Engineering strain and application thereof in preparation of pneumocandins

Abstract

The invention discloses an engineering strain and application of the engineering strain in preparation of pneumocandin. The engineering strain does not express a GloA-A2 gene, and expresses a non-ribosomal peptide synthetase gene CENRPS-A2 or CCNRPS-A2, so that the proportion of PB0-ser in a fermentation product can be adjusted, the generation of PB0-ser and PB0 is controlled from the source, and the cost for separating PB0-ser and PB0 is reduced.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an engineered strain and its application in the preparation of nimocontin. Background Technology

[0002] Pneumocandins belong to the echinocandin class of antibiotics. These antibiotics non-competitively inhibit β-(1,3)-D-glucan synthase, which is involved in fungal cell wall synthesis. Due to their mechanism of action, this class of antibiotics has the advantage of low toxicity and side effects. Currently, marketed echinocandin antibiotics include caspofungin, anidoxurine, and micafungin, primarily used for the treatment of deep fungal infections.

[0003] Pneumocandin is a series of structurally similar compounds produced by the filamentous fungus *G. lozoyensis*. Among them, pneumocandin B0 (PB0) exhibits good antifungal activity and can be chemically modified to produce caspofungin. PB0 (as shown in Formula I below) serves as an intermediate in the synthesis of caspofungin, and its purity requires strict control. Due to the complexity of microbial metabolism, this strain produces a series of structurally similar compounds along with PB0. Among these, pneumocandin B0 contains a serine analogue PB0. 0-ser The structural difference between (as shown in Formula II) and PB0 lies in the second amino acid residue on the ring. PB0 is Thr. The two have similar chemical structures, so it is very necessary to control their production from the source through strain modification.

[0004]

[0005]

[0006] PB 0-ser Separating it from PB0 is quite difficult, and in production, it is separated using a normal chromatographic column, which is costly. Summary of the Invention

[0007] This invention verifies that replacing the A2 domain of the nonribosomal peptide synthase GloA (GloA-A2) in the existing strain G. lozoyensis ATCC 74030 with the nucleotide sequence of the nonribosomal peptide synthase domain (CENRPS-A2, CE-A2) from the existing strains Coleophoma empetri (C. empetri) F-11899 or C. empetri SIPI 1284 can reduce PB. 0-serThe content of GloA-A2 gene was increased; in addition, the GloA-A2 gene was replaced with a non-ribosomal peptide synthase domain (CCNRPS-A2, abbreviated as CC-A2) derived from the existing strain Coleophoma crateriformis no. 738, and the main fermentation product PB was reduced. 0-ser Based on this, the present invention provides an engineered strain and its application in the preparation of nimocontin.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0009] A first aspect of the present invention is to provide a polypeptide fragment comprising a sequence as shown in SEQ ID NO:18 or SEQ ID NO:26.

[0010] In some embodiments of the present invention, the amino acid sequence of the polypeptide fragment is shown as SEQ ID NO:18, SEQ ID NO:22 or SEQ ID NO:26.

[0011] A second aspect of the present invention is to provide a recombinant protein comprising the polypeptide fragment described in the first aspect of the present invention.

[0012] In some embodiments of the present invention, the amino acid sequence of the recombinant protein is shown as SEQ ID NO:20, SEQ ID NO:24 or SEQ ID NO:28.

[0013] A third aspect of the present invention is to provide a nucleic acid encoding a polypeptide fragment as described in the first aspect of the present invention or a recombinant protein as described in the second aspect of the present invention.

[0014] In some embodiments of the present invention, the nucleic acid sequence encoding the polypeptide fragment is as shown in SEQ ID NO:19, SEQ ID NO:23 or SEQ ID NO:27; and / or

[0015] The nucleic acid sequence encoding the recombinant protein is shown in SEQ ID NO:21, SEQ ID NO:25 or SEQ ID NO:29.

[0016] A fourth aspect of the present invention is to provide a recombinant vector containing the nucleic acid described in the third aspect of the present invention.

[0017] In some embodiments of the present invention, the 5' and 3' ends of the nucleic acid encoding the polypeptide fragment have the 5' and 3' ends of the GloA-A2 gene, for use in homologous recombination of the nucleic acid encoding the polypeptide fragment with the GloA-A2 gene.

[0018] A fifth aspect of the present invention is to provide a combination of recombinant vectors, the combination comprising the recombinant vectors described in the fourth aspect of the present invention, namely a first vector; and a second vector containing nucleic acid encoding a Cas protein.

[0019] In some embodiments of the present invention, the Cas protein is Cas 9, Cas 12a, or Cas 13.

[0020] In some specific embodiments of the present invention, the second vector is plasmid pDHt / sk-PC.

[0021] A sixth aspect of the present invention is to provide a gene editing system, the gene editing system comprising:

[0022] (1) The nucleic acid described in the third aspect of the present invention or the recombinant vector described in the fourth aspect of the present invention;

[0023] (2) Cas protein such as Cas 9, Cas 12a or Cas 13; nucleic acid encoding the Cas protein; or a second vector containing nucleic acid encoding the Cas protein, such as plasmid pDHt / sk-PC;

[0024] Optional additions include (3) sgRNA targeting the gloA gene.

[0025] In some embodiments of the present invention, the sgRNA targets the A2 domain of the GloA protein, and the gene sequence of the sgRNA is, for example, as shown in SEQ ID NO:30; and / or, the sgRNA is located in a vector, i.e., a third vector; and / or, the gloA gene is derived from the filamentous fungus G. lozoyensis, for example, G. lozoyensis ATCC 74030.

[0026] A seventh aspect of the present invention is to provide a genetically engineered strain that does not express the GloA-A2 gene, but expresses the non-ribosomal peptide synthase gene CENRPS-A2 or CCNRPS-A2.

[0027] In some embodiments of the present invention, the CENRPS-A2 or CCNRPS-A2 is integrated into the GloA-A2 gene at a specific site.

[0028] In some specific embodiments of the present invention, the nonribosomal peptide synthase gene CENRPS-A2 is derived from Coleophoma empetri F-11899 or Coleophoma empetri SIPI 1284; and / or, the nonribosomal peptide synthase gene CCNRPS-A2 is derived from Coleophoma crateriformis no.738.

[0029] In some specific embodiments of the present invention, the starting strain of the engineered strain is G. lozoyensis, for example G. lozoyensis ATCC 74030; and / or, the nucleotide sequence of the CENRPS-A2 is as shown in SEQ ID NO:19 or SEQ ID NO:23; and / or, the nucleotide sequence of the CENRPS-A2 is as shown in SEQ ID NO:27.

[0030] The eighth aspect of the present invention is to provide a method for preparing the genetically engineered strain described in the seventh aspect of the present invention, obtained by editing the filamentous fungus G. lozoyensis (e.g., G. lozoyensis ATCC 74030) using the gene editing system described in the sixth aspect of the present invention.

[0031] The ninth aspect of the present invention is to provide the polypeptide fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the nucleic acid described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the combination described in the fifth aspect of the present invention, the gene editing system described in claim 6, or the genetically engineered strain described in the seventh aspect of the present invention in the preparation of Numococtin B0 or ​​Numococtin B. 0-ser Applications in [the context of the text].

[0032] The tenth aspect of the present invention is to provide a method for preparing Numococtin B0 or ​​Numococtin B. 0-ser A method comprising culturing the genetically engineered strain of claim 7 in a culture medium, fermenting it, and producing Numococtin BO or Numococtin B. 0-ser .

[0033] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0034] The reagents and raw materials used in this invention are all commercially available.

[0035] The positive and progressive effects of this invention are as follows:

[0036] This invention modulates the PB content in fermentation products by disabling the expression of the A2 domain of the nonribosomal peptide synthase GloA in the existing strain G. lozoyensis ATCC 74030 and expressing the nonribosomal peptide synthase gene CENRPS-A2 or CCNRPS-A2. 0-ser The ratio, controlling PB from the source 0-ser The generation of PB0 reduces the separation of PB. 0-ser And the cost of PB0. Attached Figure Description

[0037] Figure 1The HPLC chromatogram of G. lozoyensis (PC) strain is shown.

[0038] Figure 2 The HPLC chromatogram of G. lozoyensis ATCC 74030 strain.

[0039] Figure 3 This is the pAgG plasmid map.

[0040] Figure 4 The HPLC chromatogram of the control strain Gl-pAgG is shown.

[0041] Figure 5 This is a map of plasmid pAgG-sgRNA-gloA-A2.

[0042] Figure 6 The image shows the spectrum of plasmid pAgG-CE-A2-1.

[0043] Figure 7 The HPLC chromatogram of G. lozoyensis (CE-A2)-1 strain is shown.

[0044] Figure 8 The image shows the spectrum of plasmid pAgG-CEA2-2.

[0045] Figure 9 The HPLC chromatogram of G. lozoyensis (CE-A2)-2 strain is shown.

[0046] Figure 10 This is the spectrum of plasmid pAgG-CC-A2.

[0047] Figure 11 The HPLC chromatogram of G. lozoyensis (CC-A2) strain. Detailed Implementation

[0048] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0049] The primers used in the embodiments of the present invention are shown in Table 1 below.

[0050] Table 1 Primer sequences

[0051]

[0052] 2. Preparation of the culture medium used in this invention

[0053] 1. Seed culture medium formula (1L):

[0054] 20g glucose, 10g soybean meal powder, KH2PO4. pH value 5.5; sterilize at 121℃ for 20 minutes.

[0055] 2. Fermentation medium formula (1L):

[0056] Mannitol 100g, cottonseed meal powder 5g, soybean meal powder 10g, K2HPO4 4g, CaCO3 1g, L-Pro 20g. pH value 6.5; sterilize at 121℃ for 20 minutes.

[0057] 3. HPLC detection method

[0058] Add four times the volume of ethanol to the fermentation broth, sonicate for 30 min to extract the product to the extracellular space, take 1 mL of the extract, centrifuge at 12,000 rpm for 10 min, filter the supernatant through a 0.2 μm filter membrane, and perform HPLC analysis. The analytical method is as follows:

[0059] Chromatographic column: YMC J SPHERE ODS-M80 column (4.6×250mm)

[0060] Detection wavelength: 210nm

[0061] Column temperature: 30℃

[0062] Flow rate: 1.5 mL / min

[0063] The gradient methods are shown in Table 2 below:

[0064] Table 2 Gradient elution program

[0065]

[0066] Example 1: Construction of strain G. lozoyensis (PC)

[0067] The plasmid pDHt / sk-PC (Addgene plasmid#92125; http: / / n2t.net / addgene:92125; RRID:Addgene_92125) was introduced into G. lozoyensis ATCC 74030 strain using Agrobacterium-mediated transformation (AMT) to obtain the engineered strain G. lozoyensis (PC).

[0068] To detect the PB of the Cas9-expressing strain G. lozoyensis (PC) 0-serThe content was determined by inoculating the culture medium into 20 mL of seed culture medium and incubating it at 25°C and 220 rpm for 4 days. Then, the culture medium was inoculated into 30 mL of fermentation culture medium at a 10% inoculation rate and incubated at 25°C and 220 rpm for 10 days.

[0069] The content of PB0 and PB in the fermentation broth was determined by HPLC. 0-ser / (PB 0-ser +PB0).

[0070] The results are as follows Figure 1-2 As shown, the results indicated that the PB0 yield in the fermentation products of the control strain G. lozoyensis ATCC 74030 and strain G. lozoyensis (PC) was 2528.7 mg / L. 0-ser / (PB 0-ser +PB0) was 2.4% ± 0.1%.

[0071] Example 2: Construction of plasmid pAgG and strain Gl-pAgG

[0072] Construction of pAgG: Using pAg1-H3 (Novopro, V014855) as a template, PCR was performed with primers Ptrpc-F / R to obtain the trpC promoter fragment. Using plasmid pEGFP-N2 (accession number U57608.1 in the NCBI database) as a template, PCR was performed with primers NeoR-F / R to obtain the Neo fragment of the G418 resistance gene. Using pAg1-H3 as a template, PCR was performed with primers Ttrpc-F / R to obtain the trpC terminator fragment. Using the trpC promoter fragment, the Neo fragment of the G418 resistance gene, and the trpC terminator fragment as templates, overlap PCR was performed with primers Ptrpc-F and Ttrpc-R to obtain the G418 resistance gene expression cassette. This expression cassette was ligated into the HindIII / SpeI linearized pAg1-H3 vector to finally obtain the plasmid pAg1-HG. pAg1-HG was self-ligated after single digestion with EcoRI to obtain the plasmid pAgG (e.g., pAg1-HG). Figure 3 (As shown).

[0073] The plasmid pAgG was introduced into G. lozoyensis strain 74030 using AMT to obtain the engineered strain Gl-pAgG.

[0074] To detect the PB of the control strain Gl-pAgG 0-ser The content was determined by inoculating the culture medium into 20 mL of seed culture medium and incubating it at 25°C and 220 rpm for 4 days. Then, the culture medium was inoculated into 30 mL of fermentation culture medium at a 10% inoculation rate and incubated at 25°C and 220 rpm for 10 days.

[0075] The content of PB0 and PB in the fermentation broth was determined by HPLC. 0-ser / (PB 0-ser +PB0).

[0076] The results are as follows Figure 4 As shown, the results indicated that the PB0 yield in the fermentation product of strain Gl-pAgG was 2530 mg / L, and PB... 0-ser / (PB 0-ser +PB0) was 2.4% ± 0.1%.

[0077] Example 3: Construction of plasmid pAgG-CE-A2-1 and strain G. lozoyensis (CE-A2)-1

[0078] Constructing an sgRNA expression plasmid targeting the GloA-A2 gene: Using the genome of strain G. lozoyensis ATCC 74030 as a template, 5S rRNA and gloA-A2-sgRNA gene sequences (SEQ ID NO:30) were amplified using 5S-F / R. The complete gene sequence was synthesized. The 5S rRNA and gloA-A2-sgRNA fragments were homologously recombinated with the Bgl II and EcoRI-linearized vector pAgG to obtain the plasmid pAgG-sgRNA-gloA-A2 (e.g., pAgG-sgRNA-gloA-A2). Figure 5 (As shown).

[0079] The plasmid pAgG-CE-A2-1 was constructed by replacing the GloA-A2 gene with the CENRPS-A2-1 gene, i.e., replacing 365 amino acids (the sequence is shown in SEQ ID NO:18, and the nucleotide sequence encoding it is shown in SEQ ID NO:19). Figure 6(as shown in SEQ ID NO:20) to express the recombinant protein GLNRPS-CEA2-1 (encoding the nucleotide sequence shown in SEQ ID NO:21) in the final constructed strain. The specific steps are as follows: Using the genome of G. lozoyensiATCC 74030 as a template, PCR was performed using primer pairs gloA-A2-LF / R and gloA-A2-RF-1 / R-1 to obtain two homologous arms, arm-1 and arm-2, for homologous double crossover at the target gene. Using the genome of the existing strain C. empetri F-11899 as a template, PCR was performed using primer pairs CE-A2-F / CE-A2-R-1 to obtain fragment CE-A2-1. The three fragments arm-1, arm-2, and CE-A2-1 were homologously recombinated with the vector pAgG-sgRNA-gloA-A2, which was linearized by Bgl II and EcoRI, to obtain plasmid pAgG-CE-A2-1, which replaces the A2 domain of the GloA protein with the CE-A2 domain.

[0080] The plasmid pAgG-CE-A2-1 was introduced into the G. lozoyensis (PC) strain using AMT. The resulting transformants were verified by PCR to obtain the engineered strain G. lozoyensis (CE-A2)-1.

[0081] To confirm the PB of G. lozoyensis(CE-A2)-1 strain 0-ser The content was determined by inoculating the culture medium into 20 mL of seed culture medium and incubating it at 25°C and 220 rpm for 4 days. Then, the culture medium was inoculated into 30 mL of fermentation culture medium at a 10% inoculation rate and incubated at 25°C and 220 rpm for 10 days.

[0082] The content of PB0 and PB in the fermentation broth was determined by HPLC. 0-ser / (PB 0-ser +PB0).

[0083] The results are as follows Figure 7 As shown, the results indicated that the yield of PB0 in the fermentation product of G. lozoyensis (CE-A2)-1 was 2578 mg / L, and PB... 0-ser / (PB 0-ser +PB0) was 1.6% ± 0.1%.

[0084] Example 4: Construction of plasmid pAgG-CE-A2-2 and strain G. lozoyensis(CE-A2)-2

[0085] Construct plasmid pAgG-CE-A2-2 by replacing the GloA-A2 gene with the CENRPS-A2-2 gene, i.e., replacing 537 amino acids (sequence shown in SEQ ID NO:22, nucleotide sequence encoding it shown in SEQ ID NO:23). Figure 8 (as shown in SEQ ID NO:24) to express the recombinant protein GLNRPS-CEA2-2 (encoding the nucleotide sequence shown in SEQ ID NO:25) in the final constructed strain. The specific procedures are as follows: Using the genome of G. lozoyensis ATCC74030 as a template, PCR was performed using primer pairs gloA-A2-LF / R and gloA-A2-RF-2 / R-2 to obtain two homologous arms, arm-1 and arm-3, for homologous double crossover at the target gene; using the genome of the existing strain C. empetri SIPI 1284 as a template, PCR was performed using primer pairs CE-A2-F / CE-A2-R-2 to obtain fragment CE-A2-2; the three fragments arm-1, arm-3, and CE-A2-2 were homologously recombinated with the vector pAgG-sgRNA-gloA-A2 linearized with Bgl II and EcoRI to obtain plasmid pAgG-CE-A2-2, which replaces the A2 domain of the GloA protein with the CE-A2 domain.

[0086] The plasmid pAgG-CE-A2-2 was introduced into the G. lozoyensis (PC) strain using AMT. The transformed strain was verified by PCR to obtain the engineered strain G. lozoyensis (CE-A2)-2.

[0087] To confirm the PB of G. lozoyensis(CE-A2)-2 strain 0-ser The content was determined by inoculating the culture medium into 20 mL of seed culture medium and incubating it at 25°C and 220 rpm for 4 days. Then, the culture medium was inoculated into 30 mL of fermentation culture medium at a 10% inoculation rate and incubated at 25°C and 220 rpm for 10 days.

[0088] The content of PB0 and PB in the fermentation broth was determined by HPLC. 0-ser / (PB 0-ser +PB0).

[0089] The results are as follows Figure 9 As shown, the results indicated that the yield of PB0 in the fermentation product of G. lozoyensis (CE-A2)-2 was 2463 mg / L, and PB... 0-ser / (PB 0-ser +PB0) was 1.6% ± 0.1%.

[0090] Example 5: Construction of plasmid pAgG-CC-A2 and strain G. lozoyensis (CC-A2)

[0091] A plasmid pAgG-CC-A2 was constructed by replacing the GloA-A2 gene with the CCNRPS-A2 gene, specifically by replacing 365 amino acids (sequence SEQ ID NO:26, the nucleotide sequence encoding it is shown in SEQ ID NO:27), so that the recombinant protein GLNRPS-CCA2 (the nucleotide sequence encoding it is shown in SEQ ID NO:29) with the amino acid sequence shown in SEQ ID NO:28 was expressed in the final strain. The specific procedures are as follows: Using the genome of *G. lozoyensis* ATCC 74030 as a template, PCR was performed using primer pairs gloA-A2-LF / R and gloA-A2-RF-1 / R-1 to obtain two homologous arms, arm-1 and arm-2, for homologous double crossover at the target gene; the full CC-A2 gene was synthesized; the three fragments arm-1, arm-2, and CC-A2 were homologously recombinated with the vector pAgG-sgRNA-gloA-A2 linearized with Bgl II and EcoRI to obtain the plasmid pAgG-CC-A2, which replaces the GloA protein A2 domain with the CC-A2 domain (e.g., gloA-A2-RF-1 / R-1). Figure 10 (As shown).

[0092] The plasmid pAgG-CC-A2 was introduced into the G. lozoyensis (PC) strain using AMT. The resulting transformants were verified by PCR to obtain the engineered strain G. lozoyensis (CC-A2).

[0093] To confirm the PB of G. lozoyensis (CC-A2) strain 0-ser The content was determined by inoculating the culture medium into 20 mL of seed culture medium and incubating it at 25°C and 220 rpm for 4 days. Then, the culture medium was inoculated into 30 mL of fermentation culture medium at a 10% inoculation rate and incubated at 25°C and 220 rpm for 10 days.

[0094] Detection of PB in fermentation broth using HPLC 0-ser The content of.

[0095] The results are as follows Figure 11 As shown, the results indicate that PB in the fermentation products of G. lozoyensis (CC-A2) contains... 0-ser The yield was 2118 mg / L.

[0096] The sequences used in this invention are as follows:

[0097] 1. A partial amino acid sequence of the A2 domain of the CENRPS protein encoded by gene fragment CENRPS-A2-1 (SEQ ID NO:18):

[0098] QEQTKIRPNATAICAWDGDLTYGELDTQATLLARYLTSLGACSQRMIGVCMDKSKFAGISMLAILKAGAVVVPLGVNHPQGRIEGIVDDTETDIILVDEKQRERLAILRAKLITVDADLLKELPVLADKQVLQNSTEPHHAAWIIYTSGSTGKPKGVVLQHVALCSSIKAHGARFGMNSKTR MLQFAAHTFDACIQDYFTTLSWGGVVCVPSENDRMSDLTAVMHEMGVTFATLTSTVARLINPQEVPSMQLIALVGEPVKADVVEQWLGHATVLNAYGPSECSIHSSCGEPLIDATKSSVIGTGMGTRLWVVDTDYNQLCPIGAPGELLIEGPLLAREYLNDAKKTRAAFVSDPRFAQNFDLPS

[0099] 2. Nucleotide sequence of gene fragment CENRPS-A2-1 (SEQ ID NO:19):

[0100] CAAGAACAAACCAAGATTCGCCCCAACGCAACTGCCATTTGCGCATGGGATGGTGACCTCACTTATGGTGAGCTCGATACACAAGCCACATTGTTAGCCCGATACTTGACAAGCCTTGGTGCGTGTTCGCAGCGGATGATTGGTGTTTGCATGGACAAGTCCAAGTTTGCAGGCATCTCGATGCTTGCTATATTGAAAGCTGGAGCAGTAGTGGTACCACTCGGCGTCAATCATCCACAAGGGCGTATAGAGGGCATTGTTGACGACACCGAAACAGACATAATTTTGGTGGATGAGAAACAGCGAGAAAGACTCGCCATCCTAAGAGCCAAGCTGATCACCGTCGACGCAGATCTGCTAAAGGAACTTCCCGTCCTTGCGGATAAGCAAGTACTACAAAATAGCACCGAACCGCACCATGCTGCATGGATCATTTATACTTCCGGTAGCACTGGCAAGCCAAAGGGAGTTGTGTTACAGCATGTTGCCCTTTGCAGCAGTATAAAAGCCCACGGTGCTAGATTCGGCATGAATTCCAAGACCAGGATGTTGCAATTTGCAGCGCATACATTTGATGCTTGCATTCAAGATTATTTCACCACATTGTCCTGGGGGGGTGTGGTGTGCGTGCCAAGCGAGAACGACCGAATGAGCGACTTGACAGCCGTTATGCACGAAATGGGAGTCACTTTTGCTACACTTACCTCCACTGTAGCACGACTGATCAACCCTCAGGAGGTGCCATCAATGCAACTAATCGCTCTCGTAGGCGAGCCAGTCAAAGCGGACGTCGTGGAGCAGTGGCTGGGCCATGCCACAGTTCTCAATGCCTACGGACCCTCGGAATGCTCCATCCATTCCTCATGTGGTGAACCCCTCATAGATGCAACAAAATCATCAGTTATTGGTACAGGAATGGGGACAAGACTATGGGTTGTCGACACCGACTACAATCAACTCTGCCCA ATAGGAGCTCCTGGAGAATTGCTCATT GAAGGTCCACTATTAGCACGAGAATATCTCAATGATGCTAAGAAAACCAGAGCTGCATTTGTTTCGGATCCTCGTTTCGCACAAAACTTCGACCTGCCGTCC

[0101] 3. The amino acid sequence of the recombinant protein GLNRPS-CEA2-1 obtained by replacing the amino acid sequence of the A2 domain of the GLOA protein with a partial amino acid sequence of the A2 domain of the CENRPS protein (SEQ ID NO:20):

[0102]

[0103] 4. Nucleotide sequence encoding recombinant protein GLNRPS-CEA2-1 (SEQ ID NO:21):

[0104]

[0105] 5. The amino acid sequence of the A2 domain of the CENRPS protein encoded by gene fragment CENRPS-A2-2 (SEQ ID NO: 22):

[0106] SNWSKTTPVRIERCIHELVQEQTKIRPNATAICAWDGDLTYGELDTQATLLARYLTSLGACSQRMIGVCMDKSKFAGISMLAILKAGAVVVPLGVNHPQGRIEGIVDDTETDIILVDEKQRERLAILRAKLITV DADLLKELPVLADKQVLQNSTEPHHAAWIIYTSGSTGKPKGVVLQHVALCSSIKAHGARFGMNSKTRMLQFAAHTFDACIQDYFTTLSWGGVVCVPSENDRMSDLTAVMHEMGVTFATLTSTVARLINPQEVPS MQLIALVGEPVKADVVEQWLGHATVLNAYGPSECSIHSSCGEPLIDATKSSVIGTGMGTRLWVVDTDYNQLCPIGAPGELLIEGPLLAREYLNDAKKTRAAFVSDPRFAQNFDLPSGTRMYRTGDLVKQNEDSS ITHLGRRDTQIKIRGQRVEVGEIEYQIAQYPGIRTVAVELLEQDRNGSQVILTAAIEFTEDSEHRHGAMTPSGILMPTPALSLAFEMLRGSLFQVLPIYMLPSMYVAIADMPLNLNGKLDRRAVRDLLQSMNPDE

[0107] 6. Nucleotide sequence of gene fragment CENRPS-A2-2 (SEQ ID NO:23):

[0108]

[0109] 7. The amino acid sequence of the recombinant protein GLNRPS-CEA2-2 obtained by replacing the amino acid sequence of the A2 domain of the GLOA protein with the amino acid sequence of the A2 domain of the CENRPS protein (SEQ ID NO:24):

[0110]

[0111] 8. Nucleotide sequence encoding recombinant protein GLNRPS-CEA2-2 (SEQ ID NO:25):

[0112]

[0113] 9. The amino acid sequence of the A2 domain of the CCNRPS protein encoded by gene fragment CCNRPS-A2 (SEQ ID NO:26):

[0114] QEQAKIRPNATAICAWDGQLTYGELDTEATLLARYLTSLGACSQQMIGVCMDKSKFAGISMLAVLKAGAVVIPLGVNHPQARIEGIVHDTETDIILVDEKQRERLSSLKAKLIIVNADLLKEQPPLADKQMLQNSTEPHHAAWIIYTSGSTGKPKGVVLQHVALCSSIKAHGARFGMDSNTR MLQFAAHTFDICIQDYFTTLSWGGVVCVPSENDRMSDLTAVMRQMRVTFATLTSTVARLINPQEVPSMQQIALVGEPVKADVVEQWLGYTTVLNAYGPSECSIHSSCGEPLLDATKSSVIGTGMGTRLWVVDTDYNQLCPIGAPGELLIEGPLLAREYLNDAKKTRAAFVSDPRFAQNFELPS

[0115] 10. Nucleotide sequence of gene fragment CCNRPS-A2 (SEQ ID NO:27):

[0116]

[0117] 11. The amino acid sequence (SEQ ID NO:28) of the recombinant protein GLNRPS-CCA2 obtained by replacing the amino acid sequence of the A2 domain of the GLOA protein with the amino acid sequence of the A2 domain of the CCNRPS protein:

[0118]

[0119] 12. Nucleotide sequence encoding recombinant protein GLNRPS-CCA2 (SEQ ID NO:29):

[0120]

[0121] 13. gloA-A2-sgRNA gene sequence (SEQ ID NO:30):

[0122] GTGCATACATGAACTGGTACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0123] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A polypeptide fragment, characterized in that, The polypeptide fragment includes sequences as shown in SEQ ID NO:18 or SEQ ID NO:26; Preferably, the amino acid sequence of the polypeptide fragment is as shown in SEQ ID NO:18, SEQ ID NO:22 or SEQ ID NO:

26.

2. A recombinant protein, characterized in that, The recombinant protein comprises the polypeptide fragment as described in claim 1; Preferably, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:20, SEQ ID NO:24 or SEQ ID NO:

28.

3. A nucleic acid, characterized in that, Encoding the polypeptide fragment as described in claim 1 or the recombinant protein as described in claim 2; Preferably, the nucleic acid sequence encoding the polypeptide fragment is as shown in SEQ ID NO:19, SEQ ID NO:23 or SEQ ID NO:27; and / or The nucleic acid sequence encoding the recombinant protein is shown in SEQ ID NO:21, SEQ ID NO:25 or SEQ ID NO:

29.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid as described in claim 3; Preferably, the 5' and 3' ends of the nucleic acid encoding the polypeptide fragment have the 5' and 3' ends of the GloA-A2 gene, for use in homologous recombination of the nucleic acid encoding the polypeptide fragment with the GloA-A2 gene.

5. A combination of recombinant vectors, characterized in that, The combination includes the recombinant vector as described in claim 4, i.e., the first vector; and a second vector containing nucleic acid encoding the Cas protein; Preferably, the Cas protein is Cas 9, Cas 12a, or Cas 13; More preferably, the second vector is plasmid pDHt / sk-PC.

6. A gene editing system, characterized in that, The gene editing system includes: (1) The nucleic acid as described in claim 3 or the recombinant vector as described in claim 4; (2) Cas protein such as Cas 9, Cas 12a or Cas 13; nucleic acid encoding the Cas protein; or a second vector containing nucleic acid encoding the Cas protein, such as plasmid pDHt / sk-PC; Optional additions include (3) sgRNAs targeting the gloA gene; Preferably, the sgRNA targets the A2 domain of the GloA protein, and the gene sequence of the sgRNA is, for example, as shown in SEQ ID NO:30; and / or, the sgRNA is located in a vector, i.e., a third vector; and / or, the gloA gene is derived from the filamentous fungus G. lozoyensis, for example, G. lozoyensis ATCC 74030.

7. A genetically engineered strain, characterized in that, The genetically engineered strain does not express the GloA-A2 gene, but expresses the non-ribosomal peptide synthase gene CENRPS-A2 or CCNRPS-A2; Preferably, the CENRPS-A2 or CCNRPS-A2 is integrated into the GloA-A2 gene at a specific site; More preferably, the nonribosomal peptide synthase gene CENRPS-A2 is derived from Coleophoma empetri F-11899 or Coleophoma empetri SIPI 1284; and / or, the nonribosomal peptide synthase gene CCNRPS-A2 is derived from Coleophoma crateriformis no.

738. More preferably, the starting strain of the engineered strain is G. lozoyensis, such as G. lozoyensis ATCC. 74030; and / or, the nucleotide sequence of the CENRPS-A2 is as shown in SEQ ID NO:19 or SEQ ID NO:23; and / or, the nucleotide sequence of the CCNRPS-A2 is as shown in SEQ ID NO:

27.

8. A method for preparing the genetically engineered strain as described in claim 7, characterized in that, Editing filamentous fungi G. lozoyensis (e.g., G. lozoyensis ATCC) using the gene editing system as described in claim 6 74030) was obtained.

9. The polypeptide fragment of claim 1, the recombinant protein of claim 2, the nucleic acid of claim 3, the recombinant vector of claim 4, the combination of claim 5, the gene editing system of claim 6, or the genetically engineered strain of claim 7 in the preparation of Numococtin BO or Numococtin B 0-ser Applications in [the field].

10. A method for preparing Numococtin B0 or ​​Numococtin B 0-ser The method is characterized by, The method includes culturing the genetically engineered strain as described in claim 7 using a culture medium, fermenting it, and producing Numococtin BO or Numococtin B. 0-ser .

Images