method according to claim 42, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

44. A method for increasing the yield of natamycin fermentation by *Streptomyces natamycin*, characterized in that, include: By overexpression in the *Streptomyces natalata*: ACCA2, ACCE, ACCB, LPPβ and DGAT , The ACCA2 A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 12, The ACCB A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 13, The ACCE A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 14, The LPPβ A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 16, The DGAT A polypeptide encoding the amino acid sequence shown in SEQ ID NO: 15, Increase the lipid content in Streptomyces natamata, wherein Streptomyces natamata has the potential to synthesize natamycin.

45. The method according to claim 44, characterized in that, The lipid is a triglyceride.

46. ​​The method according to claim 44, characterized in that, The increase in lipid content in *Streptomyces natalata* is achieved by increasing at least one of the following: the amount of triglyceride synthesis, the size of lipid droplets, and the content of unsaturated fatty acids.

47. The method according to claim 44, characterized in that, The overexpression is achieved by introducing a construct into the *Streptomyces natalata*, the construct comprising the target gene to be overexpressed and a regulatory promoter, the regulatory promoter being operatively linked to the target gene.

48. The method according to claim 47, characterized in that, The expression promoter is pHXT1.

49. The method according to claim 48, characterized in that, The nucleotide sequence of PHXT1 is shown in SEQ ID NO:

28.

50. Use of the natamycin Streptomyces according to any one of claims 1 to 7 in increasing the yield of natamycin fermentation.

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