Mutant microorganism with enhanced ability to produce nicotinamide mononucleotide and method for producing nicotinamide mononucleotide using same

Abstract

The present invention relates to a mutant microorganism with an enhanced ability to produce nicotinamide mononucleotide and a method for producing nicotinamide mononucleotide, using same. The mutant microorganism can produce nicotinamide mononucleotide with a high yield by enhancing, weakening or inhibiting the expression of enzymes, such as nicotinate phosphoribosyltransferase, involved in nicotinamide mononucleotide production pathways.

Description

Mutant microorganism with improved nicotinamide mononucleotide production ability and method for producing nicotinamide mononucleotide using the same

[0001] The present invention relates to a mutant microorganism having improved nicotinamide mononucleotide production ability and a method for producing nicotinamide mononucleotide using the same.

[0002] Population aging is rapidly progressing worldwide, and it is projected that by 2060, the population aged 65 and older will reach 41% of the total population. As the elderly population grows, the prevalence of age-related diseases increases, placing a heavier socioeconomic and medical burden on society.

[0003] Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme for cellular metabolism. It receives hydrogen from many substrates and dehydrogenases to form NADH2. + is the oxidized form of NAD and NADH is the reduced form of NAD. NAD + It plays a role in activating the sirtuin gene, known as the longevity gene. However, NAD in the body + As aging progresses, NAD levels decrease. + It decreases due to increased consumption of NAD, and from about age 60 onwards, the body's NAD + The figure is depleted by about half. NAD + Depletion of NAD may underlie diseases such as aging, metabolic disorders, cancer and neurodegenerative diseases. + Increasing the level of NAD in the body can improve related deficiencies. + Ways to increase your levels include exercise, diet control, maintaining a healthy diet, and NAD + Although intake, etc. is suggested, NAD + NAD is broken down in the digestive tract when taken orally. + It is effective to take it in the form of a precursor.

[0004] Nicotinamide mononucleotide (NMN) is a type of nucleic acid, NAD + It is a precursor of NAD and is a physically stable compound. Recent studies have shown that supplementation with NMN can + It has been shown to enhance biosynthesis and improve various symptoms associated with aging, such as Alzheimer's, diabetes, and vascular dysfunction.

[0005] NMN is mainly produced commercially using enzymes or yeast.

[0006] In the enzyme-based method, NMN is produced by increasing the expression level of phosphoribosyl pyrophosphate (PRPP), a precursor of NMN, using phosphoribosylpyrophosphate synthase (PRS), which is involved in PRPP production, or nicotinamide phosphoribosyltransferase (NAMPT), which produces NMN using nicotinamide (NAM) as a substrate. NMN production can be improved by using mutant forms of these enzymes with improved activity. In addition, NAM can be converted into NMN using a protein mixture solution containing several enzymes, such as NAMPT, PRS, and phosphoribulokinase (PRK).

[0007] In the yeast method, Aspergillus, which is recognized as safe, is used and NAD is produced through pyrophosphatase. + We are producing NMN that can be applied to food by converting it into NMN using .

[0008] Recently, methods have been developed to increase the amount of NMN by inhibiting or enhancing the activity of several enzymes involved in producing precursors such as NMN using yeast, but much research is still needed.

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] Korean Patent Publication No. 10-2021-0091255

[0012] Korean Patent Publication No. 10-2020-0061374

[0013] The purpose of the present invention is to provide a mutant microorganism having improved nicotinamide mononucleotide production ability.

[0014] In addition, the present invention aims to provide a method for producing nicotinamide mononucleotide using the mutant microorganism.

[0015] One aspect of the present invention provides a mutant microorganism having enhanced nicotinamide mononucleotide production ability and enhanced activity of nicotinate phosphoribosyltransferase.

[0016] The "nicotinate phosphoribosyltransferase (NPT)" used in the present invention exists in bacteria, yeast, fungi, plants, insects, mammals, etc., and is known as an enzyme that converts nicotinic acid (NA) into nicotinate mononucleotide (NaMN). Since nicotinic acid (NA) is structurally very similar to nicotinamide (NAM), the present invention suggests that nicotinate phosphoribosyltransferase (NPT) will have an enzyme activity similar to nicotinamide phosphoribosyltransferase (NAMPT) that produces NMN using nicotinamide (NAM) as a substrate.

[0017] Nicotinate phosphoribosyltransferase (NPT) in the present invention may be, but is not limited to, a polypeptide having nicotinate phosphoribosyltransferase activity that converts nicotinic acid (NA) into nicotinic acid mononucleotide (NaMN). Nucleic acid and protein sequence information for enzymes involved in NMN biosynthesis, including the nicotinate phosphoribosyltransferase (NPT), can be obtained through known sequence databases (e.g., GenBank, UniProt).

[0018] According to one specific example of the present invention, the nicotinate phosphoribosyltransferase (NPT) is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Methanosarcina mazei, Methanosarcina acetivorans, Desulforapulum autotrophicum, Methanococcoides burtonii, Chromohalobacter salexigens, Arabidopsis thaliana, Dictyostelium discoideum, Ciona intestinalis, Drosophila melanogaster, Ciona savignyi, Bacillus subtilis, Caenorhabditis elegans, Mycobacterium tuberculosis, Candida glabrata or Nakaseomyces glabratus, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica, Aspergillus fumigatus fumigatus), Neurospora crassa, Chaetomium globosum, Emericella nidulans,may be derived from one or more selected from the group consisting of, but not limited to, Aspergillus oryzae, Gibberella moniliformis, Cryptococcus neoformans, and Ustilago maydis.

[0019] The above nicotinate phosphoribosyltransferase (NPT) contains a common motif, regardless of its origin. Motifs are small structural parts that appear in the amino acid sequences of various proteins. They can be divided into structural motifs, which form a consistent structure across proteins, and sequence motifs, which have a characteristic pattern in the amino acid sequence. Motifs are commonly found in proteins with common functions, and are thought to contribute to functional expression.

[0020] The nicotinate phosphoribosyltransferase (NPT) of the present invention may include a motif consisting of 23 amino acids from positions 212 to 234 at the N-terminus of the amino acid sequence based on structural homology or sequential homology (hereinafter referred to as the '212-234 motif'). It has been reported that histidine (H232) located at position 232 of the motif is autophosphorylated by ATP to increase catalytic efficiency (Biochemistry 35, 3917-3924; Biochemistry 37, 4189-4199). Therefore, the above 212-234 motif is expected to be a part related to the activity of nicotinate phosphoribosyltransferase (NPT), and nicotinate phosphoribosyltransferase (NPT) containing this motif can be expected to efficiently convert nicotinamide (NAM) into NMN.

[0021] According to one specific example of the present invention, the nicotinate phosphoribosyltransferase (NPT) may include the 212-234 motif.

[0022] Specifically, the nicotinate phosphoribosyltransferase (NPT) may be derived from the origin shown in Table 1 below, and the amino acid sequence and 212-234 motif thereof are as shown in Table 2 below.

[0023] 번호기원유전자NCBI접근번호UniProt ID1Saccharomyces cerevisiaeNPT1NP_014852P396832Saccharomyces cerevisiae S288CNPT1CAA85352.1P396833Candida glabrata-XP_448893Q6FLK14Kluyveromyces lactis-XP_453357Q6CRT25Eremothecium gossypii-NP_983562Q75BW16Candida albicansNPT1XP_889008 / XP_889008.1A0A1D8PRI07Debaryomyces hanseniiXP_462577Q6BGU48Schizosaccharomyces pombenaprtNP_001342932Q9UTK39Yarrowia lipolytica -XP_500338Q6CG7410Aspergillus oryzae-BAE64333Q2U28211Chaetomium globosum-EAQ93453.1Q2HDL612Gibberella moniliformisNPT1AAN74808Q8J2R513Neurospora crassanic-6XP_965789Q7SI0314Aspergillus fumigatus-XP_746744Q4WAN015Emericella nidulans-XP_682385Q5ARG416Cryptococcus neoformans-XP_568039 / XP_568039.1Q5K8P017Cryptococcus neoformans-EAL18922-18Ustilago maydis 521-XP_760597 / XP_760597.1-19Methanosarcina mazeipncBWP_011034984Q8PSJ320Desulforapulum autotrophicumpncBWP_012662670C0QFM521Methanosarcina acetivoranspncBWP_011022501Q8TMW622Methanococcoides burtoniipncBWP_011498483Q12Z0523Chromohalobacter salexigenspncBWP_011506244Q1QZ1024Dictyostelium discoideumnaprtXP_647373Q55G1025Arabidopsis thalianaNAPRT1NP_195412Q8RWM226Ciona intestinalis-NM_001032533F7APS627Arabidopsis thalianaNAPRT2NP_179923Q84WV828Ciona savignyi--H2YXH929Drosophila melanogasterNaprtNP_001097077Q9VQX430Drosophila melanogasterNaprtNP_722961Q9VQX431Bacillus subtilispncBWP_003228788O3209032Caenorhabditis elegansnprt-1NP_500265Q95XX133Mycobacterium tuberculosispncB2WP_003403007P9WJI734Mycobacterium tuberculosispncB1WP_003911483P9WJI9.

[0024] 번호아미노산 서열서열번호 / 서열 상동성1MSEPVIKSLLDTDMYKITMHAAVFTNFPDVTVTYKYTNRSSQLTFNKEAINWLKEQFSYLGNLRFTEEEIEYLKQEIPYLPSAYIKYISSSNYKLHPEEQISFTSEEIEGKPTHYKLKILVSGSWKDTILYEIPLLSLISEAYFKFVDIDWDYENQLEQAEKKAETLFDNGIRFSEFGTRRRRSLKAQDLIMQGIMKAVNGNPDRNKSLLLGTSNILFAKKYGVKPIGTVAHEWVMGVASISEDYLHANKNAMDCWINTFGAKNAGLALTDTFGTDDFLKSFRPPYSDAYVGVRQDSGDPVEYTKKISHHYHDVLKLPKFSKIICYSDSLNVEKAITYSHAAKENGMLATFGIGTNFTNDFRKKSEPQVKSEPLNIVIKLLEVNGNHAIKISDNLGKNMGDPATVKRVKEELGYTERSWSGDNEAHRWT2 / 100%2MSEPVIKSLLDTDMYKITMHAAVFTNFPDVTVTYKYTNRSSQLTFNKEAINWLKEQFSYLGNLRFTEEEIEYLKQEIPYLPSAYIKYISSSNYKLHPEEQISFTSEEIEGKPTHYKLKILVSGSWKDTILMRSLTVLISEAYLIVTSTGLRNHRQAEKKAETLFDNGIRFRIHGTRRRRSLKAQDLIMQGIMKAVNGNPDRNKSLLLGTSNILFAKKYGVKPIGTVAHEWVMGVASISELDYLHANKNAMDCWINTFGAKNAGLALTDTFGTDDFLKSFRPPYSDAYVGVRQDSGDPVEYTKKISHHYHDVLKLPKFSKIICYSDSLNVEKAITYSHAAKENGMLATFGIGTNFTNDFRKKSEPQVKSEPLNIVIKLLEVNGNHAIKISDNLGKNMGDPATVKRVKEELGYTERSWSGDNEAHRWT3 / 94.19%3MSSMTPAITSLLDTDMYKITMHAAVFTNFPDARVYKFTNRTAQFHFNKRAVDWIKEQFRLLGDLTFHDDVEYLAREIPFLPAKYLHYIENGFTLKPDEKQIELDCQEIKGKEDQYDLHILVKGLWIDTILYEIPILALLSEAYFKFVDTDWDYENQLSNAKEKARLFENNLSFSEFGTRRRRSFKTQDLVMEGIMQAVKENPEKYRPLLLGTSNILFAKKYGVKPIGTVAHEWIMGIASITGDYPETNRIAMDYWIKTFGKEHAGLALTDTFGTDDFLKSFKPPYSDYYIGVRQDSGDPIKYTEKIAHHFHDVLKLPKFSKFICYSDSLNIDKAIEYGKVAEAHGIKSTFGIGTNFTNDFHKKSDPSVKSAPLNIVIKLLEVNGNHSIKISDNAGKNMGDPDTVKKVKEQLGYVERQWAGGNEAHRSAA4 / 7 0.52%4MEPVITSFLDTDMYKLTMHAAVYTHFKDVKVKYKYTNRSPQMTFNKEAVEWLKGQFLLLANIRLTTEELKYIKEAIPYLPAEYLEFISNGGFEVNPKDEIKFEAREIEGEPGHYELDISIEGLWIKTIWYEIPVLALVSEAYFKFVDTDWTYDGQVEKAYAKAKKLLDHDIVFSEFGTRRRSFKTQDLVMQGIINAAKDCDKSKYVLGTSNVLFAKKYNVNPIGTVAHEWMMGIASITNDYLNANKNAMDYWIETFGMENAGLALTDTFGTDSFLKSFYPPYSDAYIGVRQDSGDPILYTEKIAHHYNDVLKLPKFSKIICYSDSLNPDRAIEYAQVAHKHGLKATFGIGTNFTNDFQRKSDVSVKSEPLNIVIKLLEVNGNHAIKISDNLGKNMGDPETVRRVKEELGYVERKYAGDNEAHRWAA5 / 69.63%5MTSEEAAIKSLLDTDMYKLTMHAAVYVNFPDTEVVYKYTNRSAGLSFNKAAIVWLEDQVSKLATLRFTAEEVAYLKETLPFLPAQYIDYISSPECRMDPATQVQLLHEQREGEERYDLNITVTGLWKDTILYEIHMLALISEAYFKFVDTDWVLDGQVEQAYRKTRLLLDNGLVFSEFGTRRRSLLVQDLVLQGIAEAVADSGTGDTQFIGT SNVYFAKKYGVKPVGTVAHEWYMGIAALTDDYRNANKNAMDFWLNTFGSEQAGLVLTDTFGTDTFLPMFRPPYSDVYDGVRQDSGDPAVFTEKVAHHYLNVLHYPRFSKVICYSDSLNPEKALQYAEVARAHGMRASFGIGTNFTNDFRRHSAPDAKSEPLNIVFKLLTVNGRPAIKISDDLGKTMGDPEKVEEVKRELGYIERTWEGSDEAHRWKD 6 / 63.85%6MNGTEDKPVIKSFLDTDLYKLFMHAAVNKQFPDVPVKYRYTNRTPQLKLNSQAISWLKKQIEYLGDLRFSAEEILYLHRVLPQLPSDYLEYLADFKLVPSSQIKYLDNDEGDFELEIVGKWNDTILYEIPLLALVSEAYFKFVDTDWNYEGQYALAQKKAKQLISNECNFSEFGTRRRSYESQEIVIKAIKDVQIDTQSKYIAGTSNVYFAMKYDLPPIGTVAHEWYMGIASITQDYVHANKLAMDYWIDTFGAKYAGLALTDTFGTDNYLTMFVAPYVNEYSGVRQDSGDPELYAEKIARHYEKMGIAKNTKIICFSDSLNVEKCLKYKNTADKLGLISTFGIGTFFTNDFNKLSNGEKSQPMNIVIKIKEANGKPAIKISDNIGKNMGDQATVDRVKQELGYTERTWSEGDETHRWSK7 / 58.31%7MTTDIPIIITSILDTDLYKLTMHAAVVKHFPNIPVVYRYTNRTPSMVLNKEAIDWLKYQISKLSDLRVSSEELEYLRKALPQMPEVYLKYLETFQLFPDKQIKYFNDESNFEEFEIEMKGKWDETMLYEIPLLALISEAYFKFVDTDWNYDGQAERAEDKCAQLFKNECTFSEFGTRRRSFKTQDLVVKNLCDFAGKNPDKKHFLGTS NVLLAKKYNTTPIGTVAHEWFMGIASITQDYTNANKLAMDYWLDTFGPEHAGLALTDTFGTDAYLKVFTKPYTDYYTGVRQDSGDPESFAEKLADHYKKQGYPDFSKIICFDSSLNVEKCLKYKQKVNSLGLIASFGIGTFFTNDFASVSDPSTKSVPLNIVIKLKEANGNPSIKISDNLGKNMGDPAVVSRVKKELNYEHSWAEGDEEKRW D8 / 56.94%8MSEPAVVSILDTDLYKLTMLQAVLEHYPDAQVSYKYTNRSKPKMALNQEAYNWLREQIRGLNRNLHLLPEEEQWLRKNCPYLKESFYEFMEFEFDPENSISLNYDSETKDLSIFIHGLWKNTIFYEIPLLALVSESYFKFVDKDWSPEGQFEKAYEKGKRLIRAGCAFTDFGTRRRDPTQEIVLQGLMKAQEDFKGPGSFLGTSNVYFAAKYNLNVSGTVAHEWYMGIAAITQNYKQANRIASLKWVQTFGTSLLIALTDTFSTDVFLKSFTANSADDLANVFHGVRQDSGCAEEYIEKVVKHYKSIGVDPSTKVIVHSDALNVDRCIELYKYCEKCGIKSAFGIGTNLTSDFQKVSNPSEVSKPMNIVIKLFSAEGTKAVKISDDIMKNTGDRDAVIQAKHQLCLPIA9 / 45.67%9MSTITSLLDTDLYKLTMQAAVLQHFPAAQATFLFKNRTPSKQLNDDAIEWLKSEIAALGELRFTEDEIVFLQKHVGFLAEYFEYLKTCQLDPAAQVKVTVNTEGHLEIEVNGPWKDTILYEIPLLALVSEAYFKFVDKDWSYDGQSELAATKAQELIAQGCAFSEFGTRRRRSLKTHDIVIAGILEGLKSAQGNGIFTGTSNVYLAKKYNLKPIGTVAHEWMMGVAAATGDYSTANLRAMELWIQTVGDANAGVALDTGFGTESFLLDFNKPLTDIYNGVRQDSGDPLEYTKLLGDHYKQLGYEPMSKVIVYSDSLDVEKCGKYKAAAAENGLKAAFGVGTFFTNDFKRLSDGQKSTPLNIVIKIQQLNGQSCIKLSDNLSKNMGDPETVERVKRELGYVEKGDVIDESKRWN10 / 53.97%10MDQEGSPPIPEGICSLLDTDLYKLTMQCAVLKY FPDTHVTYGFTNRTPHMKLTRGAHKWLLKQMDRLANIRIRITEEIKFLKTRCPYFNDAYLDFLTTFKLKPSEQIEIKFTPVNDTGSDSDTGDVEYLVKGLWVDTILYEIPLLALTSEAYFMFSDKDWDYSCQEEKAYRKGCTLLENGCIFSEFGSRRRRDYHTHDLVMVGLMKAAEEGKRQGWKGRFTGSSNVHFAMKYGVDPVGTVAHEWYMTIAAITDDYENANENALARYWLGCFGKGVLGIALTDFGTPAFLDAFRKPIPAFTSAGAGAVSTSASGPATTNESTVQSEAETKAPITAPLRDGGARTSHETYAQAYTGVRQDSGDPVYFVKMVRDFYDREGITDKKTMVFSDSLNIEHCLEYKVIAEEAGFQPVFGVGTFFTNDFTNKSNDEKSKPLNIVIKISTANGHPAVKLSDNMGKNTGDKQKVQEVKKKLGYVEHEWEEGDESNRWSKR11 / 44.38%11MDFNSSSPYPEGVISFLDTDLYKLTMQCAVLKYYKNVPVTYAFTNRTPEKKLSRKAFRWLEDQVRKLGNISLSAELEYYLKEHCPYLSPAYLEFLSEFRLRRPREQVVLSFLPTGEDTGAESDIGDLDIKISGIWSEAILYEIPLLSLTSEAYFKFMEPDWTYEGQEDQAFEKGMRLLEAGCVFSEFGTRRRRDYHTQALVFRGLTKASKEAEKRGLSGKLSGTSNVHLAMRFNIPPVGTVAHEWFMGSAAIVGDYRKATEEALRHWVGCFGEGVLGIALTDFGTPDFLTAFSKPIEYLEPPSPTTARKPSVADSFISTSPSVASHQKPNKTYAEVFTGVRQDSGDPKTFVKIIGKFYREQGIKDKKVIVFSDSLNIDRCLEYKQVSEEAGFQPTFGVGTFLTNDFVNTKTGKKSTPLNIVIKLSSADGNPAIKISDNIGKNTGDKATVKRELGYVEKMWEGGDETARWGRED DAPKQ12 / 43.87%12MDFNSSSPFPEGVISFLDTDLYKLTMQCAVFKFFKDVPTVTYAYTNRTPDKKLSRTAFKWLEEKIRKLGNISLSTDEYLFLKHCDYLSDDYLNFLKEFRLSPREQVVATFTPVGEDNGDDSIEGDVDIQIKGTWVDTILYEIPMLALTSEAYFKFMDTDWNYEGQEKQAFEKGLLKLLEAGCITSEFGTRRRRDYHTQALVFRGLVQASKEAEKKGFPGKLSGTSNVHLAMRFNIPPVGTVAHEWFMGVAAIIGDYRSATEVALRHWVACFGNKLGIALTDFGTQEFLRAFTQPVQTIEGGFPAETFKKADGTMKTYAETFAGIRQDSGDPAEYTKWMKEFYDKQGITDKKLIVFSDSLNIERCLEYKKIAEDLGFQPTFGVGTYLTNDFVHLTTGKKSVPLNIVIKISSAAGRPAVKISDNIGKNTGDKETVEKVKQELGYVEREWKEGDETSRWGKE13 / 43.79%13MDFNSSLPHPPAGVISFLDTDLYKLTMQCAVLKYFKDVPVTYSFTNRTPEKKLSREAFVWLEEQVMKLGNISLSPEELQFLKTHCPYLTEEYLDYLSEFRLRRPREQVAVSFRPDGDSDLGDIHYDIKGNWAETILYEIPLLALTSEAYFKFMDTDWDYDGQEEKAYEKGMRLLEAGCVFSEFGTRRRRDYHTQALVFRGLTKAAKEAEKRGLTGKLSGTSNVHLAMRFNIPPVGTVAHEWFMGNAAILGDYKSATEEALSRWVGCYGPGVLGIALTDFGTPEFLRAFSKPMSASGEPVPQPRDRKISTADAFISAAKDFIKDLHPDKTYAQVFTGVRQDSGDPKEFVKLMRKFYDEQGIKDKKVIVFSDSLDIDRCLEYKEVAEAAGFQPTFGVGTYFTNDFVHKATGKKSTPLNIVIKLSSAAGNPAVKISDNVGKNTGDKATVEKVKRELGYVEKDWSEGDESARWGHDGDAATA14 / 43.29%14M SQETTPPYPEGIFSLLDTDLYKLTMQCAILKYFPDVHVTYGFTNRTPDMKLTRGAYKWLLEQMDRLANVRVTDEEIAFLKKQCPYFNHAYLRYLSTFQLKPSEQIDIKFRPVQDSGSDDDLGD IEYMVKGLWVETILYEIPLLALTSQAYFMFTDKDWDHSNQEEKAFRKGCTLLENGCIFSEFGSRRRRDYHTQDLVMQGLCRAAEEGKRQGWKGVFSGTSNVHFAMRYGVTPIGTVAHEWFMAIA AITDDYENANELALRYWLGCFGEGVLGIALTDTFGTPAFLDAFRKPIPHHTSAGVGAVATIASGASTTSESQPQTEAETKPVTAPLHENDSQHSPKTYAQVYTGVRQDSGDPTYFVKMVRDFYDNEGIKEKKTVVFSDSLNIEHCLEYKTIAEEAGFAPIFGVGTFFTNDFTNKSDGKKPLNIVIKIATANGRPAVKLSDNMGKNTGDKNMVQEVKKRLGYIEHYWEEGDESNRWAKQG15 / 43.22%15MGEHSLLPDGVFSLLDTDLYKLTMQCAILKYFPDVQVTYGFTNRTHMKLTRGAYKWMLAQMDKLANIRVTEDEIAFLKRRCPYFNTAYLDFLTNFRLKPSEQIDINFTPVNDTGSDSDFGDIDYIVKGAWVDTILYEIPLLALTSQAYFMFSDKDWNYECQEGKAYRKGYVLLENGCTFSEFGTRRRSYHTQDLVMQGLCRAAREGKAKGLPGVFTGSSNVHFAMKYDVDPVGTVAHEWYMTIAAITDDYENANEMALKYWLGCFGEGVLGIALTDFGTPAFLDAFRKPIPDYTSAGTGAVSTTAESNIQSEAETKAPITAPLSPDHPPPAVKTYAQVYAGVRQDSGDPSYFVKMARDFYDREGITGTKTVVFSDSLDIEHCLEYKVLAEEAGFKPVFGVGTFFTNDFINTTNEKSQPLNIVIKISSANGRAAVKLSDNMGKNTGDKDTVQAVKKRLGYVEHEWE EGDERNRWARK16 / 42.17%16MPDKLHLPIDDVQIPFSILDTDLYKLTMQNAVLHHFSDAHVVIKFTNRSPQMLFSKECFDWVQQRVNDLSKLKLTSEERKELSKACPYFSESYLDYLSNMQLDPVKQVKLTFIPQGSNEKGEKMCVIEGPWKDTMLYEVPVMAILSEGYFKFVDTDWDYDGQFELAKKKALDLLNPPAPTTSLSFSEFGTRRRSFKAQDIIMRGLIAGHEEYKSKGGSQGILSGTSNVYLALKYGLNPVGTIAHEWIMAVGATYGYRGANGRAMDMWEEVYPPGTKFSSPLTMLTDTYTAAIFFKDFISDPARALRWAVLRQDSGDAFKFVEDAKEAWRTIEDKAGIKRDVGPNGEEVAKGKKVIFSDGLDVEKAIKLQQGCDKAGMAASFGIGTDLTNDFRKASDPSQKSKALNMVIKLNKINGKDCIKLSDDKGKHTGSLEEVRKAQQELGINKN17 / 40.50%17MPDKLHLPIDDVQIPFSILDTDLYKLTMQNAVLHHFSDAHVVIKFTNRSPQMLFSKECFDWVQQRVNDLSKLKLTSEERKELSKACPYFSESYLDYLSNMQLDPVKQVKLTFIPQGSNEKGEKMCVIEGPWKDTMLYEVPVMAILSEGYFKFVDTDWDYDGQFELAKKKALDLLNPPAPTTSLSFSEFGTRRRSFKAQDIIMRGLIAGHEEYKSKGGSQGILSGTSNVYLALKYGLNPVGTIAHEWIMAVGATYGYRGANGRAMDMWEEVYPPGTKFSSPLTMLTDTYTAAIFFKDFISDPARALRWAVLRQDSGDAFKFVEDAKEAWRTIEDKAGIKRDVGPNGEEVAKGKKVIFSDGLDVEKAIKLQQGCDKAGMAASFGIGTDLTNDFRKASDPSQKSKALNMVIKLNKINGKDCIKLSDDKGKHTGSLEEVRKAQQELGIDKN18 / 40.50%18MCAAPSTSAPVAKHASDASPIRSILDTDLYKLTMQQAVLRHYPHTRVAYKFTNRSAATMKFTRQAMDRIRNHIDNLVHLSLSAQERAWLERSCPYLGKDYLDYLEAFRFQPKQQVQLRFLPTGEDGWGHLDLNVSGVWSDVIFYEVPLMAIVSEVYFSTIDTDWSLQDQYQQAFDKACRLTSNGIRYSEFGTRRRRSYQTHRIVLQGLMAGDLSASSGCSSGKLLGTSNVHFAQQFDLVPIGTVAHEWTMAIAALQGYAHSNLKALQLWDAVYSAPDFVANSATHDLTIALTDTFSTNVFWNDLLDNPSGIEIARRWRGLRQDSGDSKAFAQKALDAYRSIGVDPKSKVVIYSDGLDVDRCLELAAYSNHIGIGAAFGIGTSFTNDFIQLSTAQKSKPLNIVIKLDSVEHRRVVKISDDLTKNTGDPTEVLAVKRRFGIPTPSSATPADASVINHLDPGAPPNPSSHAHLGTDDATMTQVSPLDQRNVAAGVELTPPNEPRRLLSARSTSKLSSPKNVASSCAWSNCALASKLVSSIPAALIAGVIERAREGDCDCEPDAPREMCLALNAANGDDAGATAPIAGLNSEIGVPGVPGVPGEPIRLLLCANPLL19 / 39.25%19MIKSILDNDLYKFTMQMAVLELFPKAEAEYRFTNRGLQRFSREFVEELRRVIDEEISGLRLTEEEEYRWLGEKCPFLKPMYLEYLKNFRFKPEEVEICLTRENDLDMRIKGPWHSTILWEIVLMAAVSELYFTTIEKEWNGSTKNPGTPESATLESVLEAYGEKILEIGKVLEENGCLFSEFGTRRRSFELHDQVMRSLVRIKTLTGTSNVYFAKKYGVKPIGTVGHEWIMGTSALVGLRYANRFAFENWVDVYNGDLGIALTDTFGSEAFFKDMDLKLSKIYDGFRHDSGDPYTFVDRVIEHYGKMGIDPMKKVIVFSDALNAEAAVKLKKYCEGKINCSFGIGTSLTNSEFFRESPPPLNMVIKLHSINGIPVVKLSDSPEKETGERDALRVANYIVGRKG LDE20 / 37.26%20MIQSILDNDLYKFTMQQAVHMLYPRVDVEYEFINRSNTPFPKDFAQRLQVEVQGMKNFRLTPEEKEYLDKTCYFMTPVYLDFLEHYTFDPDEVTVSQTNSELSVTIKGPWYRTILWEVPLMAIISELYFVMTNARPLDEQIRVINLNKAKILSCNNIRYADFGTRRRFSSSGHEALIRDILALEHNTLIGTSNVNLARLFNIKPIGTMAHEIMFHGVLNGYRMANPTAVAAWATAFHGHLGIALDTFTTTDIFLSTFDTLHAKLFDGVRHDSGDPIAFIDRIVDHYKKLHIDPITKTIVFSDGLDIDKAVHIHNHCINRIRDSYGIGTNNLTNDVGVTPLNMVIKLAKCRTAPEKDWHNAIKLSDDKGKHTGDSEELAHCIKVLERGM21 / 36.14%21MIKSILDNDLYKFTMQMAVLELFPKAEAEYRFTNRGSHHFSEEFVEKLRRVIDEDISALMLTEDEYQWLGENCSFLKPMYLEYLKNFRFKPGEVEVCLTEEKELDIRIKGPWHSTILWEIVLMAAVSELYFTTIEKEWNGKEWDGNISATSESILTAYGEKILEIGKILEENGCLFAEFGTRRRSFELHDQVMKTLLQIETLTGTSNVFFAKKYGLKPIGTVGHEWIMGTSALIGLRYANRFAFENWVEVYKGDLGIALTDTFGSEAFFKDMDLKLSKIYDGFRHDSGDPFTFVDRVIDHYRKMGIDPMKKVIVFSDALNAEAAIRLKKYCQDKINCSFGIGTSLTNNSEFFRESPPPLNMVIKLHSVNGIPVVKLSDSPEKETGERDALRVANYIVGRKGLDE22 / 35. 41%22MIRSILDNDLYKFTMQMAVLELFPNARAEYRFINRGAQSFTNDFVNELRRRIINKDISKIALSEDEYIWLKDNCPFFKPSYIEYLKNFRFNPEEVKIVLTEDNELELCIEGPWHSSILWEIVLMSTISELYFTVTDNKGEEISASNANDPENTLMEEYSSFIGDMGKELDAKGCIFSEFGTRRRRGFKLHDKVVEVLHELDSFSGTSNVYFAKKYGVRPIGTIGHEWIMGNSALVGLRNANKFAFDNWVKVYKGDLSIALSDTFGSKPFFQNFSIGLAKIYDGVRHDSGDPIKFADEVIEHYKKLGIDPMKKVLVFSDSLHVSDAVKLKEYCSGRINCSFGIGTTLTNNPDFFSYNPPLNMVIKLHKIDGIPVVKLSDSVEKATGDKDALRVANYIFGRKGLDEQF23 / 34.13%23MLDSLLDNDFYKFTMQSAVIKRFPYARARYAFINRGEHAFPPPGFGEQLREAVDAMADLSLSVEEKRFLERTCPYLDPTYLDFLSGFRYDPSEVDIVQQGERFELRIEGLWYRTILWEVPLMALISEIWYRMGDGKRDADAIIDMRTRDKIEHYKRLGLKIAEFGTRRRYSYDVHDRVVASLRHHGGDTFSGSSNVHLAMRHGVKPIGTHAHEWFMFHGARFGFKMANSLALEHWVDVYRGDLGIALTDTFTSATFFDSFDKKFAKLFDGVRHDSGDPLEFAAATIAHYERMGIDPRTKTIIFSDALTPERVERIDAFCRGRITTAFGIGTNFTTNDVGVTPMNMVIKMTEARPEGQHWMPVIKLSDVPDKNTGDPDMIELAKRVLALSRRA24 / 33.33% / 24.08% / 23.64%26MAYAYWKSGKMEDKAVFDLFFRKNPFKGEFAIFAGLSECLKFLSNFRFSDSDSDINYLKQILPHVDPQFWEYLSGLDASQVTMSAIPEGYVVFPKVPLIRLEGPLIRLEGPLPIQLLETTLNLVNYASLVATNAARFRITAGESVQLLEFGLRRAQGPDGGLSASKYCYIGGFDGTSNVLAGKLFGIPVRGTHAHAFVNSFSSLDEIKGNVELTKSYLNKISPILNVLENETNEGELTAFLSYAFAFPTSCLCLLDTYSVIKSGLPNFLAVALALNDYGYRVIGVRLDSGDLAYLSRKCREGFNLIADKFGLPWIKQLIIVASNDINEDTLRSLKEQLVEVNGEPRMKLSEDVEKINLPGKKNVFRLYSEDGLAILDLIQLCTEKPPAVGKKILVRHPFNESKRAYVCPHKVELLYTDYWGDNCLMQSFSTLQETKQIASDSLKTLRQDIKRELNPTPYKVSVTNEMYHFLHDLWLKHAPIGELS27 / 23. 28%27MAYAYWKSGKMEDKAVFDLFFRKNPFKGEFAIFAGLSECLKFLSNFRFSDSDINYLKQILPHVDPQFWEYLSGLDASQVTMSAIPEGYVVFPKVPLIRLEGPLIRLEGPLPIQLLETTLNLVNYASLVATNAARFRITAGESVQLLEFGLRRAQGPDGGLSASKYCYIGGFDGTSNVLAGKLFGIPVRGTHAHAFVNSFSSLDEIKGNVELTKSYLNKISPILNVLENETNEGELTAFLSYAFAFFPTSCLLLDTYSVIKSGLPNFLAVALALNDYGYRVIGVRLDSGDLAYLSRKCREGFNLIADKFGLPWIKQLIIVASNDINEDTLRSLKEQLVEVNGEPRMKLSEDVEKINLPGKKNVFRLYSEDGLAILDLIQLCTEKPPAVGKKILVRHPFNESKRAYVCPHKVELLYTDYWGDNCLMQSFSTLQETKQIASDSLKTLRQDIKRELNPTPYKVSVTNEMYHFLHDLWLKHAPIGELS28 / 23.23% / 23.11% / 22.73%30MNDRELGCAGGQFMDRGRMNQNGVVQPLLTDLYQITMAYAYWKSDKTDDTAVFDLFFRNNPFHGEFTIFAGLEECLKFLDSFHYSQSDIEYLKQTLPEGIEHEFFEYLGNLTARDVTLYAIDEGTVAFPRVPIIKIEGPLIIVQLLETTLLTLVNYASLMATNAARYRMVAGKHVKLLEFGLRRAQGPDGGLSASKYSYTGGFDGTSNVLAGKLFNIPVKGTHAHAYITSFSSIGELKTRLIKHKQTGILEDLLEHAVRHRALLSHLLDVSTEESSEGELAAMVSYAIAFPDGFMALVDTYDVKRSGLLNFSAVALALNDLGYHALGIRIDSGDLAYLSCLARETFEKVAERFKVPWFNKLTIVASNDINEDTILSLNEQGHKIDCFGIGTHLVTCQRQPALGCVYKLVEINGQPRIKLSQDVEKVTMPGNKNAYRLYSADGHALIDLLQKVSEPPPAVGQKVLCRHPFQESKRAYVIPSHVESLYKVYWKSGKICQQLPTLEQVREKVQISLKTLRNDHKRTLNPTPYKVAVSDNLYNFIHDLWLQNAPIGELS31 / 22.35%31MLEYGFKDDSLSLHTDLYQINMAETYWRDGIHEKKAIFELFFRRLPFENGYAVFAGLEKAIEYLENFKFTDSDLSYLQDELGYHEDFIEYLRGLSFTGSLYSMKEGELVFNNEPIMRVEAPLVEAQLIETALLNIVNYQTLIATKAARIKGVIGDEVALEFGTRRAHEMDAAMWGARAALIGGFSATSNVRAGKRFNIPVSGTHAHALVQAYRDEYTAFKKYAETHKDCVFLVDTYDTLRSGMPNAIRVAKEFGDRINFIGIRLDSGDLAYLSKKARKMLDEAGFTDAKVIASSDLDEHTIMNLKAQGARIDVWGVGTKLITAYDQPALGAVYKLVAIEEDGKMVDTIKISSNPEKVTTPGRKKVYRIINQSNHHSEGDYIALYDEQVNDQKRLRMFHPVHTFISKFVTNFYAKDLHELIFEKGILCYQNPEISDIQQYVQDNLSLLWEEYKRISKPEEYPVDLSEDCWSNKMQRIHEVKSRIEEELEE32 / 22.12% / 20.71%33MAIRQHVGALFTDLYEVTMAQAYWAERMSGTAVFEIFFRKLPPGRSYIMAAGLADVVEFLEAFRFDEQDLRYLRGLGQFSDEFLRWLAGVRFTGDVWAAPEGTVIFPNEPAVQLIAPIIEAQLVETFVLNQIHLQSVLASKAARVVAAARGRPVVDFGARRAHGTDAACKVARTSYLAGAAGTSNLLAARQYGIPTFGTMAHSFVQAFDSEVAAFEAFARLYPATMLLVDTYDTLRGVDHVIELAKRLGNRFDVRAVRLDSGDLDELSKATRARLDTAGLEQVEIFASSGLDENRIAALLAARCPIDGFGVGTQLVVAQDAPALDMAYKLVAYDGSGRTKFSSGKVIYPGRKQVFRKLEHGVFCGDTLGEHGENLPGDPLLVPIMTNGRRIRQHAPTLDGARDWARQQIDALPPELRSLEDTGYSYPVAVSDRIVGELARLRHADTAEAHPGSNVVGAKAK RP34 / 19.91%34MGPPPAARRREGEPDNQDPAGLLTDKYELTMLAAALRDGSANRPTTFEVFARRLPTGRRYGVVAGTGRLLEALPQFRFDADACELLAQFLDPATVRYLREFRFRGDIDGYAEGELYFPGSPVLSVRGSFAECVLLETLVLSIFNHDTAIASAAARMVSAAGGRPLIEMGSRRTHERAAVAAARAAYIAGFAASSNLAAQRRYGVPAHGTAAHAFTMLHAQ HGGPTELAERAAFRAQVEALGPGTTLLVDTYDVTTGVANAVAAAGAELGAIRIDSGELGVLARQAREQLDRLGATRRIVVSGDLDEFSIAALRGEPVDSYGVGTSLVTGSGAPTANMVYKLVEVDGVPVQKRSSYKESPGGRKEALRRSRATGTITEELVHPAGRPPVIVEPHRVLTLPLVRAGQPVADTSLAAARQLVASGLRSPDGLKLAPGEPAIPTRTIPA35 / 19.79%*Underline: Motif 212-234*Sequence homology: Based on the amino acid sequence of Saccharomyces cerevisiae.

[0025] According to one specific example of the present invention, the nicotinate phosphoribosyltransferase (NPT) may be encoded by the base sequence of SEQ ID NO: 1 and may be composed of at least one of the amino acid sequences of SEQ ID NOs: 2 to 35.

[0026] The base sequence or amino acid sequence of nicotinate phosphoribosyltransferase (NPT) according to the present invention may be composed of or essentially include a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity compared to the base sequence of SEQ ID NO: 1 or any amino acid sequence of SEQ ID NOs: 2 to 35, and may have an original function. Here, “homology” or “identity” means the rate of identity (%) between a reference base sequence or amino acid sequence and any other base sequence or amino acid sequence when they are aligned and analyzed to correspond as much as possible.

[0027] As used herein, “enhanced activity” or “enhanced activity” means that the activity of a target polypeptide or protein is increased compared to its intrinsic activity. This enhanced activity includes cases where the activity of the polypeptide itself is increased compared to the activity of the polypeptide of the original microorganism, i.e., the wild type or pre-transformed microorganism, due to a nucleotide modification of the gene encoding the polypeptide; cases where the copy number of the gene encoding the polypeptide is increased; cases where the overall activity level (blood level) of the polypeptide is higher than that of the original microorganism due to increased expression or increased translation of the target gene due to a modification of the regulatory region of the gene encoding the polypeptide; cases where a foreign gene encoding the polypeptide is introduced; and combinations thereof.

[0028] Here, the nucleotide modification means a difference from the original polynucleotide sequence due to deletion, substitution, addition, or a combination thereof of all or part of the polynucleotide sequence of the gene encoding the polypeptide. The regulatory region modification means a difference from the original polynucleotide sequence due to deletion, substitution, addition, or a combination thereof of all or part of the polynucleotide sequence of an element constituting the regulatory region (regulatory sequence), such as a promoter, enhancer, or transcription terminator, and for example, it may be replaced with a strong promoter to increase the expression of the gene. Here, deletion means a change in which a base, nucleotide, polynucleotide, or nucleic acid is removed, substitution means a change in which a base, nucleotide, polynucleotide, or nucleic acid is replaced with another base, nucleotide, polynucleotide, or nucleic acid, and addition means a change in which another base, nucleotide, polynucleotide, or nucleic acid is added.

[0029] The increase in the above copy number means that one or more copies of a polynucleotide sequence of a gene encoding a polypeptide are introduced into the original microbial chromosome. The introduction of the foreign gene means that a polynucleotide sequence of a foreign gene encoding a polypeptide having the same or similar activity as the polypeptide is introduced, and the foreign gene may encode a polypeptide that may or may not exist in the original microorganism, and the introduction of the foreign gene may cause the polypeptide to be expressed and its activity to increase.

[0030] According to one specific example of the present invention, the activity enhancement of nicotinate phosphoribosyltransferase (NPT) may be achieved by nucleotide modification of a gene encoding nicotinate phosphoribosyltransferase, increasing the copy number of the gene, modifying the regulatory region of the gene, introducing a foreign gene, or a combination thereof.

[0031] According to one specific example of the present invention, the nicotinate phosphoribosyltransferase (NPT) or the gene encoding it may be derived from yeast, and for example, may be nicotinate phosphoribosyltransferase (NPT) endogenous to Saccharomyces cerevisiae, but is not limited thereto.

[0032] The mutant microorganism according to the present invention may have further weakened activity of nicotinamidase.

[0033] “Nicotinamidase” used in the present invention is an enzyme that converts nicotinamide (NAM) into nicotinic acid (NA), and by controlling the activity of nicotinamidase, production of nicotinamide mononucleotide (NMN) from nicotinamide (NAM) can be induced.

[0034] The nicotinamidase in the present invention may be a polypeptide having nicotinamidase activity encoded by the PNC1 gene, but is not limited thereto.

[0035] As used herein, “weakening of activity” or “weakening of activity” means that the activity of a target polypeptide or protein is reduced or absent compared to its intrinsic activity, and may be used interchangeably with terms such as inactivation, deficiency, reduction, and deterioration. Such weakening of activity may include, but is not limited to, cases where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism, i.e., the wild type or pre-modified microorganism, due to a nucleotide modification of the gene encoding the polypeptide; cases where the overall activity level (expression level) of the polypeptide is lower than that of the original microorganism due to inhibition of expression or translation of the target gene, etc., due to a modification of the regulatory region of the gene encoding the polypeptide; cases where the activity of the polypeptide is absent even if the gene encoding the polypeptide is expressed; and combinations thereof.

[0036] Here, the nucleotide modification means a difference from the original polynucleotide sequence due to deletion, substitution, addition, or a combination thereof of all or part of the polynucleotide sequence of the gene encoding the polypeptide. The regulatory region modification means a difference from the original polynucleotide sequence due to deletion, substitution, addition, or a combination thereof of all or part of the polynucleotide sequence of an element constituting the regulatory region, such as a promoter or enhancer, and for example, it may be replaced with a weak promoter so that the expression of the gene is reduced or suppressed.

[0037] According to one specific example of the present invention, the weakening of the activity of the nicotinamidase may be due to a nucleotide modification, a regulatory region modification, or a combination thereof of the gene encoding the nicotinamidase.

[0038] According to one specific example of the present invention, the nicotinamidase or the gene encoding it may be derived from yeast, and for example, may be a nicotinamidase endogenous to Saccharomyces cerevisiae, but is not limited thereto.

[0039] According to one specific example of the present invention, the nicotinamidase may be composed of an amino acid sequence of SEQ ID NO: 48, and the gene encoding the nicotinamidase may include a base sequence of SEQ ID NO: 49.

[0040] The amino acid sequence or base sequence of the nicotinamidase according to the present invention may be composed of or essentially include a sequence having 70% or more, 80% or more, 90% or more, 98% or more, 99% or more, or 99.9% or more homology with the amino acid sequence of SEQ ID NO: 48 or the base sequence of SEQ ID NO: 49, and must be able to maintain its original function.

[0041] Additionally, the mutant microorganism of the present invention may have further weakened activity of nicotinamide mononucleotide adenylyltransferase.

[0042] “Nicotinamide mononucleotide adenylyltransferase (NMNAT)” used in the present invention uses nicotinamide mononucleotide (NMN) as a substrate to form NAD + As an enzyme that produces NMN, the production of NMN can be increased by controlling the activity of nicotinamide mononucleotide adenylyltransferase (NMNAT).

[0043] Nicotinamide mononucleotide adenylyltransferase (NMNAT) in the present invention may be a polypeptide having nicotinamide mononucleotide adenylyltransferase activity encoded by the NMA1, NMA2, or POF1 gene, but is not limited thereto.

[0044] According to one specific example of the present invention, the weakening of the activity of nicotinamide mononucleotide adenylyltransferase (NMNAT) may be due to a nucleotide modification, a regulatory region modification, or a combination thereof of a gene encoding nicotinamide mononucleotide adenylyltransferase.

[0045] According to one specific example of the present invention, the nicotinamide mononucleotide adenylyltransferase (NMNAT) or the gene encoding it may be derived from yeast, and for example, may be nicotinamide mononucleotide adenylyltransferase (NMNAT) endogenous to Saccharomyces cerevisiae, but is not limited thereto.

[0046] According to one specific example of the present invention, the nicotinamide mononucleotide adenylyltransferase (NMNAT) may be encoded by the NMA1 gene (SEQ ID NO: 43) and composed of the amino acid sequence of SEQ ID NO: 42, encoded by the NMA2 gene (SEQ ID NO: 45) and composed of the amino acid sequence of SEQ ID NO: 44, and encoded by the POF1 gene (SEQ ID NO: 47) and composed of the amino acid sequence of SEQ ID NO: 46.

[0047] The amino acid sequence or base sequence of nicotinamide mononucleotide adenylyltransferase (NMNAT) according to the present invention may be composed of or essentially include a sequence having 70% or more, 80% or more, 90% or more, 98% or more, 99% or more, or 99.9% or more homology with the amino acid sequence of SEQ ID NO: 42, 44, or 46 or the base sequence of SEQ ID NO: 43, 45, or 47, and must be able to maintain its original function.

[0048] Additionally, the mutant microorganism of the present invention may have further enhanced activity of nicotinamide phosphoribosyltransferase.

[0049] The “nicotinamide phosphoribosyltransferase (NAMPT)” used in the present invention is an enzyme that exists in some bacteria and mammals and converts nicotinamide (NAM) into nicotinamide mononucleotide (NMN). However, yeast (e.g., Saccharomyces cerevisiae)-derived nicotinamide phosphoribosyltransferase (NAMPT) has low substrate activity toward nicotinamide (NAM), and thus there is a limit to mass-producing nicotinamide mononucleotide (NMN) from nicotinamide (NAM) in yeast. In the present invention, the production yield of nicotinamide mononucleotide (NMN) can be increased by introducing nicotinamide phosphoribosyltransferase (NAMPT) derived from Chitinophaga pinensis and Haemophilus ducreyi, which are known to have high activity of nicotinamide phosphoribosyltransferase (NAMPT) from microorganisms other than yeast (Shoji, et al., Metabolic Engineering. 2021 May 65:167-177.; Sorci, L. et al., Journal of Biological Chemistry. 2010 Dec 10;285(50):39490-9.).

[0050] In the present invention, nicotinamide phosphoribosyltransferase (NAMPT) may be a polypeptide encoded by the NAMPT gene and having nicotinamide phosphoribosyltransferase activity, but is not limited thereto.

[0051] According to one specific example of the present invention, the activity of nicotinamide phosphoribosyltransferase (NAMPT) may be enhanced by introducing a gene encoding nicotinamide phosphoribosyltransferase (NAMPT) derived from Chitinophaga pinensis or Haemophilus ducreyi.

[0052] According to one specific example of the present invention, the nicotinamide phosphoribosyltransferase (NAMPT) derived from Chitinophaga pinensis may be composed of an amino acid sequence of SEQ ID NO: 38, and a gene encoding the nicotinamide phosphoribosyltransferase (NAMPT) may include a base sequence of SEQ ID NO: 39.

[0053] According to one specific example of the present invention, the nicotinamide phosphoribosyltransferase (NAMPT) derived from Haemophilus ducreyi may be composed of an amino acid sequence of SEQ ID NO: 40, and a gene encoding the nicotinamide phosphoribosyltransferase (NAMPT) may include a base sequence of SEQ ID NO: 41.

[0054] The amino acid sequence or base sequence of nicotinamide phosphoribosyltransferase (NAMPT) according to the present invention may be composed of or essentially include a sequence having 70% or more, 80% or more, 90% or more, 98% or more, 99% or more, or 99.9% or more homology with the amino acid sequence of SEQ ID NO: 38 or 40 or the base sequence of SEQ ID NO: 39 or 41, and must be able to maintain its original function.

[0055] Additionally, the mutant microorganism of the present invention may have further enhanced activity of phosphoribosylpyrophosphate synthase.

[0056] “Phosphoribosylpyrophosphate synthase (PRS)” used in the present invention is an enzyme that converts ribose 5-phosphate (ribose-5P) into phosphoribosyl pyrophosphate (PRPP), and supplies phosphoribosyl pyrophosphate (PRPP) required for the NMN production pathway.

[0057] In the present invention, phosphoribosylpyrophosphate synthase (PRS) may be a polypeptide encoded by the PRS1 gene and having phosphoribosylpyrophosphate synthase activity, but is not limited thereto.

[0058] According to one specific example of the present invention, the enhancement of the activity of phosphoribosylpyrophosphate synthase (PRS) may be achieved by nucleotide modification of a gene encoding phosphoribosylpyrophosphate synthase, an increase in the copy number of the gene, modification of a regulatory region of the gene, introduction of a foreign gene, or a combination thereof.

[0059] According to one specific example of the present invention, the phosphoribosylpyrophosphate synthase (PRS) or the gene encoding it may be derived from yeast, and for example, it may be phosphoribosylpyrophosphate synthase (PRS) endogenous to Saccharomyces cerevisiae, but is not limited thereto.

[0060] According to one specific example of the present invention, the phosphoribosylpyrophosphate synthase (PRS) may be composed of an amino acid sequence of SEQ ID NO: 36, and a gene encoding the phosphoribosylpyrophosphate synthase (PRS) may include a base sequence of SEQ ID NO: 37.

[0061] The amino acid sequence or base sequence of phosphoribosylpyrophosphate synthase (PRS) according to the present invention may be composed of or essentially include a sequence having 70% or more, 80% or more, 90% or more, 98% or more, 99% or more, or 99.9% or more homology with the amino acid sequence of SEQ ID NO: 36 or the base sequence of SEQ ID NO: 37, and must be able to maintain its original function.

[0062] As used herein, “improved nicotinamide mononucleotide production ability” means increased productivity of nicotinamide mononucleotide (NMN) compared to the strain before mutation (parent strain). The parent strain refers to a wild type or mutant microorganism that is the target of mutation, and includes a target that is directly subject to mutation or transformed with a recombinant vector, etc.

[0063] In the present invention, the parent strain refers to a microorganism before its characteristics are changed by genetic mutation due to natural or artificial factors, and may be, for example, a microorganism that does not / cannot produce NMN due to lack of an enzyme acting in the nicotinamide mononucleotide (NMN) production pathway, or an NMN-producing microorganism that expresses an enzyme involved in the NMN production pathway or a gene encoding the enzyme. For example, the parent strain may be a wild-type Saccharomyces genus or a Saccharomyces genus mutated therefrom.

[0064] The genus Saccharomyces may be known in the art, for example, Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces Saccharomyces unisporus,These may include, but are not limited to, Saccharomyces uvarum, Saccharomyces zonatus, etc.

[0065] According to one specific example of the present invention, the mutant microorganism may be Saccharomyces cerevisiae.

[0066] The mutant microorganism having improved nicotinamide mononucleotide (NMN) production ability according to the present invention has enhanced activity of nicotinate phosphoribosyltransferase (NPT), and when the activity of at least one enzyme among nicotinamidase and nicotinamide mononucleotide adenylyltransferase (NMNAT) is further weakened, when the activity of at least one enzyme among nicotinamide phosphoribosyltransferase (NAMPT) and phosphoribosylpyrophosphate synthase (PRS) is further strengthened, or when the activity of all of these is included, the nicotinamide mononucleotide (NMN) production ability can be improved.

[0067] Specifically, the mutant microorganism having improved nicotinamide mononucleotide (NMN) production ability exhibits increased nicotinamide mononucleotide (NMN) production ability compared to the parent strain, and in particular, the nicotinamide mononucleotide (NMN) production amount is increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the parent strain, or 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, It may be increased by 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, but is not limited thereto. For example, the mutant microorganism with enhanced expression of nicotinamide mononucleotide (NMN) may have an increased nicotinamide mononucleotide (NMN) production of 0.5 times or more, specifically 0.5 to 20 times (preferably 1 to 10 times) compared to the parent strain.

[0068] A composition comprising a mutant microorganism according to the present invention can be used as a composition for producing nicotinamide mononucleotide (NMN).

[0069]

[0070] A mutant microorganism according to one specific example of the present invention can be implemented through a recombinant vector including a gene encoding nicotinate phosphoribosyltransferase (NPT) for introducing the gene into a parent strain.

[0071] Additionally, the mutant microorganism can be implemented through a recombinant vector for deleting or removing a gene encoding one or more of nicotinamidase and nicotinamide mononucleotide adenylyltransferase (NMNAT) or introducing a gene encoding one or more of nicotinamide phosphoribosyltransferase (NAMPT) and phosphoribosylpyrophosphate synthase (PRS).

[0072] The term "vector" as used herein refers to any type of nucleic acid sequence carrier structure used as a means for delivering and expressing a target gene into a host cell. Unless otherwise specified, the vector may mean one that allows the carried nucleic acid sequence to be inserted into the host cell genome and expressed and / or to be expressed independently. Such a vector includes essential regulatory sequences operably linked to allow the gene insert to be expressed, and "operably linked" means that the target gene and its regulatory sequence are functionally linked to each other in a manner that enables gene expression, and a "regulatory sequence" includes a promoter sequence for performing transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation.

[0073] The vector used in the present invention is not particularly limited as long as it is replicable in a host cell, and any vector known in the art can be used. Examples of the vector include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, phage vectors or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc., and plasmid vectors include, but are not limited to, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series.

[0074] The above vector can typically be constructed as a cloning vector or an expression vector. The expression vector can be any vector commonly used in the art to express foreign genes or proteins in plants, animals, or microorganisms, and can be constructed using various methods known in the art.

[0075] The "recombinant vector" used in the present invention can be constructed using a prokaryotic or eukaryotic cell as a host, and can replicate independently of the host cell's genome or can be integrated into the genome itself. The host cell can replicate the vector, and can include an origin of replication, which is a specific base sequence where replication begins. For example, when the vector used is an expression vector and the host is a prokaryotic cell, it typically includes a strong promoter capable of driving transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. In the case of using a eukaryotic cell as a host, the replication origin that operates in the eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno replication origin, the AAV replication origin, and the BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, a tk promoter of HSV) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0076] The above recombinant vector may include a selection marker, which is used to select transformants (host cells) transformed with the vector. Since only cells expressing the selection marker can survive in a medium treated with the selection marker, selection of transformed cells is possible. Representative examples of the selection marker include, but are not limited to, ampicillin, kanamycin, streptomycin, and chloramphenicol.

[0077] A transformant can be created by inserting the above recombinant vector into a host cell, and the transformant can be obtained by introducing the recombinant vector into an appropriate host cell. Any host cell known in the art that can stably and continuously clone or express the expression vector can be used as the host cell.

[0078] When transforming prokaryotic cells to produce recombinant microorganisms, various Enterobacteriaceae species such as E. coli DH5α, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, E. coli XL1-Blue, Corynebacterium genus, Bacillus genus such as Bacillus subtilis, Bacillus thuringiensis, Salmonella typhimurium, Serratia marcescens, and Pseudomonas genus may be used as host cells, but are not limited thereto.

[0079] When transforming eukaryotic cells to produce recombinant microorganisms, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines, can be used as host cells, but are not limited thereto.

[0080] “Transformation” as used in the present invention refers to a phenomenon in which a genetic change is artificially caused by introducing external DNA into a host cell, and “transformant” refers to a host cell into which external DNA is introduced and in which the expression of a target gene is stably maintained.

[0081] The above transformation can be performed by selecting an appropriate vector introduction technique depending on the host cell, so that the target gene or the recombinant vector containing it can be expressed within the host cell. For example, vector introduction can be performed by electroporation, heat shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. The transformed gene can be included without limitation, whether it is integrated into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed within the host cell.

[0082] The above transformant includes cells transfected, transformed, or infected with a recombinant vector according to the present invention in vivo or in vitro, and may be used as the same term as recombinant host cell, recombinant cell, or recombinant microorganism.

[0083] The transformant in the present invention may be other than a human.

[0084] Genes inserted into the recombinant vector for transformation of the present invention can be substituted into a host cell such as yeast through homologous recombination crossing over.

[0085] According to one specific example of the present invention, the host cell may be yeast, for example, the yeast may be of the genus Saccharomyces or Saccharomyces cerevisiae.

[0086]

[0087] In addition, another aspect of the present invention provides a method for producing nicotinamide mononucleotide (NMN), comprising the steps of culturing the mutant microorganism in a medium; and recovering nicotinamide mononucleotide (NMN) from the mutant microorganism or the medium in which the mutant microorganism is cultured.

[0088] The above culture can be performed using an appropriate medium and culture conditions known in the art, and those skilled in the art can easily adjust the medium and culture conditions for use. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0089] According to one specific embodiment of the present invention, the medium should meet the requirements of a specific strain in an appropriate manner and can be appropriately modified by those skilled in the art. Culture media for yeast can be found in known literature (e.g., Folia Microbiol. 38 (2), 141-146 (1993)), but are not limited thereto.

[0090] According to one embodiment of the present invention, the medium may include various carbon sources, nitrogen sources, and trace element components. Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto. Nitrogen sources that can be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture, but are not limited thereto. Sources of phosphorus that can be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or their corresponding sodium-containing salts. Additionally, the culture medium may contain, but is not limited to, metal salts required for growth, such as magnesium sulfate or iron sulfate. In addition, essential growth substances, such as amino acids and vitamins, may be included. Appropriate precursors may also be used in the culture medium. The medium or individual components may be added to the culture solution during the culturing process in a suitable manner, either batchwise or continuously, but are not limited thereto.

[0091] According to one specific example of the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be appropriately added to the microbial culture medium during cultivation to adjust the pH of the culture medium. In addition, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during cultivation. Additionally, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture medium to maintain an aerobic state of the culture medium. The temperature of the culture medium may typically be 20 to 45°C, for example, 25 to 40°C. The culture period may continue until a desired amount of useful substances is obtained, and may be, for example, 10 to 160 hours.

[0092] According to one specific example of the present invention, the step of recovering nicotinamide mononucleotide (NMN) from the cultured mutant microorganism or the medium in which the mutant microorganism is cultured may be performed by collecting or recovering the nicotinamide mononucleotide (NMN) produced from the medium using a suitable method known in the art depending on the culture method. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) may be used, but is not limited thereto.

[0093] According to one specific example of the present invention, the step of recovering nicotinamide mononucleotide (NMN) can be performed by removing biomass by low-speed centrifugation of the culture medium and separating the obtained supernatant through ion exchange chromatography.

[0094] According to one specific example of the present invention, the step of recovering nicotinamide mononucleotide (NMN) may include a process of purifying nicotinamide mononucleotide (NMN).

[0095] The mutant microorganism according to the present invention can produce nicotinamide mononucleotide at a high yield by enhancing, weakening or suppressing the expression of enzymes such as nicotinate phosphoribosyltransferase involved in the nicotinamide mononucleotide production pathway.

[0096] Figure 1 is a diagram showing the substances and enzymes involved in the typical NMN production pathway.

[0097] FIG. 2 is a diagram illustrating a process of introducing a NAMPT expression cassette including a foreign gene NAMPT while deleting the endogenous gene PNC1 in Saccharomyces cerevisiae according to one embodiment of the present invention.

[0098] FIG. 3 is a diagram illustrating a process of introducing a PDC promoter to overexpress the endogenous gene PRS1 in Saccharomyces cerevisiae according to one embodiment of the present invention.

[0099] FIG. 4 is a graph measuring the NMN production of foreign NAMPT-introduced and PNC1-deleted mutant strains (RE01-1 and RE01-2) and PRS1-overexpressing mutant strains (RE02-1 and RE02-2) according to one embodiment of the present invention.

[0100] FIG. 5 is a diagram illustrating a process of introducing a CCW12 promoter to overexpress the endogenous gene NPT1 in Saccharomyces cerevisiae according to one embodiment of the present invention.

[0101] Figure 6 is a graph measuring the NMN production of an NPT1 overexpressing mutant strain (RE03) according to one embodiment of the present invention.

[0102] FIG. 7 is a diagram illustrating a process of introducing the kanamycin antibiotic resistance gene KANMX while removing PNC1 from Saccharomyces cerevisiae according to one embodiment of the present invention.

[0103] FIG. 8 is a diagram illustrating a process for introducing an NPT1 expression cassette including NPT1 into Saccharomyces cerevisiae according to one embodiment of the present invention.

[0104] Figure 9 is a graph measuring the NMN production of an NPT1 promoter-enhanced mutant strain (YJ02) and an NPT12 copy expression mutant strain (YJ03) according to one embodiment of the present invention.

[0105] FIG. 10 is a diagram illustrating a process for introducing an NPT1 expression cassette including NPT1 while deleting PNC1 from Saccharomyces cerevisiae according to one embodiment of the present invention.

[0106] FIG. 11 is a graph measuring the NMN production of a PNC1 deletion mutant (DY2 pnc1::KanMX), an NPT12 copy expression mutant (DH01), and a PNC1 deletion and NPT12 copy expression mutant (D452-2 pnc1::NPT1) according to one embodiment of the present invention.

[0107] FIG. 12 is a diagram showing a process for removing the NMNAT-related gene POF1 from Saccharomyces cerevisiae according to one embodiment of the present invention.

[0108] FIG. 13 is a graph measuring the NMN production of an NPT1 overexpressing mutant strain (YJ02), an NPT12 copy expressing mutant strain (YJ03), and a POF1 deletion mutant strain (YJ02 POF1Δ) according to one embodiment of the present invention.

[0109] The present invention will be described in more detail below. However, this description is provided merely as an example to aid understanding of the present invention, and the scope of the present invention is not limited by this exemplary description.

[0110]

[0111] Example 1. Deletion of the endogenous gene PNC1 and introduction of the foreign gene NAMPT.

[0112] 1-1. Creation of PNC1 deletion and NAMPT introduction mutant strains

[0113] A mutant strain was created by deleting PNC1, an endogenous gene of Saccharomyces cerevisiae involved in the conversion of NAM to NA, and introducing NAMPT derived from Chitinophaga pinensis or Haemophilus ducreyi in that position (see Fig. 2).

[0114] The CRISPR / Cas9 system was used to generate mutant strains, and plasmids expressing each gRNA sequence were constructed by a polymerase chain reaction (PCR)-based fast cloning method. The gRNA sequences were designed using CHOPCHOP software (https: / / chopchop.cbu.uib.no / ), and the pRS42H-PNC1 plasmid was constructed by PCR amplification using primers Kim1703 and Kim1704. The PCR product was digested with Dpn1 restriction enzyme, transformed into E. coliTOP10 (Invitrogen), and selected on LBA (containing 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 100 μg / mL ampicillin) solid medium. The gRNA sequence of the generated plasmid was selected by confirming it by Sanger sequencing using a primer for the T3 promoter (5'-CAATTAACCCTCACTAAA-3', SEQ ID NO: 50).

[0115] To construct a NAMPT expression cassette, the base sequence of NAMPT from Chitinophaga pinensis or Haemophilus ducreyi was codon-optimized and prepared, and the CCW12 promoter with a high expression level was used. The NAMPT expression cassette was amplified by PCR using the primers Kim1716 and Kim1717 and Q5® High-Fidelity DNA Polymerase (initial denaturation: 98°C for 30 s, 30 cycles: 98°C for 5 s, 66°C for 10 s, and 72°C for 55 s, final extension: 72°C for 2 min) and introduced into the DY2 strain using the LiAc transformation method. The DY2 strain is a strain in which the xylose metabolism genes XYL1, XYL2, and XYL3 have been introduced into the background of Saccharomyces cerevisiae D452-2, and the PHO13 gene encoding phosphatase and the ALD6 gene encoding acetaldehyde dehydrogenase have been deleted (Jeong, Deokyeol, et al. PLOS ONE. 2020 Jul 27;15(7):e0236294.). The target strains were selected using the Kim549 and Kim742 primers and the colony PCR method confirmed that bands of 1.67 kb and 1.74 kb in size were produced, respectively. The strains into which Chitinophaga pinensis and Haemophilus ducreyi-derived NAMPT were introduced were named RE01-1 and RE01-2, respectively.

[0116] The genes PNC1 and NAMPT, CCW12 promoters, primers, and gRNA used are shown in Table 3 below.

[0117]

[0118]

[0119] Example 2. Overexpression of the endogenous gene PRS1

[0120] 2-1. Creation of mutant strains with PNC1 deletion, NAMPT introduction, and PRS1 overexpression

[0121] A mutant strain overexpressing the endogenous gene PRS1 of Saccharomyces cerevisiae was constructed using the PDC1 promoter with a high expression level (see Fig. 3).

[0122] The CRISPR / Cas9 system was used to create mutant strains, and plasmids expressing each gRNA sequence were constructed using a PCR-based fast cloning method. The gRNA sequences were designed using CHOPCHOP software (https: / chopchop.cbu.uib.no / ) and cloned into pRS42H-PRS1. p The plasmid was constructed by polymerase chain reaction (PCR) amplification using primers Kim1638 and Kim1639. The PCR product was digested with Dpn1 restriction enzyme, transformed into Escherichia coliTOP10 (Invitrogen), and selected on LBA (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 100 μg / mL ampicillin) solid medium. The gRNA sequence of the generated plasmid was confirmed by Sanger sequencing using a primer for the T3 promoter (5'-CAATTAACCCTCACTAAA-3', SEQ ID NO: 50) and selected.

[0123] The PDC1 promoter sequence (1.4 kb) to be inserted instead of the existing promoter of PRS1 was constructed by PCR using primers Kim1740 and Kim1737 and Q5® High-Fidelity DNA Polymerase (initial denaturation: 30 s at 98°C, 30 cycles: 5 s at 98°C, 10 s at 55°C, 30 s at 72°C, final extension: 2 min at 72°C). Plasmid pRS42H-PRS1 p And the PDC1 promoter fragment was introduced into RE01-1 and RE01-2 produced in Example 1 according to the LiAc transformation method. The target strains were selected by colony PCR using the primers Kim1746 and Kim1657, and a band of 1.4 kb in size was confirmed to appear, and these were named RE02-1 and RE02-2, respectively.

[0124] The PRS1, PDC1 promoters, primers, and gRNAs used are shown in Table 4 below.

[0125] 명칭서열 (5'-3')서열번호PRS1TGGTGCAAAATTGTGGTGCCAAAAAGGTTTATGTTGTTGCTACTCACGGTATATTCACTGGAGACTGTTTGGAAGAACTTGAAAAGTCCGATGCCATTGACACAATTGTGGTCACAAATACATATCCGATTTCTGGCGAACGCATAGCTGGGTCCAAGAAATTGGTTACTATCGATGTTTCTCCAATTTTCGCAGAATGTATCCGTCGTGATCATTATGGTGAGAGTATTTCCGTTTTATTTGACTCTTTGGCAGCTCTGTAA37PDC1 프로모터(Saccharomyces cerevisiaeS288C)CCTTTTTCTGTTAGACGGTGTCTTGATCTACTTGCTATCGTTCAACACCACCTTATTTTCTAACTATTTTTTTTTTAGCTCATTTGAATCAGCTTATGGTGATGGCACATTTTTGCATAAACCTAGCTGTCCTCGTTGAACATAGGAAAAAAAAATATATAAACAAGGCTCTTTCACTCTCCTTGCAATCAGATTTGGGTTTGTTCCCTTTATTTTCATATTTCTTGTCATATTCCTTTCTCAATTATTATTTTCTACTCATAACCTCACGCAAAATAACACAGTCAAATCAATCAAA59프라이머Kim1740CTTCAACTCTTTACTCGAAAAGTTGAAACAAGTGTCTATTGATTTCATCATACTCCATAC60Kim1737GTGAGAGTTACCAACAAAAATTTTACACTTACGCATTTTTTTTGATTGATTTGACTGTG61Kim1638GTTATTGCAGAGATGTGGTTTTAGAGCTAGAAATAGCAAG62Kim1639ACATCTCTGCAATAACCGAGATCATTTATCTTTCACTGCG63Kim1746TATGTTCCGCTGATGTGATGTG64Kim1657ACAGATCCTTAGAACGACGACG65gRNA;pRS42H-PRS1 P TCGGTTATTGCAGAGATGTGCGG66

[0126]

[0127] 2-2. Evaluation of NMN production ability of PRS1 overexpressing mutant strains

[0128] To evaluate the NMN production ability according to PRS1 overexpression, the NMN production of PRS1 overexpressing mutant strains RE02-1 and RE02-2 was compared with RE01-1 and RE01-2 of Example 1.

[0129] Each strain was inoculated into 10 ml of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) and pre-cultured at 30°C, 250 rpm, for 24 h. The pre-culture was then inoculated into fresh YPD medium at 0.5% (v / v) and the main culture was performed at 30°C, 250 rpm, for 11 h. After the main culture, 50 mg of appropriately grown cells were added to Y-PER TM After being treated with yeast protein extraction reagent (Thermo Scientific) and protease inhibitor, the product was disrupted and used to evaluate NMN production.

[0130] Cell lysates were reacted in a reaction buffer containing 20% ​​(v / v) crude extract per 0.6 mL of reaction mixture and 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM ATP, 40 mM NAM, and 5 mM PRPP. After 12 h of reaction, the mixture was filtered through a 0.45 μm filter, and the NMN production was analyzed using high-performance liquid chromatography (HPLC) (Agilent technologies 1260 infinity, Agilent technologies) equipped with a column (SupelcosilTM LC-18) and a UV detector (260 mm).

[0131] As a result, referring to Fig. 4, the overexpression of PRS1 significantly increased the amount of NMN production, and in particular, when Haemophilus ducreyi-derived NAMPT was introduced (RE02-2), the amount of NMN production increased compared to when Chitinophaga pinensis-derived NAMPT was introduced (RE02-1).

[0132]

[0133] Example 3. Overexpression of the endogenous gene NPT1

[0134] 3-1. Creation of mutant strains with PNC1 deletion, NAMPT introduction, and NPT1 overexpression

[0135] A mutant strain overexpressing NPT1, an endogenous gene of Saccharomyces cerevisiae that is expected to have high structural similarity to NAMPT, was constructed (see Fig. 5).

[0136] The CRISPR / Cas9 system was used to generate mutant strains, and plasmids expressing each gRNA sequence were constructed by a PCR-based fast cloning method. The gRNA sequences were designed using CHOPCHOP software (https: / / chopchop.cbu.uib.no / ), and the pRS42H-NPT1p plasmid was constructed by polymerase chain reaction (PCR) amplification using primers Kim1806 and Kim1807. The PCR product was digested with Dpn1 restriction enzyme, transformed into E. coliTOP10 (Invitrogen), and selected on LBA (containing 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 100 μg / mL ampicillin) solid medium. The gRNA sequence of the generated plasmid was selected by confirming it by Sanger sequencing using a primer for the T3 promoter (5'-CAATTAACCCTCACTAAA-3', SEQ ID NO: 50).

[0137] The CCW12 promoter sequence to be inserted instead of the existing promoter of NPT1 was amplified through PCR using the primers Kim1808 and Kim1809 and Q5® High-Fidelity DNA Polymerase (initial denaturation: 98°C for 30 sec, 30 cycles: 98°C for 5 sec, 59°C for 10 sec, 72°C for 20 sec, final extension: 72°C for 2 min) and introduced into RE02-1 produced in Example 2-1 according to the LiAc transformation method. The target strain was selected by colony PCR using the primers Kim549 and Kim1811, and a band of 0.65 kb in size was confirmed to appear, which was named RE03.

[0138] The NPT1, primers, and gRNA used are shown in Table 5 below.

[0139] Name order (5'-3') SEQ ID NO:NPT1CAGCTAAAGAGAATGGAATGCTAGCCACATTCGGTATTGGCACAAACTTTACTAATGATTTTCGTAAGAAGTCAGAACCCCAGGTTAAAAGTGAGCCGTTAAACATCGTTATCAAACTATTAGAAGTAAAT GGTAATCACGCTATCAAAATTTCTGATAACTTAGGTAAAAATATGGGAGATCCTGCCACTGTGAAGAGAGTGAAAGAGGAATTGGGATATACTGAACGAAGTTGGAGGTGGTGATAACGAAGCGCACAGATGGACCTAA1PrimerKim1 808CAGCAGTATCTTTTTAGACCTAATCTTGACAAAACCACCCATGAACCACAC67Kim1809CAAAAGAGACTTTATCACTGGTTCTGACATTATTGATATAGTGTTTAAGCGAATG68Kim1806TCCAGTGTAGTAGCCATGT TTTAGAGCTAGAAATAGCAAG69Kim1807TGGCTACTACACTGGAAGTGATCATTTATCTTTCACTGCG70Kim549GCATCCTTTGCCTCCGTTC71Kim1811CAAAGTTTCCGCCTTCTTCTCAG72gRNA;pRS42H-NPT1 p ACTTCCAGTGTAGTAGCCATCGG73

[0140]

[0141]

[0142] 3-2. Evaluation of NMN production ability of NPT1 overexpressing mutant strains

[0143] To evaluate the NMN production ability according to NPT1 overexpression, the NMN production of the NPT1 overexpressing mutant RE03 was compared with that of RE01-1 of Example 1 and RE02-1 of Example 2.

[0144] Each strain was inoculated into 10 ml of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) and pre-cultured at 30°C, 250 rpm, for 24 h. Afterwards, the pre-culture solution was inoculated into fresh YPD medium at 0.5% (v / v) and main culture was performed at 30°C, 250 rpm, for 11 h. After main culture, 50 mg of appropriately grown cells were used as Y-PER TM After being treated with yeast protein extraction reagent (Thermo Scientific) and protease inhibitor, the product was disrupted and used to evaluate NMN production.

[0145] Cell lysates were reacted in a reaction buffer containing 20% ​​(v / v) crude extract per 0.6 mL of reaction mixture and 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM ATP, 40 mM NAM, and 5 mM PRPP. After 12 h of reaction, the mixture was filtered through a 0.45 μm filter, and the NMN production was analyzed using high-performance liquid chromatography (HPLC) (Agilent technologies 1260 infinity, Agilent technologies) equipped with a column (SupelcosilTM LC-18) and a UV detector (260 mm).

[0146] As a result, referring to Fig. 6, the NPT1 overexpressing mutant RE03 showed the highest NMN production, and this result suggests that NAM, which is structurally similar to NA, can be converted into NMN by NPT1.

[0147]

[0148] Example 4. Overexpression of the endogenous gene NPT1

[0149] To determine whether NPT produces NMN using NAM as a substrate in the absence of NAMPT, the promoter of NPT1 endogenous to Saccharomyces cerevisiae was replaced with a strong promoter or the copy number was increased to overexpress NPT1.

[0150]

[0151] 4-1. Creation of NPT1 promoter-enhanced mutant strains

[0152] Before generating a mutant strain overexpressing NPT1, PNC1 was deleted using Saccharomyces cerevisiaeDY2 as the parent strain.

[0153] PNC1, an endogenous gene that converts NAM to NA, was deleted in such a way that the PNC1 gene was replaced by the kanamycin antibiotic resistance gene (KANMX) (see Figure 7). Genomic DNA of the PNC1 knockout strain was extracted from yeast deletion clones (Invitrogen) and used as a template. The specific kanamycin antibiotic resistance gene was amplified by PCR using the Kim1683 and Kim1686 primers and Q5® High-Fidelity DNA Polymerase. PCR was performed at 98°C for 30 s, 30 cycles (98°C for 5 s, 60°C for 10 s, and 72°C for 1 min 30 s), and 72°C for 2 min. The constructed kanamycin antibiotic resistance gene was introduced into the DY2 strain using the LiAc transformation method, and the target strain was selected by colony PCR using the primers Kim1685 and Kim284, and a band of 1.5 kb in size was confirmed to appear, which was named DY2pnc1::KanMX.

[0154] The primers used are shown in Table 6 below.

[0155] Name Sequence (5'-3') Sequence number Primer Kim1683TTTCCTCTTTCCCTACGATC74 Kim1686TTGTGAATGAGAAGTCGTAG75 Kim1685GGATGGAGCTGTTTATGGATGG76 Kim284AATTTAATCGCGGCCTCG77

[0156]

[0157] Next, a mutant strain was created that overexpresses PRS1 in the DY2pnc1::KanMX strain using the same method as Example 2-1 and overexpresses NPT1 through the CCW12 promoter using the same method as Example 3-1, and this was named YJ02.

[0158]

[0159] 4-2. Generation of NPT12 copy expression mutant strain

[0160] A mutant strain expressing 2 copies of NPT1 was constructed in Saccharomyces cerevisiae (see Fig. 8).

[0161] The pRS42H-int#4 plasmid was constructed using the same method as in Example 3-1.

[0162] The NPT1 expression cassette was amplified by PCR using the primers Kim1822 and Kim1823 and Q5® High-Fidelity DNA Polymerase using YJ02 of Example 4-1 as a template (initial transformation: 98°C for 30 sec, 30 cycles: 98°C for 5 sec, 62°C for 10 sec, 72°C for 55 sec, final extension: 72°C for 2 min). The plasmid pRS42H-int#4 and the NPT1 expression cassette were introduced into YJ02 constructed in Example 4-1 according to the LiAc transformation method. The target strain was selected by colony PCR using the primers Kim322 and Kim323, and a band of 2.5 kb in size was confirmed to appear, which was named YJ03.

[0163] The primers and gRNAs used are shown in Table 7 below.

[0164] Name order (5'-3') SEQ ID NO: Primer Kim1822CATTTCTTTTTCCTCGGGCAGAGAAACTCGCAGGCAACTTGCCACCCATGAACCACAC78Kim1823GTAACGGGATCCCTCTGTGAGGGCCGATTATGC AGGCCTAGATTGCCCTGCAAAAGCAG79Kim322GCGCATCTATTTGCCGTC80Kim323TCACGACACACCTCACTG81gRNA;pPRS42H-int#4CTCTCGAAGTGGTCACGTGCGGG82

[0165]

[0166] 4-3. Evaluation of NMN production ability of NPT1 overexpressing mutant strains

[0167] The NMN production amounts of YJ02 of Example 4-1 and YJ03 of Example 4-2 were compared.

[0168] Each strain was inoculated into 10 ml of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) and pre-cultured at 30°C, 250 rpm, for 24 h. Then, the pre-culture was inoculated into YPX medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L xylose) at 0.5% (v / v) and the main culture was performed at 30°C, 250 rpm, for 16 h. After the main culture, 50 mg of appropriately grown cells were added to Y-PER TM After being treated with yeast protein extraction reagent (Thermo Scientific) and protease inhibitor, the product was disrupted and used to evaluate NMN production.

[0169] Cell lysates were reacted in a reaction buffer containing 20% ​​(v / v) crude extract per 0.6 mL of reaction mixture and 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM ATP, 40 mM NAM, and 5 mM PRPP. After 3 h of reaction, the mixture was filtered through a 0.45 μm filter, and the NMN production was analyzed using high-performance liquid chromatography (HPLC) (Agilent technologies 1260 infinity, Agilent technologies) equipped with a column (SupelcosilTM LC-18) and a UV detector (260 mm).

[0170] As a result, referring to Fig. 9, it was shown that the YJ03 mutant strain increased NMN production compared to the YJ02 mutant strain due to an increase in the copy number by expressing 2 copies of NPT1.

[0171]

[0172] Example 5. Verification of essential mutations for high NMN production.

[0173] 5-1. Creation of PNC1 deletion and / or NPT1 overexpression mutant strains

[0174] As a PNC1 deletion mutant, DY2pnc1::KanMX produced in Example 4-1 was used.

[0175] An NPT1 overexpressing mutant was constructed using the same gRNA plasmid and NPT1 expression cassette as in Example 4-2. Plasmid pRS42H-int#4 and the NPT1 expression cassette were introduced into Saccharomyces cerevisiase D452-2 using the LiAc transformation method. The target strain was selected using the same method as in Example 4-2, and was designated DH01.

[0176] To create a mutant strain in which PNC1 is deleted and NPT1 is overexpressed, PNC1 was deleted and an NPT1 expression cassette was introduced at that location (see Fig. 10).

[0177] The same gNRA plasmid as in Example 1-1 was used.

[0178] The NPT1 expression cassette was amplified by PCR using RE03 of Example 3-1 as a template, Kim1936 and Kim1937 primers, and Q5® High-Fidelity DNA Polymerase (initial transformation: 98°C for 30 s, 30 cycles: 98°C for 5 s, 60°C for 10 s, 72°C for 65 s, final extension: 72°C for 2 min). The plasmid pRS42H-PNC1 and the NPT1 expression cassette were introduced into Saccharomyces cerevisiase D452-2 according to the LiAc transformation method. The target strain was selected by colony PCR using the primers Kim549 and kim1938, and a band of 1.79 kb in size was confirmed to appear, which was named D452-2pnc1::NPT1.

[0179] The primers and gRNAs used are shown in Table 8 below.

[0180] Name Sequence (5'-3') Sequence number Primer Kim1936TTGTTAGAAAGAATAAAATACAGTACAAAACCACCCATGAACCACAC83 Kim1937TCATCAGGTTGAAGAAGTATTATTCAGCTCTTGCCCTGCAAAAGCAG84 Kim549GCATCCTTTGCCTCCGTTC85 Kim1938GCAAGCCACCCTAGTTC86 gRNA;pRS42H-PNC1AGAAAAGCATCATACAGACGAGG87

[0181]

[0182] 5-2. Evaluation of NMN production ability of PNC1 deletion and / or NPT1 overexpression mutant strains

[0183] To evaluate the NMN production ability according to PNC1 deletion and / or NPT1 overexpression, the NMN production of the PNC1 deletion mutant of Example 4-1 (DY2pnc1::KanMX), the NPT1 overexpression mutant of Example 5-1 (DH01), and the PNC1 deletion and NPT1 overexpression mutant of Example 5-1 (D452-2pnc1::NPT1) was compared.

[0184] Each strain was inoculated into 10 ml of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) and pre-cultured at 30°C, 250 rpm, for 24 h. Afterwards, the pre-culture solution was inoculated into fresh YPD medium at 0.5% (v / v) and the main culture was performed at 30°C, 250 rpm, for 11 h. After the main culture, 50 mg of appropriately grown cells were added to Y-PER TM After being treated with yeast protein extraction reagent (Thermo Scientific) and protease inhibitor, the product was disrupted and used to evaluate NMN production.

[0185] Cell lysates were reacted in a reaction buffer containing 20% ​​(v / v) crude extract per 0.6 mL of reaction mixture and 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM ATP, 40 mM NAM, and 5 mM PRPP. After 3 h of reaction, the mixture was filtered through a 0.45 μm filter, and the NMN production was analyzed using high-performance liquid chromatography (HPLC) (Agilent technologies 1260 infinity, Agilent technologies) equipped with a column (SupelcosilTM LC-18) and a UV detector (260 mm).

[0186] As a result, referring to Figure 11, NMN was not produced in the mutant strain with PNC1 deletion alone (DY2pnc1::KanMX) and the mutant strain with NPT1 expression cassette introduced (DH01), whereas NMN was produced at high concentrations in the mutant strain with PNC1 deletion and NPT1 expression cassette introduced (D452-2pnc1::NPT1). These results suggest that PNC1 deletion and NPT1 overexpression are essential mutation factors for NMN production.

[0187]

[0188] Example 6. Deletion of the endogenous gene POF1

[0189] 6-1. Production of POF1 deletion mutant strain

[0190] NAD of NMN in Saccharomyces cerevisiae + To prevent conversion, a mutant strain was created in which POF1, one of the genes encoding NMNAT, was deleted (see Fig. 12).

[0191] The pRS42H-POF1 plasmid was constructed using the kim1766 and kim1767 primers in the same manner as in Example 4-1.

[0192] POF1 fragment was produced by PCR using Q5® High-Fidelity DNA Polymerase using Saccharomyces cerevisiae as a template and Kim1784 and Kim1785 primers, respectively (initial denaturation: 30 s at 98°C, 30 cycles: 5 s at 98°C, 10 s at 61°C, 10 s at 72°C, final extension: 2 min at 72°C).

[0193] The produced fragment was introduced into the YJ02 strain of Example 4-1 according to the LiAc transformation method. The target strain was selected by colony PCR using primers kim1798 and kim1799, and a band the size of POF1 (0.24 kb) was confirmed to appear, and this was named YJ02 POF1Δ.

[0194] The POF1 primers and gRNAs used are shown in Table 9 below.

[0195]

[0196]

[0197] 6-2. Evaluation of NMN production ability of POF1 deletion mutant strains

[0198] In order to evaluate the NMN production ability according to the presence or absence of POF1 removal, the NMN production of YJ02 of Example 4-1, YJ03 of Example 4-2, and POF1-deleted mutant YJ02 POF1Δ of Example 6-1 was compared.

[0199] Each strain was inoculated into 10 ml of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) and pre-cultured at 30°C, 250 rpm, for 24 h. Afterwards, the pre-culture solution was inoculated into fresh YPD medium at 0.5% (v / v) and the main culture was performed at 30°C, 250 rpm, for 11 h. After the main culture, 50 mg of appropriately grown cells were added to Y-PER TM After being treated with yeast protein extraction reagent (Thermo Scientific) and protease inhibitor, the product was disrupted and used to evaluate NMN production.

[0200] Cell lysates were reacted in a reaction buffer containing 20% ​​(v / v) crude extract per 0.6 mL of reaction mixture and 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM ATP, 40 mM NAM, and 5 mM PRPP. After 3 h of reaction, the mixture was filtered through a 0.45 μm filter, and the NMN production was analyzed using high-performance liquid chromatography (HPLC) (Agilent technologies 1260 infinity, Agilent technologies) equipped with a column (SupelcosilTM LC-18) and a UV detector (260 mm).

[0201] As a result, referring to Figure 13, in the mutant strain (YJ02 POF1Δ) in which POF1 was deleted, NMN production increased compared to the YJ02 strain in which POF1 was not deleted, and NMN production was similar to that of the YJ03 strain in which the number of NPT1 copies was increased. These results indicate that NAD of NMN + This suggests that it may contribute to increasing NMN production by inhibiting conversion.

[0202]

[0203] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. A mutant microorganism with enhanced nicotinamide mononucleotide production ability and enhanced activity of nicotinate phosphoribosyltransferase.

2. In claim 1, The above nicotinate phosphoribosyltransferases are selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Methanosarcina mazei, Methanosarcina acetivorans, Desulforapulum autotrophicum, Methanococcoides burtonii, Chromohalobacter salexigens, Arabidopsis thaliana, Dictyostelium discoideum, Ciona intestinalis, and Drosophila. Drosophila melanogaster, Ciona savignyi, Bacillus subtilis, Caenorhabditis elegans, Mycobacterium tuberculosis, Candida glabrata or Nakaseomyces glabratus, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica, Aspergillus fumigatus, Neurospora crassa, Chaetomium globosum, Emericella nidulans,A mutant microorganism derived from at least one selected from the group consisting of Aspergillus oryzae, Gibberella moniliformis, Cryptococcus neoformans and Ustilago maydis.

3. In claim 1, A mutant microorganism in which the activity of the nicotinate phosphoribosyltransferase is enhanced by a nucleotide modification of a gene encoding nicotinate phosphoribosyltransferase, an increase in the copy number of the gene, a modification of the regulatory region of the gene, introduction of a foreign gene, or a combination thereof.

4. In claim 1, The above mutant microorganism is a mutant microorganism in which the activity of nicotinamidase is further weakened.

5. In claim 1, The above mutant microorganism is a mutant microorganism in which the activity of nicotinamide mononucleotide adenylyltransferase is further weakened.

6. In claim 1, The above mutant microorganism is a mutant microorganism in which the activity of nicotinamide phosphoribosyltransferase is further enhanced.

7. In claim 1, The above mutant microorganism is a mutant microorganism in which the activity of phosphoribosylpyrophosphate synthase is further enhanced.

8. In claim 1, The above mutant microorganism is a mutant microorganism belonging to the genus Saccharomyces.

9. In claim 1, The above mutant microorganism is Saccharomyces cerevisiae.

10. A step of culturing the mutant microorganism of claim 1 in a medium; and A method for producing nicotinamide mononucleotide, comprising the step of recovering nicotinamide mononucleotide from the mutant microorganism or the medium in which the mutant microorganism is cultured.

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