Genetically engineered bacterium and application thereof, and production method of molecules of interest

Abstract

The invention provides a genetically engineered bacterium and application thereof, and a production method of interested molecules. The genetically engineered bacterium provided by the invention heterologously expresses a xylose metabolic pathway. According to the invention, xylose metabolic pathway expression integration is carried out in the genetically engineered bacteria, so that the genetically engineered bacteria can realize efficient production of interested molecules when xylose is used as a carbon source for fermentation, and the method has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a genetically engineered bacterium for heterologously expressing a xylose metabolic pathway and its use in producing a molecule of interest, a method for producing a molecule of interest using the genetically engineered bacterium, and a method for fermentation evaluation using the genetically engineered bacterium. Background Art

[0002] In recent years, the technology of synthesizing amino acids by cumbersome and complex chemical methods has been gradually phased out, and the production of amino acids by microbial fermentation has gradually become the current mainstream trend. It uses refined or renewable carbon sources as substrates and relies on its own metabolism to synthesize products. Microbial fermentation has the advantages of sufficient raw material sources, less environmental pollution, easy control of reaction conditions, and less equipment requirements.

[0003] Most existing fermentation industries use grains or agricultural products as raw materials to obtain carbon sources that can be directly utilized by microorganisms. However, this poses a certain threat to food security and production costs. Therefore, the development of lignocellulosic raw materials represented by corn stover, wheat straw, rice straw, bagasse, etc. as alternative carbon sources for the fermentation industry has gradually attracted attention.

[0004] Lignocellulose is mainly composed of hemicellulose, cellulose, and lignin. Among them, cellulose is a homopolymer composed of glucose; hemicellulose is a heteropolymer and can be converted into fermentable pentoses, including xylose, arabinose, galactose, mannose, etc. through chemical or biological pretreatment methods. In the hemicellulose hydrolysate, the content of xylose in pentoses reaches more than about 90% of the total fermentable sugars. Therefore, the comprehensive and efficient utilization of pentoses (especially xylose) is the key requirement for the effective utilization of lignocellulose hydrolysate.

[0005] However, due to the lack of a complete xylose metabolic pathway in some wild production strains, these wild production strains cannot completely utilize lignocellulose hydrolysate. Summary of the Invention

[0006] The purpose of the present invention is to provide a genetically engineered bacterium through metabolic engineering transformation, enabling it to efficiently produce target products by fully utilizing xylose.

[0007] The xylose metabolic pathway is mainly based on the xylose isomerization pathway. Xylose is isomerized into xylulose under the action of xylose isomerase (XI), and xylulose is converted into xylulose-5-phosphate through xylulokinase (XK). The inventors of the present invention found that by heterologously expressing the xylose metabolic pathway in a starting strain lacking a complete xylose metabolic pathway, it can achieve the utilization of xylose. Based on this, the present invention was completed.

[0008] Specifically, on one hand, the present invention provides a genetically engineered bacterium that heterologously expresses the xylose metabolic pathway.

[0009] In some preferred embodiments, the xylose metabolic pathway includes a xylose metabolism module.

[0010] In some preferred embodiments, the xylose metabolism module includes xylose isomerase, xylulokinase, and a promoter, wherein the promoter is used to regulate the expression of the xylose isomerase and the xylulokinase.

[0011] Xylose isomerase, xylulokinase, and promoters known in the art can be used. In some embodiments, the promoter includes a constitutive promoter and an inducible promoter; preferably, the constitutive promoter includes P eftu 、P gro 、P lacM and / or P sod ; the inducible promoter includes P trc and / or P tac .

[0012] In a specific embodiment, the xylose isomerase is encoded by the xylA gene and is derived from Escherichia coli BL21. The nucleotide sequence of the xylA gene is SEQ ID NO:1.

[0013] In a specific embodiment, the amino acid sequence of the xylose isomerase is SEQ ID NO:39.

[0014] In a specific embodiment, the xylulokinase is encoded by the xylB gene and is derived from Escherichia coli BL21. The nucleotide sequence of the xylB gene is SEQ ID NO:2.

[0015] In a specific embodiment, the amino acid sequence of the xylulokinase is SEQ ID NO:40.

[0016] In a specific embodiment, the promoter is P eftu , and its nucleotide sequence is SEQ ID NO:4.

[0017] In a specific embodiment, the promoter is P gro , and its nucleotide sequence is SEQ ID NO:5.

[0018] In a specific embodiment, the promoter is P lacM , and its nucleotide sequence is SEQ ID NO:6.

[0019] In a specific embodiment, the promoter is P sod, and its nucleotide sequence is SEQ ID NO:7.

[0020] In a specific embodiment, the promoter is P trc , and its nucleotide sequence is SEQ ID NO:8.

[0021] In a specific embodiment, the promoter is P tac , and its nucleotide sequence is SEQ ID NO:9.

[0022] In a specific embodiment, the nucleotide sequence of the xylose metabolism module is SEQ ID NO:3.

[0023] xylA gene sequence (SEQ ID NO:1):

[0024] atgcaagcctattttgaccagctcgatcgcgttcgttatgaaggctcaaaatcctcaaacccgttagcattccgtcactacaatcccgacgaact

[0025] ggtgttgggtaagcgtatggaagagcacttgcgttttgccgcctgctactggcacaccttctgctggaacggggcggatatgtttggtgtggg

[0026] ggcgtttaatcgtccgtggcagcagcctggtgaggcactggcgttggcgaagcgtaaagcagatgtcgcatttgagtttttccacaagttacat

[0027] gtgccattttattgcttccacgatgtggatgtttcccctgagggcgcgtcgttaaaagagtacatcaataattttgcgcaaatggttgatgtcctgg

[0028] caggcaagcaagaagagagcggcgtgaagctgctgtggggaaccgccaactgctttacaaaccctcgctacggcgcgggtgcggcgac

[0029] gaacccagatcctgaagtcttcagctgggcggcaacgcaagttgttacagcgatggaagcaacccataaattgggcggtgaaaactatgtc

[0030] ctgtggggcggtcgtgaaggttacgaaacgctgttaaataccgacttgcgtcaggagcgtgaacaactgggccgctttatgcagatggtggt

[0031] tgagcataaacataaaatcggtttccagggcacgttgcttatcgaaccgaaaccgcaagaaccgaccaaacatcaatatgattacgatgccg

[0032] cgacggtctatggcttcctgaaacagtttggtctggaaaaagagattaaactgaacattgaagctaaccacgcgacgctggcaggtcactcttt

[0033] ccatcatgaaatagccaccgccattgcgcttggcctgttcggttctgtcgacgccaaccgtggcgatgcgcaactgggctgggacaccgac

[0034] cagttcccgaacagtgtggaagagaatgcgctggtgatgtatgaaattctcaaagcaggcggtttcaccaccggtggtctgaacttcgatgcc

[0035] aaagtacgtcgtcaaagtactgataaatatgatctgttttacggtcatatcggcgcgatggatacgatggcactggcgctgaaaattgcagcgc

[0036] gcatgattgaagatggcgagctggataaacgcatcgcgcagcgttattccggctggaatagcgaattgggccagcaaatcctgaaaggcca

[0037] aatgtcactggcagatttagccaaatatgctcaggaacataatttgtctccggtgcatcagagtggtcgccaggagcaactggaaaatctggt

[0038] aaatcattatctgttcgacaaataa

[0039] Amino acid sequence of xylose isomerase (SEQ ID NO:39):

[0040] MQAYFDQLDRVRYEGSKSSNPLAFRHYNPDELVLGKRMEEHLRFAACYWHTFCWNGA

[0041] DMFGVGAFNRPWQQPGEALALAKRKADVAFEFFHKLHVPFYCFHDVDVSPEGASLKEY

[0042] INNFAQMVDVLAGKQEESGVKLLWGTANCFTNPRYGAGAATNPDPEVFSWAATQVVTA

[0043] MEATHKLGGENYVLWGGREGYETLLNTDLRQEREQLGRFMQMVVEHKHKIGFQGTLLI

[0044] EPKPQEPTKHQYDYDAATVYGFLKQFGLEKEIKLNIEANHATLAGHSFHHEIATAIALGLF

[0045] GSVDANRGDAQLGWDTDQFPNSVEENALVMYEILKAGGFTTGGLNFDAKVRRQSTDK

[0046] YDLFYGHIGAMDTMALALKIAARMIEDGELDKRIAQRYSGWNSELGQQILKGQMSLAD

[0047] LAKYAQEHNLSPVHQSGRQEQLENLVNHYLFDK

[0048] xylB gene sequence (SEQ ID NO:2):

[0049] atgtatatcgggatagatcttggcacctcgggcgtaaaagttattttgctcaacgagcagggtgaggtggttgcttcgcaaacggaaaagctg

[0050] accgtttcgcgcccgcatccactctggtcggaacaagacccggaacagtggtggcaggcaactgatcgcgcaatgaaagctctgggcgat

[0051] cagcattctctgcaggacgttaaagcattgggtattgccggccagatgcatggagcaaccttactggatgctcaacaacgggtattgcgccct

[0052] gccattttgtggaacgacgggcgctgtgcgcaagagtgcactttgctggaagcgagagttccgcaatcacgagtgattaccggcaacctgat

[0053] gatgcccggatttactgcgcctaaattgctatgggttcagcggcatgagccggagatattccgtcaaatcgacaaagtattattaccgaaagat

[0054] tacttgcgtctgcgtatgacgggggagtttgccagcgatatgtctgacgcagctggcaccatgtggctggatgtcgcaaagcgtgactggag

[0055] tgacgtcatgctgcaggcttgcgacttatctcgtgaccagatgcccgcattatacgaaggcagcgaaattactggtgctttgttacctgaagttg

[0056] cgaaagcgtggggtatggcgacggtgccagttgtcgcaggcggtggcgacaatgcagctggtgcagttggtgtgggaatggttgatgcta

[0057] atcaggcaatgttatcgctggggacgtcgggggtctattttgctgtcagcgaagggttcttaagcaagccagaaagcgccgtacatagcttttg

[0058] ccatgcgctaccgcaacgttggcatttaatgtctgtgatgctgagtgcagcgtcgtgtctggattgggccgcgaaattaaccggcctgagcaa

[0059] tgtcccagctttaatcgctgcagctcaacaggctgatgaaagtgccgagccagtttggtttctgccttatctttccggcgagcgtacgccacac

[0060] aataatccccaggcgaagggggttttctttggtttgactcatcaacatggccccaatgaactggcgcgagcagtgctggaaggcgtgggttat

[0061] gcgctggcagatggcatggatgtcgtgcatgcctgcggtattaaaccgcaaagtgttacgttgattgggggcggggcgcgtagtgagtactg

[0062] gcgtcagatgctggcggatatcagcggtcagcagctcgattaccgtacgggaggggatgtggggccagcactgggcgcagcaaggctg

[0063] gcgcagatcgcggcgaatccagagaaatcgctcattgaattgttgccgcaactaccgttagaacagtcgcatctaccagatgcgcagcgtta

[0064] tgccgcttatcagccacgacgagaaacgttccgtcgcctctatcagcaacttctgccattaatggcgtaa

[0065] Amino acid sequence of xylulokinase (SEQ ID NO:40):

[0066] MYIGIDLGTSGVKVILLNEQGEVVASQTEKLTVSRPHPLWSEQDPEQWWQATDRAMKAL

[0067] GDQHSLQDVKALGIAGQMHGATLLDAQQRVLRPAILWNDGRCAQECTLLEARVPQSRVI

[0068] TGNLMMPGFTAPKLLWVQRHEPEIFRQIDKVLLPKDYLRLRMTGEFASDMSDAAGTMW

[0069] LDVAKRDWSDVMLQACDLSRDQMPALYEGSEITGALLPEVAKAWGMATVPVVAGGGD

[0070] NAAGAVGVGMVDANQAMLSLGTSGVYFAVSEGFLSKPESAVHSFCHALPQRWHLMSV

[0071] MLSAASCLDWAAKLTGLSNVPALIAAAQQADESAEPVWFLPYLSGERTPHNNPQAKGVF

[0072] FGLTHQHGPNELARAVLEGVGYALADGMDVVHACGIKPQSVTLIGGGARSEYWRQMLA

[0073] DISGQQLDYRTGGDVGPALGAARLAQIAANPEKSLIELLPQLPLEQSHLPDAQRYAAYQP

[0074] RRETFRRLYQQLLPLMA

[0075] Promoter P eftu Sequence (SEQ ID NO:4):

[0076] agatcagtaggcgcgtagggtaagtggggtagcggcttgttagatatcttgaaatcggctttcaacagcattgatttcgatgtatttagctggcc

[0077] gttaccctgcgaatgtccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatgcgctag

[0078] atctgtgtgctcagtcttccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtccaggag

[0079] gacataca

[0080] Promoter P gro Sequence (SEQ ID NO:5):

[0081] agtttggctgccatgtgaatttttagcaccctcaacagttgagtgctggcactctcgggggtagagtgccaaataggttgtttgacacacagttg

[0082] ttcacccgcgacgacggctgtgctggaaacccacaaccggcacacacaaaatttttctcat

[0083] Promoter P lacM Sequence (SEQ ID NO:6):

[0084] tgagctgtttacaattaatcatcgtgtggtaccatgtgtggaattg

[0085] Promoter P sod Sequence (SEQ ID NO:7):

[0086] taaataggtcggctgaaaaatttcgttgcaatatcaacaaaaaggcctatcattgggaggg

[0087] Promoter P trc Sequence (SEQ ID NO:8):

[0088] ttgacaattaatcatccggctcgtataatg

[0089] Promoter P tac Sequence (SEQ ID NO:9):

[0090] ttgacaattaatcatcggctcgtataatg

[0091] Nucleotide sequence of the xylose metabolism module (P eftu -xylAB) (SEQ ID NO:3):

[0092] agatcagtaggcgcgtagggtaagtggggtagcggcttgttagatatcttgaaatcggctttcaacagcattgatttcgatgtatttagctggcc

[0093] gttaccctgcgaatgtccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatgcgctag

[0094] atctgtgtgctcagtcttccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtccaggag

[0095] gacatacaatgcaagcctattttgaccagctcgatcgcgttcgttatgaaggctcaaaatcctcaaacccgttagcattccgtcactacaatccc

[0096] gacgaactggtgttgggtaagcgtatggaagagcacttgcgttttgccgcctgctactggcacaccttctgctggaacggggcggatatgttt

[0097] ggtgtgggggcgtttaatcgtccgtggcagcagcctggtgaggcactggcgttggcgaagcgtaaagcagatgtcgcatttgagtttttccac

[0098] aagttacatgtgccattttattgcttccacgatgtggatgtttcccctgagggcgcgtcgttaaaagagtacatcaataattttgcgcaaatggttg

[0099] atgtcctggcaggcaagcaagaagagagcggcgtgaagctgctgtggggaaccgccaactgctttacaaaccctcgctacggcgcgggt

[0100] gcggcgacgaacccagatcctgaagtcttcagctgggcggcaacgcaagttgttacagcgatggaagcaacccataaattgggcggtgaa

[0101] aactatgtcctgtggggcggtcgtgaaggttacgaaacgctgttaaataccgacttgcgtcaggagcgtgaacaactgggccgctttatgca

[0102] gatggtggttgagcataaacataaaatcggtttccagggcacgttgcttatcgaaccgaaaccgcaagaaccgaccaaacatcaatatgatta

[0103] cgatgccgcgacggtctatggcttcctgaaacagtttggtctggaaaaagagattaaactgaacattgaagctaaccacgcgacgctggcag

[0104] gtcactctttccatcatgaaatagccaccgccattgcgcttggcctgttcggttctgtcgacgccaaccgtggcgatgcgcaactgggctggg

[0105] acaccgaccagttcccgaacagtgtggaagagaatgcgctggtgatgtatgaaattctcaaagcaggcggtttcaccaccggtggtctgaac

[0106] ttcgatgccaaagtacgtcgtcaaagtactgataaatatgatctgttttacggtcatatcggcgcgatggatacgatggcactggcgctgaaaat

[0107] tgcagcgcgcatgattgaagatggcgagctggataaacgcatcgcgcagcgttattccggctggaatagcgaattgggccagcaaatcctg

[0108] aaaggccaaatgtcactggcagatttagccaaatatgctcaggaacataatttgtctccggtgcatcagagtggtcgccaggagcaactgga

[0109] aaatctggtaaatcattatctgttcgacaaataacggctaactgtgcagtccgttggcccggttatcggtagcgataccgggcatttttttaagga

[0110] acgatcgatatgtatatcgggatagatcttggcacctcgggcgtaaaagttattttgctcaacgagcagggtgaggtggttgcttcgcaaacgg

[0111] aaaagctgaccgtttcgcgcccgcatccactctggtcggaacaagacccggaacagtggtggcaggcaactgatcgcgcaatgaaagctc

[0112] tgggcgatcagcattctctgcaggacgttaaagcattgggtattgccggccagatgcatggagcaaccttactggatgctcaacaacgggtat

[0113] tgcgccctgccattttgtggaacgacgggcgctgtgcgcaagagtgcactttgctggaagcgagagttccgcaatcacgagtgattaccggc

[0114] aacctgatgatgcccggatttactgcgcctaaattgctatgggttcagcggcatgagccggagatattccgtcaaatcgacaaagtattattac

[0115] cgaaagattacttgcgtctgcgtatgacgggggagtttgccagcgatatgtctgacgcagctggcaccatgtggctggatgtcgcaaagcgt

[0116] gactggagtgacgtcatgctgcaggcttgcgacttatctcgtgaccagatgcccgcattatacgaaggcagcgaaattactggtgctttgttac

[0117] ctgaagttgcgaaagcgtggggtatggcgacggtgccagttgtcgcaggcggtggcgacaatgcagctggtgcagttggtgtgggaatgg

[0118] ttgatgctaatcaggcaatgttatcgctggggacgtcgggggtctattttgctgtcagcgaagggttcttaagcaagccagaaagcgccgtac

[0119] atagcttttgccatgcgctaccgcaacgttggcatttaatgtctgtgatgctgagtgcagcgtcgtgtctggattgggccgcgaaattaaccgg

[0120] cctgagcaatgtcccagctttaatcgctgcagctcaacaggctgatgaaagtgccgagccagtttggtttctgccttatctttccggcgagcgta

[0121] cgccacacaataatccccaggcgaagggggttttctttggtttgactcatcaacatggccccaatgaactggcgcgagcagtgctggaaggc

[0122] gtgggttatgcgctggcagatggcatggatgtcgtgcatgcctgcggtattaaaccgcaaagtgttacgttgattgggggcggggcgcgtag

[0123] tgagtactggcgtcagatgctggcggatatcagcggtcagcagctcgattaccgtacgggaggggatgtggggccagcactgggcgcag

[0124] caaggctggcgcagatcgcggcgaatccagagaaatcgctcattgaattgttgccgcaactaccgttagaacagtcgcatctaccagatgcg

[0125] cagcgttatgccgcttatcagccacgacgagaaacgttccgtcgcctctatcagcaacttctgccattaatggcgtaa

[0126] The xylose metabolism module can be constructed using plasmid vectors known in the art. In a specific embodiment, the construction of the xylose metabolism module is carried out on the plasmid vector pEC-XK99E, wherein the xylose metabolism module is located at the multiple cloning site of the plasmid vector pEC-XK99E and is regulated by the P trc promoter carried by the plasmid vector pEC-XK99E itself.

[0127] In another specific embodiment, the construction of the xylose metabolism module is carried out on the plasmid vector pEC-XK99E(DP), wherein the plasmid vector pEC-XK99E(DP) is based on the plasmid vector pEC-XK99E as a template, and the operator gene lacI and its promoter sequence are deleted by whole plasmid PCR, and at the same time the P trc promoter sequence is deleted.

[0128] Xylose metabolism pathways (e.g., xylose metabolism modules) provided by the present invention with different copy numbers can be integrated into the genome of the starting strain. For the integration site, the condition that the deletion or absence of this site has no negative impact on the production and metabolism of the genetically engineered bacterium should be met. In some embodiments, the xylose metabolism pathway is integrated into one, two, three or more sites in the genome of the genetically engineered bacterium. In some specific embodiments, the xylose metabolism pathway is integrated into 1-5 sites in the genome of the genetically engineered bacterium.

[0129] In some preferred embodiments, when the genetically engineered bacterium is Corynebacterium glutamicum, the integration sites are selected from at least one of the cg1507-cg1524 gene locus, the cg1890 gene locus, and the cg1895 gene locus of Corynebacterium glutamicum.

[0130] The inventors of the present invention found that as the copy number of the xylose metabolism pathway (e.g., xylose metabolism module) in the genome of the starting strain increases, due to the excessive consumption of carbon sources by the strain or the energy consumption of other non-product synthesis pathways, the conversion rate of xylose to the target product no longer shows an obvious upward trend.

[0131] In addition, in order to further improve the consumption of xylose and the yield of the target product, the inventors further modified the genetically engineered bacterium. Specifically, in some embodiments, the genetically engineered bacterium also heterologously expresses the phosphoketolase pathway.

[0132] In some preferred embodiments, the method for heterologously expressing the phosphoketolase pathway is to integrate the phosphoketolase gene into one, two, three or more sites in the genome of the genetically engineered bacterium. In some specific embodiments, the phosphoketolase gene is integrated into 1 to 5 sites in the genome of the genetically engineered bacterium.

[0133] In some preferred embodiments, the deletion or absence of the site has no negative impact on the production and metabolism of the genetically engineered bacterium.

[0134] In a specific embodiment, the amino acid sequence of the phosphoketolase is SEQ ID NO: 10, and its number in Genbank (Gene ID) is 69713549; the phosphoketolase is encoded by the pket gene and is derived from Lactococcus lactis, and the nucleotide sequence of the pket gene is SEQ ID NO: 11.

[0135] Amino acid sequence of phosphoketolase (SEQ ID NO: 10):

[0136] MTEYNSEAYLKKLDKWWRAATYLGAGMIFLKENPLFSVTGTPIKAENLKANPIGHWGT

[0137] VSGQTFLYAHANRLINKYNQKMFYMGGPGHGGQAMVVPSYLDGSYTEAYPEITQDLEG

[0138] MSRLFKRFSFPGGIGSHMTAQTPGSLHEGGELGYVLSHATGAILDQPEQIAFAVVGDGEA

[0139] ETGPLMTSWHSIKFINPKNDGAILPILDLNGFKISNPTLFARTSDVDIRKFFEGLGYSPRYIE

[0140] NDDIHDYMAYHKLAAEVFDKAIEDIHQIQKDAREDNRYQNGEIPAWPIVIARLPKGWGG

[0141] PRYNDWSGPKFDGKGMPIEHSFRAHQVPLPLSSKNMGTLPEFVKWMTSYQPETLFNAD

[0142] GSLKEELRDFAPKGEMRMASNPVTNGGVDSSNLVLPDWQEFANPISENNRGKLLPDTND

[0143] NMDMNVLSKYFAEIVKLNPTRFRLFGPDETMSNRFWEMFKVTNRQWMQVIKNPNDEFI

[0144] SPEGRIIDSQLSEHQAEGWLEGYTLTGRTGVFASYESFLRVVDSMLTQHFKWIRQAADQK

[0145] WRHDYPSLNVISTSTVFQQDHNGYTHQDPGMLTHLAEKKSDFIRQYLPADGNTLLAVFD

[0146] RAFQDRSKINHIVASKQPRQQWFTKEEAEKLATDGIATIDWASTAKDGEAVDLVFASAGA

[0147] EPTIETLAALHLVNEVFPQAKFRYVNVLELGRLQKKKGALNQERELSDEEFEKYFGPSGT

[0148] PVIFGFHGYEDLIESIFYQRGHDGLIVHGYREDGDITTTYDMRVYSELDRFHQAIDAMQV

[0149] LYVNRKVNQGLAKAFIDRMERTLVKHFEVTRNEGVDIPEFTEWVWSDLKK

[0150] pket gene sequence (SEQ ID NO:11):

[0151] atgacagaatataattcagaagcttatttgaaaaagcttgataaatggtggcgagcagcaacttatcttggagcaggaatgatcttcttgaaaga

[0152] aaatcctttgttctctgtgacaggtactccaattaaagcggaaaaccttaaagccaatcctattgggcactgggggacggtttcaggacaaactt

[0153] tcctctatgctcatgctaatcgtctaatcaataaatataatcaaaagatgttttacatgggtggccccggacatggtggacaagctatggttgttcc

[0154] ttcttatcttgatggctcatatacagaagcttatccagagattactcaagatttggaaggcatgtcacgtttgtttaaacgtttctcatttcctggagg

[0155] aatagggtcacatatgacagcacaaacccctggttcacttcatgaaggaggtgagttgggttatgtgctatcacatgcaacaggggctattctt

[0156] gaccaacctgaacaaattgcttttgctgttgttggggatggagaagctgaaactggaccgttgatgacaagttggcactctattaaattcattaat

[0157] cctaagaatgacggggcaattttaccaattcttgatttaaatggttttaaaatttcaaatcctactttgttcgctcgaacttcagatgttgatattcgta

[0158] aattctttgaaggactgggttactcacctcgttatattgaaaatgatgatattcatgattacatggcttatcataaattagcagctgaagtttttgata

[0159] aagcgattgaagacattcatcaaattcagaaagatgcgcgtgaagataatcgttatcaaaatggagagattccagcttggccaattgttatcgc

[0160] acgtttaccaaaaggttggggtggtccacgttataatgattggtcaggtcctaaatttgacggtaagggaatgccgattgaacatagtttccgtg

[0161] cgcatcaagttccacttccgttatcttctaaaaatatgggaactttaccagaatttgtaaaatggatgacttcttaccaaccagaaactttatttaatg

[0162] ctgatggaagtttgaaagaagagttgcgtgattttgcaccaaaaggtgagatgcgaatggcttcaaaccctgtaacaaatgggggagttgatt

[0163] cttctaatttggttttaccagattggcaagaatttgcaaatccaatttctgaaaataatcgaggaaaattactccctgatacaaatgacaatatggat

[0164] atgaatgttttgtcaaaatattttgctgaaatagtcaaacttaatcctacgcgtttccgtttgtttggtcctgatgaaaccatgtctaatcgtttttggg

[0165] aaatgtttaaggtgacgaatcgtcaatggatgcaagtcataaaaaatccaaatgatgaatttatctcacctgagggtcgcattattgattctcaatt

[0166] atcagaacaccaggcagaaggttggcttgaaggttatactttaactggacgtacaggagtatttgcaagttatgaatcattcttgcgagtcgtgg

[0167] attcaatgttaactcaacatttcaaatggattcgtcaagcagcagaccaaaaatggcgccatgattatccttcgcttaatgttatttcgacttcaact

[0168] gttttccaacaagaccataacggttatactcaccaagatcctggaatgttgactcatttggcagaaaagaaatctgattttatcagacaatacctg

[0169] ccagctgatggaaatactttgctagccgtatttgaccgtgcttttcaagatagaagtaaaattaatcatattgtagcctctaaacaacctcgtcaac

[0170] aatggtttactaaagaagaagctgaaaaattggcgactgacggaattgcaacaattgattgggcttcaacggctaaagatggagaagcagta

[0171] gatttagtttttgcctcagcaggagctgagcctacaattgaaacactagcagctttacatcttgtaaacgaagttttcccacaggcgaaattccgt

[0172] tatgtgaatgtgcttgaactgggtcggttgcaaaagaaaaaaggcgctctaaaccaagaacgtgaactctcagatgaagaatttgaaaaatac

[0173] tttggcccttcaggcactccagtaatttttggattccatgggtatgaagatttaatcgaatccattttctatcaaagaggacacgatggtttgattgtt

[0174] catggttaccgtgaagatggtgacatcacgacgacttatgatatgcgggtttactctgagcttgaccgtttccaccaagcgattgatgccatgca

[0175] agttctttatgtcaaccgaaaagttaatcaaggtctagcgaaagctttcattgaccgaatggaacggacactagttaaacactttgaagtgacaa

[0176] gaaatgaaggagttgatattcctgagtttactgaatgggtttggtctgatttaaagaaatag

[0177] Acetate kinase catalyzes the formation of by-product acetate from acetyl phosphate, thereby causing the ineffective conversion of carbon sources and reducing the conversion rate of carbon sources to end products. Therefore, to avoid this catalytic reaction, in some preferred embodiments, the genetically engineered bacterium does not express acetate kinase, for example, by knocking out the ackA gene.

[0178] In some preferred embodiments, when the genetically engineered bacterium is Corynebacterium glutamicum, the site where the phosphoketolase gene is integrated into the genome of the genetically engineered bacterium is the ackA gene locus, and the Gene ID of the ackA gene in Genbank is 3344522.

[0179] In some preferred embodiments, on the basis of the genetically engineered bacterium expressing one or more copies (such as two, three or more copies) of the xylose metabolic pathway, the phosphoketolase pathway is also heterologously expressed.

[0180] For the genetically engineered bacterium of the present invention, the starting strain can be any production strain that can be fermented and cultured, as long as it lacks a complete xylose metabolic pathway or needs to strengthen the xylose metabolic pathway. Those skilled in the art can select the corresponding production strain that can produce the target product according to the target product. In some embodiments, the starting strain is Corynebacterium glutamicum, preferably Corynebacterium glutamicum CathS141 (which is deposited in the China Center for Type Culture Collection (CCTCC), deposit number CCTCC NO: M 20211495). This strain has good performance in tolerating the inhibitors produced by lignocellulose pretreatment and has good adaptability to the hydrolysate system obtained by hydrolyzing agricultural and forestry waste. Fermentation using the hydrolysate system obtained by hydrolyzing agricultural and forestry waste can achieve a relatively high L-lysine yield and productivity.

[0181] In another aspect, the present invention also provides the use of phosphoketolase in the fermentation production of a molecule of interest by Corynebacterium glutamicum, wherein the amino acid sequence of phosphoketolase is as shown in SEQ ID NO: 10. Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum CathS141.

[0182] In some embodiments, the molecule of interest is at least one of an amino acid, a protein, a terpene compound, and an aromatic alcohol.

[0183] In a specific embodiment, the molecule of interest is L-lysine.

[0184] In another aspect, the present invention also provides the use of a nucleic acid molecule encoding phosphoketolase (i.e., the pket gene) in the fermentation production of a molecule of interest by Corynebacterium glutamicum, wherein the sequence of the pket gene is as shown in SEQ ID NO: 11. Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum CathS141.

[0185] In some embodiments, the molecule of interest is at least one of an amino acid, a protein, a terpene compound, and an aromatic alcohol.

[0186] In a specific embodiment, the molecule of interest is L-lysine.

[0187] In another aspect, the present invention also provides the use of the genetically engineered bacterium in the production of a molecule of interest, where the molecule of interest is a molecule that can be produced by the genetically engineered bacterium through fermentation culture.

[0188] In some embodiments, the molecule of interest is at least one of an amino acid, a protein, a terpene compound, and an aromatic alcohol.

[0189] In some preferred embodiments, the genetically engineered bacterium is Corynebacterium glutamicum, and the molecule of interest is L-lysine.

[0190] In a specific embodiment, the genetically engineered bacterium is Corynebacterium glutamicum CathS141.

[0191] In another aspect, the present invention also provides a method for producing a molecule of interest, which includes:

[0192] Providing the genetically engineered bacterium;

[0193] Using a xylose-containing substance as a carbon source to perform fermentation culture on the genetically engineered bacterium to obtain a fermentation product, and the fermentation product contains the molecule of interest;

[0194] Optionally, separating, extracting, and / or purifying the molecule of interest from the fermentation product.

[0195] In some embodiments, the genetically engineered bacterium is Corynebacterium glutamicum, and the molecule of interest is L-lysine.

[0196] In some preferred embodiments, the genetically engineered bacterium is Corynebacterium glutamicum CathS141.

[0197] In some embodiments, the xylose-containing substance is a hydrolysate obtained by hydrolyzing agricultural and forestry waste (for example, lignocellulose hydrolysate), and the agricultural and forestry waste contains a large amount of lignocellulose. Since the content of xylose in the hydrolysate is relatively high, the xylose metabolic pathway can enable the genetically engineered bacterium to better utilize the xylose in the hydrolysate.

[0198] In some preferred embodiments, the agricultural and forestry waste is selected from at least one of straw, rice straw, rice husk, bagasse, wood, and wood chips; the straw includes corn straw, wheat straw, and cotton straw.

[0199] In some preferred embodiments, the hydrolysate obtained from the hydrolysis treatment of the agricultural and forestry waste is a straw hydrolysate. The straw hydrolysate can be formed through dry dilute acid pretreatment, biological detoxification treatment, and enzymatic hydrolysis. The specific treatment steps are as described in Chinese Patent Application No. 202111662200.X, the full text of which is incorporated herein by reference.

[0200] It can be understood that in the fermentation culture, in addition to using the xylose-containing substance as a carbon source, nitrogen sources, inorganic salts, and trace elements known in the art can also be added as components of the fermentation medium.

[0201] Compared with the prior art, the present invention has the following beneficial effects:

[0202] (1) The genetically engineered bacterium provided by the present invention integrates the expression of the xylose metabolic pathway, enabling the strain to utilize xylose and achieving efficient production of molecules of interest during fermentation using the lignocellulosic raw materials of agricultural and forestry waste as a carbon source, having good industrial application prospects;

[0203] (2) By further introducing the phosphoketolase pathway exogenously, the genetically engineered bacterium provided by the present invention can further improve the utilization rate of xylose and enhance the synthesis of molecules of interest;

[0204] (3) The present invention applies phosphoketolase and / or the nucleic acid molecule encoding phosphoketolase to the fermentation production of Corynebacterium glutamicum for the first time;

[0205] (4) The genetically engineered bacterium provided by the present invention has good application prospects in the biological fermentation production of agricultural waste. Especially when applied to fermenting straw hydrolysate as a substrate to produce L-lysine, it can completely consume xylose, not only reducing the difficulty in subsequent separation and extraction but also achieving the full utilization of lignocellulosic resources, which is beneficial to environmental protection while increasing the yield of L-lysine. Detailed Embodiments

[0206] The present invention will be further described below through specific examples. The examples described in the present invention are only for the purpose of illustrating the present invention and do not limit the scope of the present invention. Unless otherwise specified, the raw materials and materials used in the following examples are commercially available products, and the methods and conditions used are known methods and conventional conditions in the art.

[0207] Materials and Methods:

[0208] I. Strains and Vectors

[0209] Starting strain: Corynebacterium glutamicum CathS141 (taxonomic name: Corynebacterium glutamicum CathS141), which is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: M20211495. The name of the culture is CathS141, and the public can obtain it from Shanghai Kaisai Biotechnology Co., Ltd.;

[0210] Escherichia coli DH5α is used for the construction of engineering plasmids, and Escherichia coli BL21 is used to provide the xylose metabolism genes xylA and xylB from Escherichia coli.

[0211] II. Construction method of integration / knockout plasmid

[0212] Using the genome of Corynebacterium glutamicum CathS141 as a template, the upstream and downstream homologous arms of 500 - 1500 bp of the target site to be integrated / knocked out are amplified by PCR, and then the upstream homologous arm - integration fragment - downstream homologous arm or upstream homologous arm - downstream homologous arm is ligated to the pK18mobsacB plasmid by enzymatic digestion / homologous recombination ligation.

[0213] The specific method for constructing gene integration / gene knockout engineering strains is as follows: Culture Corynebacterium glutamicum CathS141 using EPO medium to prepare competent cells, and electrotransform the constructed integration plasmid or knockout plasmid into Corynebacterium glutamicum CathS141. Use a 2 mm electroporation cuvette, with an electric shock voltage of 2.8 KV and shock twice (or use a 1 mm electroporation cuvette, with an electric shock voltage of 1.8 KV and shock twice), and then spread it on an LBHIS plate containing kanamycin resistance. Perform PCR identification on the grown colonies to determine the completion of one homologous recombination. Culture the strain that has completed one recombination in an LBHIS medium containing kanamycin resistance for overnight culture, and then inoculate it into a medium containing 10% sucrose for subculture. After subculturing in this way twice, take the diluted bacterial solution and spread it on a plate containing 10% sucrose. Through sucrose marker screening, the grown colonies are verified by PCR again, and after gene sequencing, the correct strain is the successfully constructed strain.

[0214] III. Medium

[0215] EPO medium: 5 g / L glucose, 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 1 mL / L Tween - 80, pH 7.0, sterilize at 121 °C for 20 min, and add 4 g / L of sterile isoniazid and 25 g / L of glycine after sterilization.

[0216] LBHIS medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 18.5 g / L brain heart infusion, 91 g / L sorbitol, pH 7.0, sterilized at 121 °C for 20 min.

[0217] Seed medium: 20 g / L glucose, 10 g / L peptone, 0.02 g / L zinc sulfate heptahydrate, 1 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L potassium dihydrogen phosphate, 3 g / L urea, 0.5 g / L magnesium sulfate, 0.5 g / L corn steep liquor.

[0218] Fermentation medium: 10 g / L beef extract, 15 g / L corn steep liquor, 10 g / L Angel yeast powder, 15 g / L peptone, 5 g / L dipotassium hydrogen phosphate trihydrate, 2.5 g / L potassium dihydrogen phosphate, 5 g / L urea, 25 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, 200 g / L calcium carbonate, add 40 g / L xylose as carbon source or diluted straw hydrolysate as carbon source as needed.

[0219] IV. Lysine fermentation method: Take 400 - 500 μL of the experimental strain bacterial solution preserved in glycerol tube and spread it evenly on the LBHIS solid plate overnight for activation. Use an inoculation loop to scrape the colonies and inoculate them into a 500 mL conical flask containing 50 mL of seed medium, and culture at 30 °C and 220 rpm for 7 - 8 h. Inoculate into a 500 mL conical flask containing 25 mL of fermentation medium at an inoculation amount of 10% (v / v), and ferment and culture at 35 °C and 220 rpm for 96 h. Add kanamycin (10 μg / mL) as needed. Example 1: Construction of xylose metabolism module P eftu -xylAB on plasmid vector and evaluation of fermentation performance of engineered strain

[0220] Xylose metabolism module P eftu Construction of -xylAB: Using the genome of Corynebacterium glutamicum CathS141 as a template, the promoter P (its nucleotide sequence is SEQ ID NO: 4) was amplified by PCR using primers H1 and H2; using the genome of Escherichia coli BL21 as a template, the xylAB fragment (its nucleotide sequence is SEQ ID NO: 12) was amplified by PCR using primers H3 and H4; using P eftu and xylAB as templates, and using H1 and H4 as primers, the P eftu -xylAB fusion fragment (its nucleotide sequence is SEQ ID NO: 3) was obtained by overlap extension PCR, and double digestion was performed using restriction endonucleases Ecor I and Xba I to expose sticky ends for subsequent construction of engineering plasmids. eftu

[0221] ​Using plasmid vector pEC-XK99E as the backbone, double digestion was performed using restriction enzymes Ecor I and Xba I, and then it was ligated with the double-digested P eftu -xylAB fragment using T4 DNA ligase to obtain plasmid pEC-P trc -P eftu -xylAB.

[0222] Since the xylose metabolism module P eftu -xylAB contains the promoter P eftu , and the plasmid vector pEC-XK99E backbone also contains its own P trc inducible promoter, so in plasmid pEC-P trc -P eftu -xylAB, the xylAB gene is regulated by a dual promoter. However, since dual promoter regulation inhibits gene expression, plasmid vector pEC-P trc -P eftu -xylAB was used as a template, and inverse PCR amplification was performed using primers H5 and H6 to delete the operator gene lacI and its promoter sequence, and at the same time delete the P trc promoter sequence to obtain plasmid pEC-P eftu -xylAB.

[0223] The detailed information of the primers used above is shown in Table 1.

[0224] Table 1

[0225] Primer Name Sequence H1 ccggaattcagatcagtaggcgcgtagggtaag(SEQ ID NO:13) H2 tgtatgtcctcctggacttcgtggt(SEQ ID NO:14) H3 gaagtccaggaggacatacaatgcaagcctattttgaccagctcga(SEQ ID NO:15) H4 gctctagattacgccattaatggcagaagttgctg(SEQ ID NO:16) H5 aagcggcatgcatttacgtttttcacacaggaaacagaccatggaattca(SEQ ID NO:17) H6 aacgtaaatgcatgccgcttcg(SEQ ID NO:18)

[0226] Nucleotide sequence of the xylAB fragment (SEQ ID NO:12):

[0227] atgcaagcctattttgaccagctcgatcgcgttcgttatgaaggctcaaaatcctcaaacccgttagcattccgtcactacaatcccgacgaact

[0228] ggtgttgggtaagcgtatggaagagcacttgcgttttgccgcctgctactggcacaccttctgctggaacggggcggatatgtttggtgtggg

[0229] ggcgtttaatcgtccgtggcagcagcctggtgaggcactggcgttggcgaagcgtaaagcagatgtcgcatttgagtttttccacaagttacat

[0230] gtgccattttattgcttccacgatgtggatgtttcccctgagggcgcgtcgttaaaagagtacatcaataattttgcgcaaatggttgatgtcctgg

[0231] caggcaagcaagaagagagcggcgtgaagctgctgtggggaaccgccaactgctttacaaaccctcgctacggcgcgggtgcggcgac

[0232] gaacccagatcctgaagtcttcagctgggcggcaacgcaagttgttacagcgatggaagcaacccataaattgggcggtgaaaactatgtc

[0233] ctgtggggcggtcgtgaaggttacgaaacgctgttaaataccgacttgcgtcaggagcgtgaacaactgggccgctttatgcagatggtggt

[0234] tgagcataaacataaaatcggtttccagggcacgttgcttatcgaaccgaaaccgcaagaaccgaccaaacatcaatatgattacgatgccg

[0235] cgacggtctatggcttcctgaaacagtttggtctggaaaaagagattaaactgaacattgaagctaaccacgcgacgctggcaggtcactcttt

[0236] ccatcatgaaatagccaccgccattgcgcttggcctgttcggttctgtcgacgccaaccgtggcgatgcgcaactgggctgggacaccgac

[0237] cagttcccgaacagtgtggaagagaatgcgctggtgatgtatgaaattctcaaagcaggcggtttcaccaccggtggtctgaacttcgatgcc

[0238] aaagtacgtcgtcaaagtactgataaatatgatctgttttacggtcatatcggcgcgatggatacgatggcactggcgctgaaaattgcagcgc

[0239] gcatgattgaagatggcgagctggataaacgcatcgcgcagcgttattccggctggaatagcgaattgggccagcaaatcctgaaaggcca

[0240] aatgtcactggcagatttagccaaatatgctcaggaacataatttgtctccggtgcatcagagtggtcgccaggagcaactggaaaatctggt

[0241] aaatcattatctgttcgacaaataacggctaactgtgcagtccgttggcccggttatcggtagcgataccgggcatttttttaaggaacgatcga

[0242] tatgtatatcgggatagatcttggcacctcgggcgtaaaagttattttgctcaacgagcagggtgaggtggttgcttcgcaaacggaaaagctg

[0243] accgtttcgcgcccgcatccactctggtcggaacaagacccggaacagtggtggcaggcaactgatcgcgcaatgaaagctctgggcgat

[0244] cagcattctctgcaggacgttaaagcattgggtattgccggccagatgcatggagcaaccttactggatgctcaacaacgggtattgcgccct

[0245] gccattttgtggaacgacgggcgctgtgcgcaagagtgcactttgctggaagcgagagttccgcaatcacgagtgattaccggcaacctgat

[0246] gatgcccggatttactgcgcctaaattgctatgggttcagcggcatgagccggagatattccgtcaaatcgacaaagtattattaccgaaagat

[0247] tacttgcgtctgcgtatgacgggggagtttgccagcgatatgtctgacgcagctggcaccatgtggctggatgtcgcaaagcgtgactggag

[0248] tgacgtcatgctgcaggcttgcgacttatctcgtgaccagatgcccgcattatacgaaggcagcgaaattactggtgctttgttacctgaagttg

[0249] cgaaagcgtggggtatggcgacggtgccagttgtcgcaggcggtggcgacaatgcagctggtgcagttggtgtgggaatggttgatgcta

[0250] atcaggcaatgttatcgctggggacgtcgggggtctattttgctgtcagcgaagggttcttaagcaagccagaaagcgccgtacatagcttttg

[0251] ccatgcgctaccgcaacgttggcatttaatgtctgtgatgctgagtgcagcgtcgtgtctggattgggccgcgaaattaaccggcctgagcaa

[0252] tgtcccagctttaatcgctgcagctcaacaggctgatgaaagtgccgagccagtttggtttctgccttatctttccggcgagcgtacgccacac

[0253] aataatccccaggcgaagggggttttctttggtttgactcatcaacatggccccaatgaactggcgcgagcagtgctggaaggcgtgggttat

[0254] gcgctggcagatggcatggatgtcgtgcatgcctgcggtattaaaccgcaaagtgttacgttgattgggggcggggcgcgtagtgagtactg

[0255] gcgtcagatgctggcggatatcagcggtcagcagctcgattaccgtacgggaggggatgtggggccagcactgggcgcagcaaggctg

[0256] gcgcagatcgcggcgaatccagagaaatcgctcattgaattgttgccgcaactaccgttagaacagtcgcatctaccagatgcgcagcgtta

[0257] tgccgcttatcagccacgacgagaaacgttccgtcgcctctatcagcaacttctgccattaatggcgtaa

[0258] The plasmids pEC-XK99E, pEC-P trc -P eftu -xylAB, and pEC-P eftu -xylAB were transformed into the Corynebacterium glutamicum wild-type strain CathS141 by electroporation. Positive strains were screened by PCR verification and named CathS141-P, CathS141-4, and CathS141-5, respectively. Among them, CathS141-P is a control strain containing an empty plasmid.

[0259] The engineered strains CathS141-P, CathS141-4, and CathS141-5 and the wild-type strain CathS141 were evaluated and compared in a fermentation medium with 40 g / L xylose as the sole carbon source. The results showed that the wild-type strain CathS141 could not utilize xylose and could not produce lysine, and the control strain CathS141-P containing an empty plasmid also could not utilize xylose and could not produce lysine; while the strains CathS141-4 and CathS141-5 both showed the ability to utilize xylose to produce lysine, and the strain CathS141-5 showed better performance (see Table 2).

[0260] Table 2

[0261] Strain Xylose Consumption Rate (%) Lysine Yield (g / L) Lysine / Xylose Conversion Rate (%) CathS141 0 0 0 CathS141-P 0 0 0 CathS141-4 39.0 8.99 57.6 CathS141-5 43.3 10.76 62.1

[0262] Example 2: Xylose metabolic module P with different copy numbers eftuIntegration of -xylAB into the Genome of Corynebacterium glutamicum and Evaluation of the Fermentation Performance of Engineered Strains

[0263] 1. Construction of Gene Editing Plasmids

[0264] Based on the research in Example 1, on the plasmid vector, when xylAB is regulated by a single promoter P eftu it shows better xylose consumption ability and lysine production ability. However, the use of plasmid vectors inevitably increases the material cost of antibiotics on the one hand, and on the other hand, plasmid vectors have great instability during multiple fermentation passages. Therefore, in this example, xylose metabolism modules P with different copy numbers are integrated into the genome of the Corynebacterium glutamicum starting strain eftu -xylAB.

[0265] Integration of the xylose metabolism module P eftu -xylAB at the CGP3 locus of the Corynebacterium glutamicum CathS141 genome (the position of this locus in the genome is cg1507 - cg1524, with a size of approximately 187.3 kbp, and the deletion or absence of this locus fragment will not have a negative impact on the growth and metabolism of Corynebacterium glutamicum strains): Using the Corynebacterium glutamicum CathS141 genome as a template, fragment CGP3-UP was obtained by PCR amplification using primers H7 and H8, and fragment CGP3-DW was obtained by PCR amplification using primers H9 and H10; using the P eftu -xylAB fusion fragment as a template, the P eftu -xylAB fragment without restriction enzyme sites was amplified by PCR using primers H11 and H12; using fragment CGP3-UP, fragment CGP3-DW, and P eftu -xylAB as templates, the CGP3-UP-P eftu -xylAB-CGP3-DW fusion fragment was obtained by overlap extension PCR using primers H7 and H10; using the plasmid vector pK18mobsacB as a backbone, it was double-digested with restriction enzymes BamH I and Hind III, and then ligated with the CGP3-UP-P eftu -xylAB-CGP3-DW fragment using homologous recombinase to obtain plasmid pK18-CGP3-AB. The plasmid pK18-CGP3-AB was transformed into Corynebacterium glutamicum CathS141 competent cells by electroporation, and strains with correct homology were screened by PCR verification and named CathS141-6.

[0266] The xylose metabolism module P eftuIntegration of -xylAB at the cg1890 locus of the Corynebacterium glutamicum CathS141-6 genome (deletion or absence of the fragment at this locus will not have a negative impact on the growth and metabolism of Corynebacterium glutamicum strains): Using the Corynebacterium glutamicum CathS141 genome as a template, fragment 1890-UP was obtained by PCR amplification using primers H13 and H14, and fragment 1890-DW was obtained by PCR amplification using primers H15 and H16; using fragment 1890-UP, fragment 1890-DW, and P eftu -xylAB as a template, the 1890-UP-P eftu -xylAB-1890-DW fusion fragment was obtained by overlap extension PCR using primers H13 and H16; using the plasmid vector pK18mobsacB as a backbone, double digestion was performed using restriction enzymes BamH I and Hind III, and then it was ligated with the 1890-UP-P eftu -xylAB-1890-DW fragment using homologous recombinase to obtain plasmid pK18-1890-AB. The plasmid pK18-1890-AB was introduced into competent cells of Corynebacterium glutamicum CathS141-6 by electroporation, and the strains with correct homology were screened by PCR verification and named CathS141-7.

[0267] Xylose metabolism module P eftu Integration of -xylAB at the cg1895 locus of the Corynebacterium glutamicum CathS141-7 genome (deletion or absence of the fragment at this locus will not have a negative impact on the growth and metabolism of Corynebacterium glutamicum strains): Using the Corynebacterium glutamicum CathS141 genome as a template, fragment 1895-UP was obtained by PCR amplification using primers H17 and H18, and fragment 1895-DW was obtained by PCR amplification using primers H19 and H20; using fragment 1895-UP, fragment 1895-DW, and P eftu -xylAB as a template, the 1895-UP-P eftu -xylAB-1895-DW fusion fragment was obtained by overlap extension PCR using primers H17 and H20; using the plasmid vector pK18mobsacB as a backbone, double digestion was performed using restriction enzymes BamH I and Hind III, and then it was ligated with the 1895-UP-P eftuThe -xylAB-1895-DW fragment was ligated to obtain the plasmid pK18-1895-AB. The plasmid pK18-1895-AB was transformed into the competent cells of Corynebacterium glutamicum CathS141-7 by electroporation, and the strains with correct homology were screened by PCR verification and named CathS141-8.

[0268] The detailed information of the primers used above is shown in Table 3.

[0269] Table 3

[0270]

[0271]

[0272] The engineered strains CathS141-6, CathS141-7 and CathS141-8 and the starting strain CathS141 were evaluated and compared in a fermentation medium with 40 g / L xylose as the sole carbon source. The results showed that the starting strain CathS141 could not utilize xylose and could not produce lysine, while CathS141-6, CathS141-7 and CathS141-8 all showed the ability to utilize xylose to produce lysine. Among them, the strain CathS141-8 containing three copies of P eftu -xylAB showed the best performance (see Table 4).

[0273] Table 4

[0274] Strain Xylose Consumption Rate (%) Lysine Yield (g / L) Lysine / Xylose Conversion Rate (%) CathS141 0 0 0 CathS141-6 39.6 9.16 57.8 CathS141-7 51.4 13.39 65.1 CathS141-8 66.8 17.94 67.1

[0275] Example 3: Integration of the phosphoketolase gene pket at the ackA gene locus of the genome of strain CathS141-6 and evaluation of the fermentation performance of the engineered strain

[0276] Integration of phosphoketolase gene pket at the ackA gene locus in the genome of Corynebacterium glutamicum CathS141-6: Using the genome of Corynebacterium glutamicum CathS141 as a template, fragment ackA-UP was obtained by PCR amplification using primers H21 and H22, and fragment ackA-DW was obtained by PCR amplification using primers H23 and H24; Using the genome of Lactococcus lactis as a template, the pket fragment was amplified by PCR using primers H25 and H26; Using the ackA-UP fragment, ackA-DW fragment and pket fragment as templates, and using H21 and H24 as primers, the ackA-UP-pket-ackA-DW fusion fragment was obtained by overlap extension PCR; Using plasmid vector pK18mobsacB as a backbone, it was double digested with restriction enzymes BamH I and Hind III, and ligated with the ackA-UP-pket-ackA-DW fragment using homologous recombinase to obtain plasmid pK18-ackA-pket. The plasmid pK18-ackA-pket was transformed into competent cells of Corynebacterium glutamicum CathS141-6 by electroporation, and the strains with correct homology were screened by PCR verification and named CathS141-6-pket. The detailed information of the primers used above is shown in Table 5.

[0277] Table 5

[0278] Primer Name Primer Sequence H21 aattcgagctcggtacccggggatcctcgaaccagtcatgagcgccg(SEQ ID NO:33) H22 tctgaattatattctgtcattagctgcgtcctcctgcctgaa(SEQ ID NO:34) H23 ggtctgatttaaagaaatagctctcctggttaggatccaccacaaatc(SEQ ID NO:35) H24 gtaaaacgacggccagtgccaagcttccagcgacccacacgtagtcc(SEQ ID NO:36) H25 atgacagaatataattcagaagcttatttgaaaaagcttg(SEQ ID NO:37) H26 ctatttctttaaatcagaccaaacccattcagtaaac(SEQ ID NO:38)

[0279] The engineered strains CathS141-6 and CathS141-6-pket were evaluated and compared in a fermentation medium with 40 g / L xylose as the sole carbon source. The results showed that compared with the engineered strain CathS141-6, the xylose consumption ability of the engineered strain CathS141-6-pket that synchronously integrated and expressed the phosphoketolase pathway was further improved. Correspondingly, its lysine conversion rate was also further enhanced. It is speculated that the expression and integration of the PKET pathway strengthened the consumption of xylose and the synthesis of products by promoting the metabolic conversion of the end product (xylulose-5-phosphate) of the xylose isomerization pathway. (See Table 6).

[0280] Table 6

[0281] Strain Xylose Consumption Rate (%) Lysine Yield (g / L) Lysine / Xylose Conversion Rate (%) CathS141-6 39.6 9.16 57.8 CathS141-6-pket 45.3 11.2 61.8

[0282] Example 4: Integration of phosphoketolase gene pket at the ackA gene locus in the genome of strain CathS141-8 and evaluation of the fermentation performance of the engineered strain

[0283] It can be obtained from Example 2 that when the xylose metabolic module P eftuWhen the copy number of -xylAB in the genome increases to 3, although the consumption rate of xylose increases by more than 15 percentage points compared to that with 2 copy numbers, its ability to convert xylose into lysine only increases by 2 percentage points. This indicates that further increasing the copy number may cause the strain to consume too much carbon source for growth or energy consumption in other non-product synthesis pathways, thereby imposing a certain pressure on fermentation production. To further improve the consumption of xylose and the ability to produce lysine, referring to the results of Example 3, this example further conducts engineering transformation of the strain.

[0284] Integration of the phosphoketolase gene pket at the ackA gene locus in the genome of Corynebacterium glutamicum CathS141-8: The constructed plasmid pK18-ackA-pket was transformed into the competent cells of Corynebacterium glutamicum CathS141-8 by electroporation, and the strain with correct homology was screened by PCR verification and named CathS141-9.

[0285] The engineered strains CathS141-8 and CathS141-9 were evaluated and compared in a fermentation medium with 40 g / L xylose as the sole carbon source. The results showed that based on the strain CathS141-8 containing a three-copy xylose metabolism module, after additionally integrating and expressing the phosphoketolase pathway, the xylose consumption ability of the strain CathS141-9 was further improved, and its lysine conversion rate was also further enhanced. This indicates that under the condition of the joint catalysis of the PKET pathway and the PPP pathway for the metabolic conversion of xylulose-5-phosphate, the xylose consumption and lysine synthesis abilities of the strain were further increased (see Table 7).

[0286] Table 7

[0287] Strain Xylose Consumption Rate (%) Lysine Yield (g / L) Lysine / Xylose Conversion Rate (%) CathS141 0 0 0 CathS141-8 66.8 17.94 67.1 CathS141-9 72.2 20.33 70.4

[0288] Example 5: Comparison of the fermentation performance of the engineered strain CathS141-9 and the control strain in lignocellulose hydrolysate

[0289] The collected wheat straw was subjected to dry dilute acid pretreatment, biological detoxification and enzymatic hydrolysis, and finally the wheat straw hydrolysate (wheat straw hydrolysate) containing high concentrations of glucose and xylose was separated. The presence of xylose in the hydrolysate will, on the one hand, cause certain obstacles to the subsequent separation and extraction process, and on the other hand, a large amount of fermentable carbon source is not effectively utilized, resulting in a certain amount of resource waste. Therefore, the xylose-utilizing strain CathS141-9 obtained by the above transformation and the starting strain CathS141 were used as substrates for fermentation comparison with wheat straw hydrolysate to verify the industrial application performance of the genetically engineered bacteria.

[0290] In this example, the diluted hydrolyzate was used as the carbon source, with a glucose concentration of 53 g / L and a xylose concentration of 15.7 g / L. The starting strain CathS141 used in the present invention is tolerant to the toxic inhibitors in the straw hydrolyzate and can grow normally and produce lysine.

[0291] The engineered strain CathS141-9 with xylose utilization ability constructed in the present invention was fermented and compared with the control strain CathS141 lacking xylose utilization function. The results showed that strain CathS141 could only utilize glucose but not xylose in the hydrolyzate at all, and its lysine was all converted from glucose; strain CathS141-9 could not only completely utilize glucose in the hydrolyzate but also completely utilize xylose in the hydrolyzate. In addition, the lysine yield of strain CathS141-9 was significantly higher than that of the control strain CathS141 (see Table 8).

[0292] Table 8

[0293] Strain Glucose Consumption Rate (%) Xylose Consumption Rate (%) Lysine Yield (g / L) CathS141 100 0 34.8 CathS141-9 100 100 44.5

[0294] Obviously, the above examples are only for clear illustration and not limitations on the implementation modes. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation modes here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium heterologously expresses the xylose metabolic pathway.

2. The genetically engineered bacterium according to claim 1, wherein The xylose metabolic pathway includes a xylose metabolism module; Preferably, the xylose metabolism module includes xylose isomerase, xylulokinase, and a promoter.

3. The genetically engineered bacterium according to claim 2, characterized in that, The xylose isomerase is encoded by the xylA gene, and the nucleotide sequence of the xylA gene is SEQ ID NO:1; and / or, The amino acid sequence of the xylose isomerase is SEQ ID NO:39; and / or, The xylulokinase is encoded by the xylB gene, and the nucleotide sequence of the xylB gene is SEQ ID NO:2; and / or, The amino acid sequence of the xylulokinase is SEQ ID NO:40; and / or, The nucleotide sequence of the xylose metabolism module is SEQ ID NO:

3.

4. The genetically engineered bacterium according to claim 2, wherein The promoter includes a constitutive promoter and an inducible promoter; Preferably, the constitutive promoter includes P eftu , P gro , P lacM , P sod , whose nucleotide sequences are SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 in sequence; and / or, The inducible promoter includes P trc and P tac , and their nucleotide sequences are SEQ ID NO:8 and SEQ ID NO:9 in sequence.

5. The genetically engineered bacterium according to any one of claims 1-4, characterized in that, The xylose metabolic pathway is integrated into one, two, three, or more sites in the genome of the genetically engineered bacterium; Preferably, the genetically engineered bacterium is Corynebacterium glutamicum, and the site is selected from at least one of the cg1507-cg1524 gene locus, the cg1890 gene locus, and the cg1895 gene locus in the genome of Corynebacterium glutamicum.

6. The genetically engineered bacterium according to any one of claims 1-5, characterized in that, The genetically engineered bacterium also heterologously expresses the phosphoketolase pathway; Preferably, the method for heterologously expressing the phosphoketolase pathway is to integrate the phosphoketolase gene into one, two, three, or more sites in the genome of the genetically engineered bacterium; More preferably, the genetically engineered bacterium is Corynebacterium glutamicum, and the site is the ackA gene locus.

7. The genetically engineered bacterium according to claim 6, wherein The amino acid sequence of the phosphoketolase is SEQ ID NO:10; and / or, The phosphoketolase is encoded by the pket gene, and the nucleotide sequence of the pket gene is SEQ ID NO:

11.

8. The genetically engineered bacterium according to any one of claims 1-7, characterized in that, The starting strain of the genetically engineered bacterium is a strain lacking a complete xylose metabolic pathway or a strain that needs to strengthen the xylose metabolic pathway; Preferably, the starting strain is Corynebacterium glutamicum; More preferably, the starting strain is Corynebacterium glutamicum CathS141.

9. Use of the genetically engineered bacterium according to any one of claims 1-8 in the production of a molecule of interest, wherein the molecule of interest is a molecule that can be produced by the genetically engineered bacterium through fermentation culture; Preferably, the molecule of interest is at least one of an amino acid, a protein, a terpene compound, and an aromatic alcohol; More preferably, the genetically engineered bacterium is Corynebacterium glutamicum, and the molecule of interest is L-lysine.

10. A method for producing a molecule of interest, characterized in that, Comprising: Providing the genetically engineered bacterium according to any one of claims 1-8; Using a xylose-containing substance as a carbon source to perform fermentation culture on the genetically engineered bacterium to obtain a fermentation product, and the fermentation product contains the molecule of interest; Optionally, separating, extracting, and / or purifying the molecule of interest from the fermentation product.

11. The production method according to claim 10, characterized in that, The starting strain of the genetically engineered bacterium is Corynebacterium glutamicum; and / or, The xylose-containing substance is a hydrolysate obtained by hydrolyzing agricultural and forestry waste; and / or, The molecule of interest is at least one of amino acids, proteins, terpenoids, and aromatic alcohols.

12. Use of phosphoketolase and / or a nucleic acid molecule encoding phosphoketolase in the fermentative production of a molecule of interest by Corynebacterium glutamicum; Preferably, the amino acid sequence of the phosphoketolase is SEQ ID NO: 10; and / or, The nucleic acid molecule encoding phosphoketolase is the pket gene, and the nucleotide sequence of the pket gene is SEQ ID NO: 11.

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