Enzymes, enzyme compositions, expression cassettes, recombinant plasmids and strains for producing DFL

Abstract

The present application relates to the field of microbial fermentation, and particularly relates to enzymes, enzyme compositions, expression frames, recombinant plasmids and strains for producing DFL. The present application provides a recombinant Escherichia coli engineering strain for expressing phosphomannanase gene (manB), mannose-1-phosphoguanosine transglycosylase gene (manC), GDP mannose 4,6-dehydratase gene (gmd), GDP-L-fucose synthase gene (wcaG), alpha-1,2 fucosyltransferase and alpha-1,3-fucosyltransferase genes in series. The inventors greatly improve the yield of the DFL production engineering strain by selecting high-efficiency key enzymes alpha-1,3-fucosyltransferase from different sources and expressing them in series with different strengths of alpha-1,2-fucosyltransferase, and reduce the generation of byproduct 2'-FL, and improve the economic type of the fermentation method for synthesizing DFL.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation, in particular to enzymes, enzyme compositions, expression cassettes, recombinant plasmids and strains for producing DFL. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are a group of nutrients derived from human milk, which are second only to lactose and fat in content. The HMO family is considered to be a very effective prebiotic, which plays a positive role in maintaining the intestinal health of infants and promoting brain development. Its obvious health benefits enable it to be approved for use in food, such as infant formula and food, and consumer health products.

[0003] The structures of HMOs are diverse, and the most well-known and widely studied include 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3FL), which together account for 1 / 3 of the total HMO fraction, both of which are oligosaccharides of three monosaccharides obtained by fucosylation of one molecule of lactose and one molecule of fucose at different sites. While difucosyllactose (DFL), whose core structure is a tetrasaccharide obtained by fucosylation of lactose at C2' and C3 positions. Due to the slight difference in structure between DFL and 2'-FL and 3FL, DFL has unique antibacterial activity, such as strong antibacterial activity against group B streptococcus such as Streptococcus agalactiae.

[0004] Chemical synthesis of DFL requires chemical reagents, harsh reaction conditions, is not environmentally friendly, and is not economical. In contrast, biological synthesis method only needs mild conditions and low-cost reagents, which provides a more economical and environmentally friendly strategy for the synthesis of DFL. The currently reported biological pathways for the synthesis of DFL are: (1) through the catalysis of fuckinase / GDP-focalpyrophospylase (Fkp), GDP-L-fucose is converted from fucose, which is called a salvage pathway, but it has the disadvantages of expensive substrate and low yield. (2) De novo synthesis pathway: a-1, 2 fucosyltransferase catalyzes the connection of one molecule of lactose at the reducing end and one molecule of fucose at the non-reducing end to form 2'FL, and then a-1, 3 fucosyltransferase continues to catalyze the connection of 2'FL and one molecule of fucose residue at a-1, 3 to synthesize DFL. In contrast, the de novo synthesis pathway has the advantage of low-cost substrate. Therefore, it is of great significance to construct a recombinant microorganism for efficient de novo synthesis of DFL for the functional research and production application of DFL.

[0005] So far, only a few studies have reported the microbial synthesis of DFL, and the use of expensive fucose as a substrate has a low yield of only 5.1 g / L, which hinders the functional research and application of DFL. In recent years, with the rapid development of synthetic biology technology, microbial synthesis has become the mainstream method for synthesizing fucosylated human milk oligosaccharides. For example, a high-yield DFL construction strain was successfully obtained by expressing a suitable alpha-1,3-fucosyltransferase and other metabolic engineering methods based on the reported high-yield 2'FL engineering strain. In a 5L fermenter, the engineered strain produced 33.45g / L of DFL, which is the highest titer reported to date.

[0006] Fucosyltransferase can synthesize DFL with GDP-L-fucose and lactose as substrates, and is one of the key limiting factors for synthesizing DFL. However, fucosyltransferase has the defects of low heterologous expression, poor catalytic activity, and single source. The yield of DFL synthesized by the current microbial method is low, and a large amount of 2'FL and 3FL byproducts are generated. SUMMARY

[0007] Therefore, the present application provides enzymes, enzyme compositions, expression frames, recombinant plasmids and strains for producing DFL. The present application screens high-efficiency alpha-1,3-fucosyltransferase from different sources, and effectively improves DFL while reducing the generation of intermediate byproducts by different combinations of the existing alpha-1,2-fucosyltransferase in the applicant's laboratory. Finally, a DFL-producing E. coli engineering strain is obtained.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides alpha-1,3-fucosyltransferase derived from Bacteroides gallinaceum;

[0010] The alpha-1,3-fucosyltransferase has:

[0011] (I) the amino acid sequence shown in SEQ ID No. 1; and / or

[0012] (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0013] (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II).

[0014] In a second aspect, the present application also provides an enzyme composition comprising an alpha-1,2-fucosyltransferase and the alpha-1,3-fucosyltransferase.

[0015] In some embodiments of the present application, the alpha-1,2-fucosyltransferase is derived from Helicobacter pylori;

[0016] The alpha-1,2-fucosyltransferase has:

[0017] (I) an amino acid sequence as set forth in SEQ ID No. 2; and / or

[0018] (II) a sequence in which one or more amino acids in the amino acid sequence as set forth in (I) are substituted, deleted, added and / or replaced; or

[0019] (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence as set forth in any one of (I) or (II).

[0020] In some embodiments of the present application, the enzyme composition has:

[0021] (I) an amino acid sequence as set forth in SEQ ID No. 3; and / or

[0022] (II) a sequence in which one or more amino acids in the amino acid sequence as set forth in (I) are substituted, deleted, added and / or replaced; or

[0023] (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence as set forth in any one of (I) or (II).

[0024] In some embodiments of the present application, the enzyme composition further comprises one or more of ManB, ManC, Gmd or WcaG.

[0025] The ManB, ManC, Gmd or WcaG respectively has:

[0026] (i) the ManB has an amino acid sequence as set forth in SEQ ID No. 4; and / or

[0027] the ManC has an amino acid sequence as set forth in SEQ ID No. 5; and / or

[0028] the Gmd has an amino acid sequence as set forth in SEQ ID No. 6; and / or

[0029] the WcaG has an amino acid sequence as set forth in SEQ ID No. 7; or

[0030] (ii) a sequence of one or more amino acids substituted, deleted, added and / or replaced based on the amino acid sequence as set forth in (i); or

[0031] (iii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence as set forth in any one of (i) or (ii).

[0032] In a third aspect, the present application provides a nucleic acid molecule encoding any one of:

[0033] (A) the alpha-1, 3-fucosyltransferase; and / or

[0034] (B) the enzyme composition.

[0035] In a fourth aspect, the present application provides an expression cassette comprising the nucleic acid molecule.

[0036] In a fifth aspect, the present application provides a recombinant plasmid comprising the expression cassette.

[0037] In some embodiments of the present application, the recombinant plasmid further comprises a vector backbone, which includes but is not limited to one or more of pAC series, pGEX series and / or pET series;

[0038] Preferably, the vector backbone of the recombinant plasmid includes but is not limited to one or more of pAC, pGEX-5x and / or pET28a;

[0039] More preferably, the vector backbone of the recombinant plasmid includes but is not limited to pAC and / or pGEX-5x.

[0040] More preferably, the pAC is pACYCDuet-1, with the catalog number of BioVector-0147603.

[0041] In some embodiments of the present application, the recombinant plasmid comprises: pAC-CBGW and / or pGEX-FL20-FL59.

[0042] In some embodiments of the present application, the pAC-CBGW is used to express the ManB, the ManC, the Gmd and the WcaG in tandem using the pAC;

[0043] The pGEX-FL20-FL59 is used to express the FL20-FL59 by using the pGEX-5x.

[0044] In some embodiments of the present application, the recombinant plasmid:

[0045] (A) the pAC-CBGW and the pGEX-FL20-FL59 respectively have the nucleotide sequences as shown in SEQ ID No. 8, 14; or

[0046] (B) the nucleotide sequences encoding the same proteins as the nucleotide sequences shown in (A) but different from the nucleotide sequences shown in (A) due to the degeneracy of genetic code; or

[0047] (C) the nucleotide sequences obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequences shown in (A) or (B), and the nucleotide sequences functionally identical or similar to the nucleotide sequences shown in (A) or (B); or

[0048] (D) the nucleotide sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequences of any one of (A)-(C).

[0049] In a sixth aspect, the present application provides a strain, wherein the strain:

[0050] (I) expresses the enzyme or the combination of enzymes; and / or

[0051] (II) is transformed or transfected with the expression cassette; and / or

[0052] (III) is transformed or transfected with the recombinant plasmid.

[0053] In some embodiments of the present application, the strain includes, but is not limited to, one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae;

[0054] Preferably, the strain includes Escherichia coli;

[0055] More preferably, the strain includes Escherichia coli W3110 and / or Escherichia coli K-12 MG1655.

[0056] In some embodiments of the present application, the strain includes:

[0057] (I) expresses the FL20-FL59, the ManB, the ManC, the Gmd and the WcaG; and / or

[0058] transformed with said pGEX-FL20-FL59 and said pAC-CBGW.

[0059] In a seventh aspect, the present application provides a method for constructing said strain, said method comprising:

[0060] (I) expressing said enzyme or said combination of enzymes; and / or

[0061] (II) transforming or transfecting said expression cassette; and / or

[0062] (III) transforming or transfecting said recombinant plasmid.

[0063] In an eighth aspect, the present application provides the use of any of the following in the synthesis of 3-fucosyllactose:

[0064] (i) said enzyme; and / or

[0065] (ii) said expression cassette; and / or

[0066] (iii) said recombinant plasmid;

[0067] (iv) said strain;

[0068] (v) the strain obtained by said method for constructing.

[0069] In some embodiments of the present application, said enzyme comprises said FL59;

[0070] said recombinant plasmid comprises said pGEX-FL59;

[0071] said strain comprises any of the following:

[0072] (1) expressing said FL59; and / or

[0073] (2) transformed with said pGEX-FL59.

[0074] In a ninth aspect, the present application provides the use of any of the following in the synthesis of difucosyllactose:

[0075] (i) said combination of enzymes; and / or

[0076] (ii) said expression cassette; and / or

[0077] (iii) said recombinant plasmid;

[0078] (iv) said strain;

[0079] (v) the strain obtained by said method for constructing.

[0080] In some embodiments of the present application, said combination of enzymes comprises:

[0081] the FL20-FL59, the ManB, the ManC, the Gmd and the WcaG;

[0082] the plasmids include the pGEX-FL20-FL59 and the pAC-CBGW;

[0083] the strain is any one of the following:

[0084] i) expressing the FL20-FL59, the ManB, the ManC, the Gmd and the WcaG; and / or

[0085] ii) being transformed with the pGEX-FL20-FL59 and the pAC-CBGW.

[0086] In a tenth aspect, the present application provides a method for synthesizing 3-fucosyllactose and / or difucosyllactose, which comprises picking the strain or the strain obtained by the construction method, fermentation, collecting the fermentation broth, and obtaining 3-fucosyllactose and / or difucosyllactose.

[0087] The present application provides a recombinant Escherichia coli engineering strain for expressing phosphomannanase gene (manB), mannose-1-phosphate guanylyltransferase gene (manC), GDP-mannose 4,6-dehydratase gene (gmd), GDP-L-fucose synthase gene (wcaG), α-1,2 fucosyltransferase and α-1,3-fucosyltransferase in series. The inventors greatly improve the yield of DFL production engineering strain by expressing the preferred high-efficiency key enzyme α-1,3-fucosyltransferase from different sources and in series with different strengths of α-1,2-fucosyltransferase, and reduce the generation of byproduct 2'-FL, thereby improving the economy of fermentation method for synthesizing DFL. BRIEF DESCRIPTION OF DRAWINGS

[0088] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, brief descriptions will be given below to the drawings needed to be used in the embodiments or prior art descriptions.

[0089] Figure 1 pAC-CBGW plasmid is shown;

[0090] Figure 2 pGEX-FL46 plasmid is shown;

[0091] Figure 3 pGEX-FL20 plasmid is shown;

[0092] Figure 4 pGEX-FL35 plasmid is shown;

[0093] Figure 5 pGEX-FL59 plasmid

[0094] Figure 6 SDS detection of target gene expression level of strain 2FL-A

[0095] Figure 7 SDS detection of target gene expression level of strain 2FL-B

[0096] Figure 8 SDS detection of target gene expression level of strain 3FL-A

[0097] Figure 9 SDS detection of target gene expression level of strain 3FL-B

[0098] Figure 10 Strain 72h flask fermentation yield

[0099] Figure 11 pGEX-FL46-FL59 plasmid

[0100] Figure 12 pGEX-FL20-FL59 plasmid

[0101] Figure 13 DFL production strain 72h flask yield

[0102] Figure 14 Electrophoresis result of Example 1. DETAILED DESCRIPTION

[0103] The present application discloses enzymes, enzyme compositions, expression frames, recombinant plasmids and strains for producing DFL, and those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0104] The present application comprises the following technical solutions:

[0105] A high-efficiency α-1,3-fucosyltransferase FL59, which is derived from Bacteroides gallinaceum. Its amino acid sequence is shown in SEQ ID No. 1. It is expressed in series with α-1,2-fucosyltransferase FL20, which is derived from Helicobacter pylori, and its amino acid sequence is shown in SEQ ID No. 2, to obtain a series-expressed fucosyltransferase FL20-FL59, and its amino acid sequence is shown in SEQ ID No. 3. A series-expressed phosphomannanase gene (manB), mannose-1-phosphate guanylyltransferase gene (manC), GDP-mannose 4,6-dehydratase gene (gmd), and GDP-L-fucose synthase gene (wcaG) are derived from Escherichia coli K-12 MG1655. Their amino acid sequences are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7, respectively.

[0106] Amino acid sequence of high-efficiency α-1,3-fucosyltransferase FL59 (shown in SEQ ID No. 1): FMCDCLSIILLVKMKKIYLKFVDFWDGFDTISNFIVDALSIQYEVVLSNEPDYLFYSCFGTSHLEYDCIKIMFIGENIVPDFNVCDYAIGFNYIDFGDRYLRLPLYAIYDGFSNLQNKKIDVNKALDRKFCSIVVSNNKWADPIRETFFKLLSSYKKVDSGGRAWNNIGGPVDNKLDFISQYKFNIAFENSRVLGYTTEKIMEPMQVNSIPVYWGNPLVGKDFNVDSFVNAHDFDSLERLVEYIIELDSSKDKYLEMLEKPWLLDKTYLDWKQLLLNFINNIMMKSYKDAKYLVNYGHAGKYRNEQRFWGRCERKFKLQRIIEYYSQLFDRK;

[0107] Amino acid sequence of alpha-1,2-fucosyltransferase FL20 (as shown in SEQ ID No. 2): FMITCFLKGGLGNQLFEIFTTISYAIKSNQNFSFLYSRTLNGSPERPTYWNSFLLCLKMFTVHKLPENMHIMSEENFNYKELPINTSKNIILDGYFQSYKYFEYYFENICKLIQLDKQKTMIQQKHLYNYKNMISMHFRIGDYKHKQNYHPVMNLQFYKNSIQHIINITDKLDLKFLYFCEDEDVEEVSDMIEQLQHSFEKCKFISANSNSNIKTNIEDWEQMLMMSLCQHNIIPNSTFSWWGAYFNSNTEKIVCYPDVWFGPAVSKRIVDDLFPESWTKISCH;

[0108]

[0109] Phosphomannanase (ManB) amino acid sequence (as shown in SEQ ID No. 4):

[0110] MKKLTCFKAYDIRGKLGEELNEDIAWRIGRAYGEFLKPKTIVLGGDVRLTSETLKLALAKGLQDAGV

[0111] DVLDIGMSGTEEIYFATFHLGVDGGIEVTASHNPMDYNGMKLVREGARPISGDTGLRDVQRLAEAND

[0112] FPPVDETKRGRYQQINLRDAYVDHLFGYINVKNLTPLKLVINSGNGAAGPVVDAIEARFKALGAPVEL

[0113] IKVHNTPDGNFPNGIPNPLLPECRDDTRNAVIKHGADMGIAFDGDFDRCFLFDEKGQFIEGYYIVGLLA

[0114] EAFLEKNPGAKIIHDPRLSWNTVDVVTAAGGTPVMSKTGHAFIKERMRKEDAIYGGEMSAHHYFRDF

[0115] AYCDSGMIPWLLVAELVCLKDKTLGELVRDRMAAFPASGEINSKLAQPVEAINRVEQHFSREALAVDRTDGISMTFADWRFNLRTSNTEPVVRLNVESRGDVPLMEARTRTLLTLLNE;

[0116] Mannose-1-phosphate guanosyltransferase (ManC) amino acid sequence (as shown in SEQ ID No. 5): MAQSKLYPVVMAGGSGSRLWPLSRVLYPKQFLCLKGDLTMLQTTICRLNGVEMAQSKLYPVVMAGGSGSRLWPLSRVLYPKQFLCLKGDLTMLQTTICRLNGVECESPVVICNEQHRFIVAEQLRQLNKLTENIILEPAGRNTAPAIALAALAAKRHSPESDPLMLVLAADHVIADEDAFRAAVRNAMPYAEAGKLVTFGIVPDLPETGYGYIRRGEVSAGEQDMVAFEVAQFVEKPNLETAQAYVASGEYYWNSGMFLFRAGRYLEELKKYRPDILDACEKAMSAVDPDLNFIRVDEEAFLACPEESVDYAVMERTADAVVVPMDAGWSDVGSWSSLWEISAHTAEGNVCHGDVINHKTENSYVYAESGLVTTVGVKDLVVVQTKDAVLIADRNAVQDVKKVVEQIKADGRHEHRVHREVYRPWGKYDSIDAGDRYQVKRITVKPGEGLSVQMHHHRAEHWVVVAGTAKVTIDGDIKLLGENESIYIPLGATHCLENPGKIPLDLIEVRSGSYLEEDDVVRFADRYGRV;

[0117] GDP-mannose 4,6-dehydratase (Gmd) amino acid sequence (as shown in SEQ ID No. 6):

[0118] MSKVALITGVTGQDGSYLAEFLLEKGYEVHGIKRRASSFNTERVDHIYQDPHTCNPKFHLHYGDLSD

[0119] TSNLTRILREVQPDEVYNLGAMSHVAVSFESPEYTADVDAMGTLRLLEAIRFLGLEKKTRFYQASTSE

[0120] LYGLVQEIPQKETTPFYPRSPYAVAKLYAYWITVNYRESYGMYACNGILFNHESPRRGETFVTRKITR

[0121] AIANIAQGLESCLYLGNMDSLRDWGHAKDYVKMQWMMLQQEQPEDFVIATGVQYSVRQFVEMAA

[0122] AQLGIKLRFEGTGVEEKGIVVSVTGHDAPGVKPGDVIIAVDPRYFRPAEVETLLGDPTKAHEKLGWKPEITLREMVSEMVANDLEAAKKHSLLKSHGYDVAIALES;

[0123] GDP-L-fucose synthase (WcaG) amino acid sequence (as shown in SEQ ID No. 7):

[0124] MSKQRVFIAGHRGMVGSAIRRQLEQRGDVELVLRTRDELNLLDSRAVHDFFASERIDQVYLAAAKV

[0125] GGIVANNTYPADFIYQNMMIESNIIHAAHQNDVNKLLFLGSSCIYPKLAKQPMAESELLQGTLEPTNEP

[0126] YAIAKIAGIKLCESYNRQYGRDYRSVMPTNLYGPHDNFHPSNSHVIPALLRRFHEATAQNAPDVVVW

[0127] GSGTPMREFLHVDDMAAASIHVMELAHEVWLENTQPMLSHINVGTGVDCTIRELAQTIAKVVGYKGRVVFDASKPDGTPRKLLDVTRLHQLGWYHEISLEAGLASTYQWFLENQDRFRG.

[0128]

[0129]

[0130]

[0131]

[0132] Nucleotide sequence of plasmid pGEX-FL59 (as set forth in SEQ ID No. 12):

[0133] TCGAATAGCTGACGTGCCACGTGGTTACGAAGACCGCAGTCCGTCGGTAGCCTTCGACACCATAC

[0134] CGACACGTCCAGCATTTAGTGACGTATTAAGCACAGCGAGTTCCGCGTGAGGGCAAGACCTATTA

[0135] CAAAAAACGCGGCTGTAGTATTGCCAAGACCGTTTATAAGACTTTACTCGACAACTGTTAATTAG

[0136] TAGCCGAGCATATTACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCATAAGTACA

[0137] GGGGATATGATCCAATAACCTTTTAATTCCCGGAACACGTTGGGTGAGCTGAAGAAAACCTTATA

[0138] GAACTTCTTTTTATACTTCTCGTAAACATACTCGCGCTACTTCCACTATTTACCGCTTTGTTTTTCA

[0139] AACTTAACCCAAACCTCAAAGGGTTAGAAGGAATAATATAACTACCACTACAATTTAATTGTGTC

[0140] AGATACCGGTAGTATGCAATATATCGACTGTTCGTGTTGTACAACCCACCAACAGGTTTTCTCGC

[0141] ACGTCTCTAAAGTTACGAACTTCCTCGCCAAAACCTATAATCTATGCCACAAAGCTCTTAACGTA

[0142] TATCATTTCTGAAACTTTGAGAGTTTCAACTAAAAGAATCGTTCGATGGACTTTACGACTTTTACA

[0143] AGCTTCTAGCAAATACAGTATTTTGTATAAATTTACCACTAGTACATTGGGTAGGACTGAAGTAC

[0144] AACATACTGCGAGAACTACAACAAAATATGTACCTGGGTTACACGGACCTACGCAAGGGTTTTA

[0145] ATCAAACAAAATTTTTTGCATAACTTCGATAGGGTGTTTAACTATTCATGAACTTTAGGTCGTTCA

[0146] TATATCGTACCGGAAACGTCCCGACCGTTCGGTGCAAACCACCACCGCTGGTAGGAGGTTTTAGC

[0147] CTAGACTAGCTTCCAGCACCCTAGGGGCTTAAGTACACACTAACAGACTCGTAATAAGACGACC

[0148] ACTTTTACTTTTTTTAGATGGACTTTAAGCACCTAAAGACCCTACCAAAACTATGGTAATCATTAA

[0149] AATAGCACCTGCGGGACTCGTAAGTCATACTTCAACACGACTCGTTACTTGGCCTAATAGACAAA

[0150] ATATCAACGAAACCATGGTCGGTAGACCTTATACTAACATAATTTTAGTACAAGTAGCCGCTTTT

[0151] ATAGCAAGGCCTAAAATTACACACGCTAATACGTTAACCAAAATTAATATAGCTGAAGCCGCTA

[0152] GCAATAGACGCGGACGGCGACATACGTTAAATACTACCGAAATCATTAGACGTCTTATTTTTTTA

[0153] GCTGCAATTGTTTCGGGACCTAGCGTTTAAAACATCATAACAACACTCATTATTGTTCACCCGTCT

[0154] GGACCTTATTATAACCGCCAGGCCAACTATTATTTGACCTAAAATAATCGGTCATGTTCAAATTA

[0155] GGACCTTATTATAACCGCCAGGCCAACTATTATTTGACCTAAAATAATCGGTCATGTTCAAATTA

[0156] TAGCGTAAACTTTTGTCGGCACAAGACCCAATATGGTGGCTTTTTTAATACCTTGGCTACGTCCAC

[0157] TTATCGTAAGGCCACATAACCCCGTTAGGCGACCAACCATTTCTAAAATTACAACTATCGAAGCA

[0158] ATTGCGGGTACTAAAACTATCAGACCTTGCAGACCAACTTATATAATAACTTGACCTATCATCGT

[0159] TCCTATTTATAGACCTTTACGACCTTTTTGGCACCGACGACCTATTTTGGATAGACCTAACCTTTG

[0160] TCGACGACGACTTAAAATAATTATTGTAATACTACTTCTCGATGTTCCTACGGTTTATAGACCACT

[0161] TAATACCAGTACGGCCATTTATAGCATTACTTGTCGCAAAAACCCCAGCGACGCTTGCATTTAAA

[0162] TTTGACGTCGCGTAATAACTTATGATATCAGTCGACAAACTGGCATTTATTATTCGCCGGCGTAG

[0163] CACTGACTGACTGCTAGACGGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTC

[0164] GAGGGCCTCTGCCAGTGTCGAACAGACATTCGCCTACGGCCCTCGTCTGTTCGGGCAGTCCCGCG

[0165] CAGTCGCCCACAACCGCCCACAGCCCCGCGTCGGTACTGGGTCAGTGCATCGCTATCGCCTCACA

[0166] TATTAAGAACTTCTGCTTTCCCGGAGCACTATGCGGATAAAAATATCCAATTACAGTACTATTATT

[0167] ACCAAAGAATCTGCAGTCCACCGTGAAAAGCCCCTTTACACGCGCCTTGGGGATAAACAAATAA

[0168] AAAGATTTATGTAAGTTTATACATAGGCGAGTACTCTGTTATTGGGACTATTTACGAAGTTATTAT

[0169] AACTTTTTCCTTCTCATACTCATAAGTTGTAAAGGCACAGCGGGAATAAGGGAAAAAACGCCGTA

[0170] AAACGGAAGGACAAAAACGAGTGGGTCTTTGCGACCACTTTCATTTTCTACGACTTCTAGTCAAC

[0171] CCACGTGCTCACCCAATGTAGCTTGACCTAGAGTTGTCGCCATTCTAGGAACTCTCAAAAGCGGG

[0172] GCTTCTTGCAAAAGGTTACTACTCGTGAAAATTTCAAGACGATACACCGCGCCATAATAGGGCAC

[0173] AACTGCGGCCCGTTCTCGTTGAGCCAGCGGCGTATGTGATAAGAGTCTTACTGAACCAACTCATG

[0174] AGTGGTCAGTGTCTTTTCGTAGAATGCCTACCGTACTGTCATTCTCTTAATACGTCACGACGGTAT

[0175] TGGTACTCACTATTGTGACGCCGGTTGAATGAAGACTGTTGCTAGCCTCCTGGCTTCCTCGATTGG

[0176] CGAAAAAACGTGTTGTACCCCCTAGTACATTGAGCGGAACTAGCAACCCTTGGCCTCGACTTACT

[0177] TCGGTATGGTTTGCTGCTCGCACTGTGGTGCTACGGACGTCGTTACCGTTGTTGCAACGCGTTTGA

[0178] TAATTGACCGCTTGATGAATGAGATCGAAGGGCCGTTGTTAATTATCTGACCTACCTCCGCCTATT

[0179] TCAACGTCCTGGTGAAGACGCGAGCCGGGAAGGCCGACCGACCAAATAACGACTATTTAGACCT

[0180] CGGCCACTCGCACCCAGAGCGCCATAGTAACGTCGTGACCCCGGTCTACCATTCGGGAGGGCAT

[0181] AGCATCAATAGATGTGCTGCCCCTCAGTCCGTTGATACCTACTTGCTTTATCTGTCTAGCGACTCT

[0182] ATCCACGGAGTGACTAATTCGTAACCATTGACAGTCTGGTTCAAATGAGTATATATGAAATCTAA

[0183] CTAAATTTTGAAGTAAAAATTAAATTTTCCTAGATCCACTTCTAGGAAAAACTATTAGAGTACTG

[0184] GTTTTAGGGAATTGCACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTAGTTTCCTA

[0185] GAAGAACTCTAGGAAAAAAAGACGCGCATTAGACGACGAACGTTTGTTTTTTTGGTGGCGATGG

[0186] TCGCCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAGGCTTCCATTGACCGAAGTCGT

[0187] CTCGCGTCTATGGTTTATGACAGGAAGATCACATCGGCATCAATCCGGTGGTGAAGTTCTTGAGA

[0188] CATCGTGGCGGATGTATGGAGCGAGACGATTAGGACAATGGTCACCGACGACGGTCACCGCTAT

[0189] TCAGCACAGAATGGCCCAACCTGAGTTCTGCTATCAATGGCCTATTCCGCGTCGCCAGCCCGACT

[0190] TGCCCCCCAAGCACGTGTGTCGGGTCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGT

[0191] CGCACTCGATACTCTTTCGCGGTGCGAAGGGCTTCCCTCTTTCCGCCTGTCCATAGGCCATTCGCC

[0192] GTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTTGCGGACCATAGAAATATCAG

[0193] GACAGCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCCGCCTC

[0194] GGATACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGA

[0195] GTGTACAAGAAAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGGAAACTCACTC

[0196] GACTATGGCGAGCGGCGTCGGCTTGCTGGCTCGCGTCGCTCAGTCACTCGCTCCTTCGCCTTCTCG

[0197] CGGACTACGCCATAAAAGAGGAATGCGTAGACACGCCATAAAGTGTGGCGTATTTAAGGCTGTG

[0198] GTAGCTTACCACGTTTTGGAAAGCGCCATACCGTACTATCGCGGGCCTTCTCTCAGTTAAGTCCC

[0199] ACCACTTACACTTTGGTCATTGCAATATGCTACAGCGTCTCATACGGCCACAGAGAATAGTCTGG

[0200] CAAAGGGCGCACCACTTGGTCCGGTCGGTGCAAAGACGCTTTTGCGCCCTTTTTCACCTTCGCCG

[0201] CTACCGCCTCGACTTAATGTAAGGGTTGGCGCACCGTGTTGTTGACCGCCCGTTTGTCAGCAACG

[0202] ACTAACCGCAACGGTGGAGGTCAGACCGGGACGTGCGCGGCAGCGTTTAACAGCGCCGCTAATT

[0203] TAGAGCGCGGCTAGTTGACCCACGGTCGCACCACCACAGCTACCATCTTGCTTCGCCGCAGCTTC

[0204] GGACATTTCGCCGCCACGTGTTAGAAGAGCGCGTTGCGCAGTCACCCGACTAGTAATTGATAGGC

[0205] GACCTACTGGTCCTACGGTAACGACACCTTCGACGGACGTGATTACAAGGCCGCAATAAAGAAC

[0206] TACAGAGACTGGTCTGTGGGTAGTTGTCATAATAAAAGAGGGTACTTCTGCCATGCGCTGACCCG

[0207] CACCTCGTAGACCAGCGTAACCCAGTGGTCGTTTAGCGCGACAATCGCCCGGGTAATTCAAGACA

[0208] GAGCCGCGCAGACGCAGACCGACCGACCGTATTTATAGAGTGAGCGTTAGTTTAAGTCGGCTATC

[0209] GCCTTGCCCTTCCGCTGACCTCACGGTACAGGCCAAAAGTTGTTTGGTACGTTTACGACTTACTCC

[0210] CGTAGCAAGGGTGACGCTACGACCAACGGTTGCTAGTCTACCGCGACCCGCGTTACGCGCGGTA

[0211] ATGGCTCAGGCCCGACGCGCAACCACGCCTATAGAGCCATCACCCTATGCTGCTATGGCTTCTGT

[0212] CGAGTACAATATAGGGCGGCAATTGGTGGTAGTTTGTCCTAAAAGCGGACGACCCCGTTTGGTCG

[0213] CACCTGGCGAACGACGTTGAGAGAGTCCCGGTCCGCCACTTCCCGTTAGTCGACAACGGGCAGA

[0214] GTGACCACTTTTCTTTTTGGTGGGACCGCGGGTTATGCGTTTGGCGGAGAGGGGCGCGCAACCGG

[0215] CTAAGTAATTACGTCGACCGTGCTGTCCAAAGGGCTGACCTTTCGCCCGTCACTCGCGTTGCGTT

[0216] AATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTGAAATACGAAGGCCGAGCATA

[0217] CAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCCTAA

[0218] GTGACCGGCAGCAAAATGTTGCAGCACTGACCCTTTTGGGACCGCAATGGGTTGAATTAGCGGA

[0219] ACGTCGTGTAGGGGGAAAGCGGTCGACCGCATTATCGCTTCTCCGGGCGTGGCTAGCGGGAAGG

[0220] GTTGTCAACGCGTCGGACTTACCGCTTACCGCGAAACGGACCAAAGGCCGTGGTCTTCGCCACGG

[0221] CCTTTCGACCGACCTCACGCTAGAAGGACTCCGGCTATGACAGCAGCAGGGGAGTTTGACCGTCT

[0222] ACGTGCCAATGCTACGCGGGTAGATGTGGTTGCATTGGATAGGGTAATGCCAGTTAGGCGGCAA

[0223] ACAAGGGTGCCTCTTAGGCTGCCCAACAATGAGCGAGTGTAAATTACAACTACTTTCGACCGATGTCCTTCCGGTCTGCGCTTAATAAAAACTACCGCAACCTTAA.

[0224]

[0225] Nucleotide sequence of plasmid pGEX-FL20-FL59 (as set forth in SEQ ID No. 14):

[0226] TCGAATAGCTGACGTGCCACGTGGTTACGAAGACCGCAGTCCGTCGGTAGCCTTCGACACCATAC

[0227] CGACACGTCCAGCATTTAGTGACGTATTAAGCACAGCGAGTTCCGCGTGAGGGCAAGACCTATTA

[0228] CAAAAAACGCGGCTGTAGTATTGCCAAGACCGTTTATAAGACTTTACTCGACAACTGTTAATTAG

[0229] TAGCCGAGCATATTACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCATAAGTACA

[0230] GGGGATATGATCCAATAACCTTTTAATTCCCGGAACACGTTGGGTGAGCTGAAGAAAACCTTATA

[0231] GAACTTCTTTTTATACTTCTCGTAAACATACTCGCGCTACTTCCACTATTTACCGCTTTGTTTTTCA

[0232] AACTTAACCCAAACCTCAAAGGGTTAGAAGGAATAATATAACTACCACTACAATTTAATTGTGTC

[0233] AGATACCGGTAGTATGCAATATATCGACTGTTCGTGTTGTACAACCCACCAACAGGTTTTCTCGC

[0234] ACGTCTCTAAAGTTACGAACTTCCTCGCCAAAACCTATAATCTATGCCACAAAGCTCTTAACGTA

[0235] TATCATTTCTGAAACTTTGAGAGTTTCAACTAAAAGAATCGTTCGATGGACTTTACGACTTTTACA

[0236] AGCTTCTAGCAAATACAGTATTTTGTATAAATTTACCACTAGTACATTGGGTAGGACTGAAGTAC

[0237] AACATACTGCGAGAACTACAACAAAATATGTACCTGGGTTACACGGACCTACGCAAGGGTTTTA

[0238] ATCAAACAAAATTTTTTGCATAACTTCGATAGGGTGTTTAACTATTCATGAACTTTAGGTCGTTCA

[0239] TATATCGTACCGGAAACGTCCCGACCGTTCGGTGCAAACCACCACCGCTGGTAGGAGGTTTTAGC

[0240] CTAGACTAGCTTCCAGCACCCTAGGGGCTTAAGTACTAGTGGACGAAAGACTTTCCACCAGACCC

[0241] ATTAGTCGACAAACTTTAAAAATGGTGGTAATCAATGCGTTAATTCTCATTAGTCTTGAAATCAA

[0242] AGGACATATCGGCATGGGACTTACCGTCAGGCCTTGCGGGCTGGATAACCTTATCGAAAGACGA

[0243] CACAGACTTTTACAAATGGCACGTATTTGACGGCCTTTTATACGTATAATACTCGCTTCTTTTGAA

[0244] GTTAATGTTCCTTGACGGCTAATTATGGTCATTTTTATAATAGGACCTGCCGATAAAAGTCTCAAT

[0245] ATTTATAAAGCTCATGATGAAGCTCTTGTAAACGTTTGACTAAGTCGACCTATTTGTCTTTTGGTA

[0246] CTAAGTCGTCTTTGTAGACATATTAATGTTCTTATACTAGTCGTACGTGAAAGCATAACCGCTAAT

[0247] ATTTGTATTTGTCTTAATGGTAGGCCACTACTTAGACGTCAAAATATTTTTATCGTAGGTCGTATA

[0248] GTAGTTGTAATGGCTATTTGACCTAGACTTTAAAGACATAAAGACACTTCTACTTCTGCAACTTCT

[0249] TCACTCACTATACTAACTTGTCGACGTCGTATCAAAACTTTTTACGTTTAAATAGTCGCGGTTATC

[0250] GTTATCGTTATAATTTTGGTTATAGCTCCTGACCCTTGTCTACGACTACTACTCAGACACGGTCGT

[0251] ATTATAATAAGGCTTATCGTGGAAATCGACCACCCCGCGGATAAAATTATCATTATGGCTTTTTT

[0252] AGCACACGATAGGCCTACACACCAAACCGGGCCGTCAATCGTTTGCATAACACCTACTAGACAA

[0253] AGGCCTTTCAACCTGGTTTTAATCAACAGTAATTCAGCCATGGGAGCTCAGAAACTGTTAATTAG

[0254] TAGCCGAGCATATTACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCATAAGTACA

[0255] GGGGATATGATCCAATAACCTTTTAATTCCCGGAACACGTTGGGTGAGCTGAAGAAAACCTTATA

[0256] GAACTTCTTTTTATACTTCTCGTAAACATACTCGCGCTACTTCCACTATTTACCGCTTTGTTTTTCA

[0257] AACTTAACCCAAACCTCAAAGGGTTAGAAGGAATAATATAACTACCACTACAATTTAATTGTGTC

[0258] AGATACCGGT AGTATGCAAT ATATCGACTG TTCGTGTTGT ACAACCCACC A ACAGGTTTTCTCGC

[0259] ACGTCTCTAA AGTTACGAACTTCCTCGCC AAAACCTATA ATCTATGCCA CA AAGCTCTTAACGTA

[0260] TATCATTTCT GAAACTTTGA GAGTTTCAAC TAAAAGAATC GTTCGATGGA CT TTACGACTTTTACA

[0261] AGCTTCTAGC AAATACAGTA TTTTGTATAA ATTTACCACT AGTACATTGG GTAGGACTGA AGTAC

[0262] AACATACTGC GAGAACTACA AC AAAATATGT ACCTGGGTTAC ACGGACCTAC GCAAGGGTTTTA

[0263] ATCAAACAAA ATTTTTTGCA TAACTTCGAT AGGGTGTTTA ACTATTCATG AACTTTAGGC GTTCA

[0264] TATATCGTAC CGGAAACGTC CCGACCGTTC GGTGCAAACC ACCACCGCTG GTAGGAGGTT TTAGC

[0265] CTAGACTAGC TTCCAGCACC CTAGGGGCTT AAGTACACAC TAACAGACTC GTAATAAGAC GACC

[0266] ACTTTTACTT TTTTAGATGG ACTTTAAGCA CCTAAAGACC CTACCAAAAC TATGGTAATC ATTAA

[0267] AATAGCACCT GCGGGACTCG TAAGTCATAC TTCAACACGA CTCGTTACTT GGCCTAATAG ACAAA

[0268] ATATCAACGA AACCATGGTC GGTAGACCTT ATACTAACATA TTTTAGTACA AGTAGCCGCT TTT

[0269] ATAGCAAGGCCTAAAATTACACACGCTAATACGTTAACCAAAATTAATATAGCTGAAGCCGCTA

[0270] GCAATAGACGCGGACGGCGACATACGTTAAATACTACCGAAATCATTAGACGTCTTATTTTTTTA

[0271] GCTGCAATTGTTTCGGGACCTAGCGTTTAAAACATCATAACAACACTCATTATTGTTCACCCGTCT

[0272] AGGCTAAGCGCTTTGGAAAAAATTTGACGACTCATCAATATTCTTCCAACTATCACCACCAGCGC

[0273] GGACCTTATTATAACCGCCAGGCCAACTATTATTTGACCTAAAATAATCGGTCATGTTCAAATTA

[0274] TAGCGTAAACTTTTGTCGGCACAAGACCCAATATGGTGGCTTTTTTAATACCTTGGCTACGTCCAC

[0275] TTATCGTAAGGCCACATAACCCCGTTAGGCGACCAACCATTTCTAAAATTACAACTATCGAAGCA

[0276] ATTGCGGGTACTAAAACTATCAGACCTTGCAGACCAACTTATATAATAACTTGACCTATCATCGT

[0277] TCCTATTTATAGACCTTTACGACCTTTTTGGCACCGACGACCTATTTTGGATAGACCTAACCTTTG

[0278] TCGACGACGACTTAAAATAATTATTGTAATACTACTTCTCGATGTTCCTACGGTTTATAGACCACT

[0279] TAATACCAGTACGGCCATTTATAGCATTACTTGTCGCAAAAACCCCAGCGACGCTTGCATTTAAA

[0280] TTTGACGTCGCGTAATAACTTATGATATCAGTCGACAAACTGGCATTTATTCGCCGGCGTAGCAC

[0281] TGACTGACTGCTAGACGGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTCGAG

[0282] GGCCTCTGCCAGTGTCGAACAGACATTCGCCTACGGCCCTCGTCTGTTCGGGCAGTCCCGCGCAG

[0283] TCGCCCACAACCGCCCACAGCCCCGCGTCGGTACTGGGTCAGTGCATCGCTATCGCCTCACATAT

[0284] TAAGAACTTCTGCTTTCCCGGAGCACTATGCGGATAAAAATATCCAATTACAGTACTATTATTAC

[0285] CAAAGAATCTGCAGTCCACCGTGAAAAGCCCCTTTACACGCGCCTTGGGGATAAACAAATAAAA

[0286] AGATTTATGTAAGTTTATACATAGGCGAGTACTCTGTTATTGGGACTATTTACGAAGTTATTATAA

[0287] CTTTTTCCTTCTCATACTCATAAGTTGTAAAGGCACAGCGGGAATAAGGGAAAAAACGCCGTAAA

[0288] ACGGAAGGACAAAAACGAGTGGGTCTTTGCGACCACTTTCATTTTCTACGACTTCTAGTCAACCC

[0289] ACGTGCTCACCCAATGTAGCTTGACCTAGAGTTGTCGCCATTCTAGGAACTCTCAAAAGCGGGGC

[0290] TTCTTGCAAAAGGTTACTACTCGTGAAAATTTCAAGACGATACACCGCGCCATAATAGGGCACAA

[0291] CTGCGGCCCGTTCTCGTTGAGCCAGCGGCGTATGTGATAAGAGTCTTACTGAACCAACTCATGAG

[0292] TGGTCAGTGTCTTTTCGTAGAATGCCTACCGTACTGTCATTCTCTTAATACGTCACGACGGTATTG

[0293] GTACTCACTATTGTGACGCCGGTTGAATGAAGACTGTTGCTAGCCTCCTGGCTTCCTCGATTGGCG

[0294] AAAAAACGTGTTGTACCCCCTAGTACATTGAGCGGAACTAGCAACCCTTGGCCTCGACTTACTTC

[0295] GGTATGGTTTGCTGCTCGCACTGTGGTGCTACGGACGTCGTTACCGTTGTTGCAACGCGTTTGATA

[0296] ATTGACCGCTTGATGAATGAGATCGAAGGGCCGTTGTTAATTATCTGACCTACCTCCGCCTATTTC

[0297] AACGTCCTGGTGAAGACGCGAGCCGGGAAGGCCGACCGACCAAATAACGACTATTTAGACCTCG

[0298] GCCACTCGCACCCAGAGCGCCATAGTAACGTCGTGACCCCGGTCTACCATTCGGGAGGGCATAG

[0299] CATCAATAGATGTGCTGCCCCTCAGTCCGTTGATACCTACTTGCTTTATCTGTCTAGCGACTCTAT

[0300] CCACGGAGTGACTAATTCGTAACCATTGACAGTCTGGTTCAAATGAGTATATATGAAATCTAACT

[0301] AAATTTTGAAGTAAAAATTAAATTTTCCTAGATCCACTTCTAGGAAAAACTATTAGAGTACTGGT

[0302] TTTAGGGAATTGCACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTAGTTTCCTAGA

[0303] AGAACTCTAGGAAAAAAAGACGCGCATTAGACGACGAACGTTTGTTTTTTTGGTGGCGATGGTCG

[0304] CCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAGGCTTCCATTGACCGAAGTCGTCTC

[0305] GCGTCTATGGTTTATGACAGGAAGATCACATCGGCATCAATCCGGTGGTGAAGTTCTTGAGACAT

[0306] CGTGGCGGATGTATGGAGCGAGACGATTAGGACAATGGTCACCGACGACGGTCACCGCTATTCA

[0307] GCACAGAATGGCCCAACCTGAGTTCTGCTATCAATGGCCTATTCCGCGTCGCCAGCCCGACTTGC

[0308] CCCCCAAGCACGTGTGTCGGGTCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGTCGC

[0309] ACTCGATACTCTTTCGCGGTGCGAAGGGCTTCCCTCTTTCCGCCTGTCCATAGGCCATTCGCCGTC

[0310] CCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTTGCGGACCATAGAAATATCAGGAC

[0311] AGCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCCGCCTCGGA

[0312] TACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGAGTG

[0313] TACAAGAAAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGGAAACTCACTCGAC

[0314] TATGGCGAGCGGCGTCGGCTTGCTGGCTCGCGTCGCTCAGTCACTCGCTCCTTCGCCTTCTCGCGG

[0315] ACTACGCCATAAAAGAGGAATGCGTAGACACGCCATAAAGTGTGGCGTATTTAAGGCTGTGGTA

[0316] GCTTACCACGTTTTGGAAAGCGCCATACCGTACTATCGCGGGCCTTCTCTCAGTTAAGTCCCACC

[0317] ACTTACACTTTGGTCATTGCAATATGCTACAGCGTCTCATACGGCCACAGAGAATAGTCTGGCAA

[0318] AGGGCGCACCACTTGGTCCGGTCGGTGCAAAGACGCTTTTGCGCCCTTTTTCACCTTCGCCGCTAC

[0319] CGCCTCGACTTAATGTAAGGGTTGGCGCACCGTGTTGTTGACCGCCCGTTTGTCAGCAACGACTA

[0320] ACCGCAACGGTGGAGGTCAGACCGGGACGTGCGCGGCAGCGTTTAACAGCGCCGCTAATTTAGA

[0321] GCGCGGCTAGTTGACCCACGGTCGCACCACCACAGCTACCATCTTGCTTCGCCGCAGCTTCGGAC

[0322] ATTTCGCCGCCACGTGTTAGAAGAGCGCGTTGCGCAGTCACCCGACTAGTAATTGATAGGCGACC

[0323] TACTGGTCCTACGGTAACGACACCTTCGACGGACGTGATTACAAGGCCGCAATAAAGAACTACA

[0324] GAGACTGGTCTGTGGGTAGTTGTCATAATAAAAGAGGGTACTTCTGCCATGCGCTGACCCGCACC

[0325] TCGTAGACCAGCGTAACCCAGTGGTCGTTTAGCGCGACAATCGCCCGGGTAATTCAAGACAGAG

[0326] CCGCGCAGACGCAGACCGACCGACCGTATTTATAGAGTGAGCGTTAGTTTAAGTCGGCTATCGCC

[0327] TTGCCCTTCCGCTGACCTCACGGTACAGGCCAAAAGTTGTTTGGTACGTTTACGACTTACTCCCGT

[0328] AGCAAGGGTGACGCTACGACCAACGGTTGCTAGTCTACCGCGACCCGCGTTACGCGCGGTAATG

[0329] GCTCAGGCCCGACGCGCAACCACGCCTATAGAGCCATCACCCTATGCTGCTATGGCTTCTGTCGA

[0330] GTACAATATAGGGCGGCAATTGGTGGTAGTTTGTCCTAAAAGCGGACGACCCCGTTTGGTCGCAC

[0331] CTGGCGAACGACGTTGAGAGAGTCCCGGTCCGCCACTTCCCGTTAGTCGACAACGGGCAGAGTG

[0332] ACCACTTTTCTTTTTGGTGGGACCGCGGGTTATGCGTTTGGCGGAGAGGGGCGCGCAACCGGCTA

[0333] AGTAATTACGTCGACCGTGCTGTCCAAAGGGCTGACCTTTCGCCCGTCACTCGCGTTGCGTTAATT

[0334] ACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTGAAATACGAAGGCCGAGCATACAAC

[0335] ACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCCTAAGTGA

[0336] CCGGCAGCAAAATGTTGCAGCACTGACCCTTTTGGGACCGCAATGGGTTGAATTAGCGGAACGTC

[0337] GTGTAGGGGGAAAGCGGTCGACCGCATTATCGCTTCTCCGGGCGTGGCTAGCGGGAAGGGTTGT

[0338] CAACGCGTCGGACTTACCGCTTACCGCGAAACGGACCAAAGGCCGTGGTCTTCGCCACGGCCTTT

[0339] CGACCGACCTCACGCTAGAAGGACTCCGGCTATGACAGCAGCAGGGGAGTTTGACCGTCTACGT

[0340] GCCAATGCTACGCGGGTAGATGTGGTTGCATTGGATAGGGTAATGCCAGTTAGGCGGCAAACAA

[0341] GGGTGCCTCTTAGGCTGCCCAACAATGAGCGAGTGTAAATTACAACTACTTTCGACCGATGTCCTTCCGGTCTGCGCTTAATAAAAACTACCGCAACCTTAA.

[0342] Amino acid sequence of pGEX-FL46 (as shown in SEQ ID No. 15):

[0343] EFMQYKIVEFTGGLGNQMFQYAFAKSLEHHLQMPILLDKTWFEKEGYSIPFSLDIFNIDLPYATQEQIQKARAIEGKPKLIRSILKRVGFPRVTYVETFAYKEEYLKPNSFAYFRGYFQNPKYFQSCASKIKALFAPPPIKSAINKHKLQQILQSPNSVFIHIRRGDYVQLSWQLDIEYFKKAVDVIAHKIENPQFFLFCADREFAHNLDLGYPFVDMTSENITRDNHFEDLILMMHCQNGIISNSTYSWWAAYLINNPQKIVIAPTPWLFGNDEIVCEDWIKIKVAGIKR*

[0344] The amino acid sequence of pGEX-FL35 (as shown in SEQ ID No. 16):

[0345] MKSLKIKFVDFWPGFNPNDNFITNALKGYEIVITDTPDYLFFSIFGYSHLKYNCVKIMFVGENIVPDFNLCDYAMGFDFLNFGDRYMRLPLFLICDNFKELSVTKDFSPKRMLNRKFCSIVVSNAQVSNPIRERFFRLLSEYKQVDSGGRLWNNVGGPVADKQKFISGYKFNIAFENSAVLGYTTEKIMDAMTANTLPIYWGNPWVGRDFNKRSFVNVNSFASLEKAVEYIVDLDTNDERYLEMMQEPWVNDVSIFDWEDKLCAFLAHIVEKPFAEAQYLVDDGMQKLYKQNMKTLAFVNEKLKVPRLISAYKKLSNGKYK*

[0346] The present application also provides a plasmid containing the above-mentioned FL20-FL59 genes expressed in series and a plasmid containing the above-mentioned manB, manC, gmd, wcaG genes. The plasmid contains a vector backbone for expressing the above-mentioned FL20-FL59 genes, preferably the vector is pGEX series such as pGEX-5x, etc., pET series such as pET28a, etc., but is not limited thereto.

[0347] The present application also provides a microorganism for expressing the above-mentioned manB, manC, gmd, wcaG, FL20-FL59 genes. For example, the above-mentioned FL20-FL59 genes are cloned in the genome of the microorganism.

[0348] The microbial host can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, preferably Escherichia coli, and more preferably Escherichia coli K-12 MG1655.

[0349] The application also provides the guiding significance of the microorganism expressing the manB, manC, gmd, wcaG and FL20-FL59 genes in the production of DFL, in particular, the production of difucosyllactose by fermentation of the microorganism.

[0350] The experimental methods in the embodiments of the application are carried out under conventional conditions, and refer to the experimental methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Fourth Edition, 2017, Science Press). The materials, reagents and the like used in the embodiments can be obtained from commercial channels.

[0351] The DNA used in the embodiments of the application is synthesized by a commercial biotechnology company, such as Kingsriver Biotech Co., Ltd., after codon optimization. The DNA sequences of manB, manC, gmd and wcaG are obtained by PCR amplification using the genomic DNA of Escherichia coli K-12 MG1655 strain as a template.

[0352] The application provides:

[0353] 1) An alpha-1,3-fucosyltransferase homologous enzyme, which is a fucosyltransferase from Bacteroides gallinaceum, responsible for the reaction of GDP-fucose and lactose (or 2'-fucose lactose) at position 3 to generate DFL.

[0354] 2) A fucosyltransferase combination that can not only produce high yield of DFL but also effectively reduce the yield of 2'FL: FL20-FL59.

[0355] 3) A recombinant plasmid combination for expressing manB, manC, gmd, wcaG, FL20, FL46, FL35 and FL59 genes, including the plasmid itself and the combination.

[0356] 4) The above-mentioned engineering strains for construction are all Escherichia coli K-12 MG1655.

[0357] The beneficial effects of the application include but are not limited to:

[0358] 1. A new efficient alpha-1,3-fucosyltransferase (FL59) is provided. FL59 is derived from Bacteroides gallinaceum, which improves the catalytic efficiency compared with FL35, and is a rate-limiting enzyme for 3-FL production. The use of FL59 can improve the conversion rate of 3-FL.

[0359] 2. The engineering strain for expressing manB / manC / gmd / wcaG and FL20-FL59 in series is constructed. The enzymes in the GDP-fucose biosynthesis pathway and the fucosyltransferases are expressed in series to balance the substrate competition among different transferases, avoid excessive accumulation of 2'FL, and synergistically improve the yield of DFL.

[0360] 3. The excellent performance of the engineering strain is verified in shake flask fermentation. The yield is increased by 5.7 times. It is reported that the yield is low, only 5.1 g / L, using expensive fucose as the substrate (Zhang A, et al. 2021. "Microbial production of human milk oligosaccharide lactodifucotetraose". Metab. Eng. 66: 12-20; Sophie Issartel et al., CN 116348610A).

[0361] 4. The process of the application is simple and controllable, the raw materials are widely available, and has good application prospect.

[0362] The raw materials and reagents used in the enzymes, enzyme compositions, expression frames, recombinant plasmids and strains for producing DFL provided by the application can be purchased from the market.

[0363] The application will be further described below in combination with examples:

[0364] Example 1 Construction and identification of recombinant chassis strain

[0365] 1. Construction of recombinant chassis strain

[0366] 1) Plasmid construction and identification of recombinant strain

[0367] The plasmid pAC (pACYCDuet-1, product number: BioVector-0147603) is 2.4 kb in size, contains a chloramphenicol resistance gene, a p15Aori replication initiation site, a tac promoter, and multiple restriction enzyme sites. The DNA fragments of manB, manC, gmd, and wcaG are connected to the pAC plasmid, and the nucleotide sequence is shown in SEQ ID No. 8, Figure 1

[0368] ​The target DNA fragment was ligated to the vector using a homologous recombination kit (manufacturer: Yixing, catalog number: 10923ES20 / 50). The ligation product was transformed into E. coli host strain DH5a using heat shock method, purchased from Thermo Fisher Scientific, catalog number 18265017. Spread on LB solid medium. Culture until the growth of transformants, pick single colony, incubate in LB liquid medium at 37°C overnight, centrifuge at 12000 rpm for 1 min, extract plasmid, and the extraction method is according to the kit (manufacturer: Yixing, catalog number: 19021ES50) instruction.

[0369] The recombinant vector was digested with EcoR I and Nde I, and the DNA fragment containing the target gene cluster was determined by electrophoresis. The sequence of the target plasmid was verified by sequencing.

[0370] 2. Construction of pAC-CBGW recombinant vector containing the chassis strain

[0371] The recombinant vector was transformed into E. coli host strain K-12 MG1655 using heat shock method, purchased from Thermo Fisher Scientific, catalog number C601003. Pick single colony of transformants.

[0372] 3. Monoclonal verification:

[0373] Pick single colony from the resistant plate, inoculate into 2 mL of LB liquid resistant medium, incubate at 37°C for 4 h, and obtain bacterial solution. Prepare PCR system using specific primers of target DNA fragment, detect PCR product by agarose gel electrophoresis, and observe the corresponding size of target band.

[0374] Primer sequence:

[0375] N-terminal ATGGCGCAGTCGAAACTCTATCCAG (as shown in SEQ ID No. 17);

[0376] C-terminal TTACCCCCGAAAGCGGTCTTGATTC (as shown in SEQ ID No. 18).

[0377] The electrophoresis gel map is shown in Figure 14 .

[0378] Example 2 Verification of expression level of pGEX-FL46, pGEX-FL20, pGEX-FL35, and pGEX-FL59 recombinant vectors containing target genes

[0379] 1) Plasmid construction

[0380] The plasmid pGEX-5x is 5.8 kb in size, contains an ampicillin resistance gene, a lactose repressor lac I gene, an ori replication initiation site, a tac promoter, and has multiple restriction enzyme sites. Commercially synthesized fucosyltransferase gene DNA fragments were ligated to the pGEX-5x plasmid. There were pGEX-FL46, pGEX-FL20, pGEX-FL35, and pGEX-FL59, respectively, and the corresponding nucleotide sequences are shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12, Figure 2 , Figure 3 , Figure 4 , Figure 5 The amino acid sequence of FL46 is shown in SEQ ID No. 15, and the amino acid sequence of FL35 is shown in SEQ ID No. 16.

[0381] 2) Construction of a single transferase-producing strain

[0382] The above recombinant vectors were transformed into the chassis strain obtained in Example 1 using the heat shock method. They were plated on LB solid medium and incubated at 37°C until the transformants grew. Single colonies were picked and named 2FL-A, 2FL-B, 3FL-A, and 3FL-B.

[0383] Three single colonies of each of the obtained strains 2FL-A, 2FL-B, 3FL-A, and 3FL-B were selected and cultured in 5 mL of LB medium until the OD600 was about 0.6-0.8. IPTG inducer was added to a final concentration of 0.3 mM and the culture was induced for 18 h. The bacterial cells were collected and resuspended with PBS buffer, then ultrasonically broken, and subjected to SDS-PAGE analysis. Each target gene was expressed (see Figures 6-9 ).

[0384] 3) Shake flask fermentation of a single transferase-producing strain

[0385] Strawberries 2FL-A, 2FL-B, 3FL-A, and 3FL-B were streaked onto plates, and 2-3 single clones were transferred to LB tubes. The inoculum was cultured at 37°C with shaking at 220 rpm for approximately 14 hours. Then, the inoculum was transferred to shake flasks at a 1% (v / v) inoculation rate and cultured at 37°C with shaking at 220 rpm for approximately 12 hours. Finally, a 4% (v / v) inoculation was transferred to fermentation shake flasks and cultured at 30°C with shaking at 220 rpm for approximately 72 hours. The shake flask culture medium consisted of: 1 g / L citric acid monohydrate, 2 g / L diammonium hydrogen phosphate, 6.75 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate heptahydrate, 7.5 g / L yeast extract, 20 g / L glycerol, 10 g / L lactose, 10 mL / L TES trace element solution, and water to a final volume of 1 L. Sampling was conducted to determine the contents of 2'-FL and 3-FL (2FL-A and 2FL-B produced 2'-FL; 3FL-A and 3FL-B produced 3FL). Fermentation results are shown in [reference needed]. Figure 10 Table 1.

[0386] 4) High-performance liquid chromatography (HPLC) detection of 2'FL and 3FL:

[0387] Sample preparation: Centrifuge 1 ml of fermentation broth at 12000 rpm for 10 min. Collect the supernatant, dilute 10-fold with mobile phase, purify by filtration through a 0.22 μmL filter membrane, and perform HPLC analysis.

[0388] HPLC detection conditions: High-performance liquid chromatography (HPLC, Agilent 1260 Infinity) was used, equipped with a Rocksil Carbohydrate ES 5u (250mm × 4.6mm) sugar column and an evaporative light detector. Elution flow rate: 0.8 mL / min; elution time: 30 min; column temperature: 40℃; mobile phase A: 70% acetonitrile (v / v), mobile phase B: 30% water (v / v); injection volume: 10 μL. Evaporative light detector temperature: 90℃; air flow rate: 2.2 L / min. This method can also be used to detect lactose and other substances in the fermentation broth.

[0389] Table 1. Yield and corresponding lactose conversion rate of strain during 72h shake-flask fermentation.

[0390] Strain name Yield g / L Conversion (molar ratio lactose) 2FL-A 7.51 0.52 2FL-B 11.39 0.84 3FL-A 3.43 0.55 3FL-B 11.50 0.88

[0391] Depend on Figure 10 As shown in Table 1, the constructed genetically engineered strain 3FL-B screened in this invention improved the lactose conversion rate of the control strain 3FL-A, thereby enabling lactose to be converted into 3-fucolactose more effectively, with a yield 3.35 times higher than that of 3FL-A.

[0392] Example 3: Construction of DEL engineered strain

[0393] 1) Plasmid construction

[0394] Commercially synthesized pGEX-FL46-FL59 and pGEX-FL20-FL59 fucosyltransferase gene DNA fragments were ligated to pGEX-5x plasmid. The corresponding nucleotide sequences are shown in SEQ ID No. 13, SEQ ID No. 14, Figure 11 、 Figure 12 .

[0395] 2) Construction of DFL production strain

[0396] The above ethnic plasmid was transformed into the chassis strain in Example 1 according to the method in Example 2 to obtain DFL-1 and DFL-2 production strains.

[0397] Example 4 Shake flask fermentation of DEL engineering strain

[0398] The strain obtained in Example 3 was subjected to shake flask fermentation according to the method in Example 2, and the results are shown in Table 2. Figure 13 、.

[0399] Table 2 72h shake flask results of DFL fermentation strain

[0400] g / L DFL yield 2FL yield DFL-1 1.159 5.741 DFL-2 6.445 3.694

[0401] As shown in Table 2, the yield of the genetically engineered strain DFL-2 constructed by the application in shake flask fermentation can reach 6.445g / L, which is 5.7 times that of the control strain. By screening a more efficient α-1,3 fucosyltransferase and selecting the most suitable α-1,2 fucosyltransferase, the effect of significantly improving the target product DFL can be achieved, and the production of byproduct 2'-FL is also effectively reduced, which facilitates the subsequent separation and purification work of the target product.

[0402] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Enzyme composition, characterized in that, comprises an α-1,2-fucosyltransferase and an α-1,3-fucosyltransferase; the α-1,3-fucosyltransferase is derived from Bacteroides gallinaceum, and has an amino acid sequence as shown in SEQ ID No. 1; the α-1,2-fucosyltransferase is derived from Helicobacter pylori, and has an amino acid sequence as shown in SEQ ID No.

2.

2. The enzyme composition of claim 1, wherein, the enzyme composition further comprises a tandemly expressed fucosyltransferase FL20-FL59, which has an amino acid sequence as shown in SEQ ID No.

3.

3. The enzyme composition of claim 2, wherein, the enzyme composition further comprises one or more of ManB, ManC, Gmd or WcaG; the ManB has an amino acid sequence as shown in SEQ ID No. 4; the ManC has an amino acid sequence as shown in SEQ ID No. 5; the Gmd has an amino acid sequence as shown in SEQ ID No. 6; the WcaG has an amino acid sequence as shown in SEQ ID No.

7.

4. A nucleic acid molecule encoding the enzyme composition according to any one of claims 1 to 3.

5. An expression cassette characterized in that, comprising the nucleic acid molecule according to claim 4.

6. A recombinant plasmid characterized in that, comprising the expression cassette according to claim 5.

7. The recombinant plasmid of claim 6, wherein the recombinant plasmid further comprises a vector backbone, which comprises but is not limited to one or more of pAC series, pGEX series and / or pET series.

8. The recombinant plasmid of claim 6, wherein the vector backbone of the recombinant plasmid is one or more of pAC, pGEX-5x and / or pET28a.

9. The recombinant plasmid of claim 6, wherein the vector backbone of the recombinant plasmid is pAC or pGEX-5x.

10. A strain, characterized in that, the strain: (I) expressing the enzyme composition according to any one of claims 1 to 3; and / or (II) transformed or transfected with the expression cassette according to claim 5; and / or (III) transformed or transfected with the recombinant plasmid according to any one of claims 6 to 9.

11. The strain of claim 10, wherein the strain is a Lactobacillus strain. the strain is one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

12. The strain of claim 10, wherein, the strain is Escherichia coli.

13. The strain of claim 10, wherein, the strain is Escherichia coli W3110 or Escherichia coli K-12 MG1655.

14. Use of any of the following in the synthesis of 3-fucosyllactose and / or difucosyllactose: (i) the enzyme composition according to any one of claims 1 to 3; and / or (ii) the expression cassette according to claim 5; and / or (iii) the recombinant plasmid according to any one of claims 6 to 9; (iv) the strain according to any one of claims 10 to 13.

15. A method for the synthesis of 3-fucosyllactose and / or difucosyllactose, characterized in that, picking the strain according to any one of claims 10 to 13, fermenting, collecting the fermentation broth, and obtaining 3-fucosyllactose and / or difucosyllactose.

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