Engineering bacterium, construction method and application thereof, and method for producing pneumocandin B0 through fermentation

Abstract

The invention relates to a gene modification technology, and discloses an engineering bacterium, a construction method and application thereof, and a method for producing pneumocandin B0 through fermentation. The engineering bacterium is obtained by genetic modification of an original strain, compared with the original strain, the engineering bacterium overexpresses a global transcriptional regulation factor GLlaeA, and the amino acid sequence of the global transcriptional regulation factor GLlaeA is as shown in SEQ ID NO: 1. The method for constructing the engineering bacteria comprises the following steps: carrying out genetic modification on an original strain so as to enable the original strain to overexpress the global transcription regulation factor GLlaeA. The yield of the pneumocandin B0 produced by fermentation of the engineering bacterium is high, and a better potential choice is provided for microbial fermentation production of the pneumocandin B0.

Description

Technical Field

[0001] The present invention relates to gene modification technology, and in particular to an engineering bacterium, a construction method and application thereof, and a method for producing pneumocandin B0 by fermentation. Background Art

[0002] Transcriptional regulatory factors are ubiquitous in filamentous fungi and can be divided into global transcriptional regulatory factors and pathway-specific transcriptional regulatory factors. Transcriptional regulatory factors are involved in the regulation of important life processes such as growth, development and secondary metabolism of filamentous fungi, especially playing a key role in the complex and multi-level secondary metabolism of filamentous fungi. Pneumocandin B0 is the precursor of the first semi-synthetic echinocandin antifungal drug caspofungin. It is a secondary metabolite produced by the fermentation of the filamentous fungus Glarealozoyensis. It can directly inhibit the activity of β-1,3-D-glucan synthase that synthesizes fungal cell walls, thereby inhibiting fungal infection and preventing the synthesis of cell walls, leading to fungal death. Compared with other antifungal substances, it has a wide range of applications, strong selectivity and lower side effects.

[0003] The methods used to increase the yield of Pneumocandin B0 produced by fermentation mainly include mutagenesis, culture medium optimization, osmotic stress strategy, gene editing technology, etc.

[0004] Merck obtained Glarealozoyensis ATCC 20957 by mutagenizing Glarea lozoyensis ATCC 20868, whose main products are pneumocandin B0 and pneumocandin C0. Glarealozoyensis ATCC 20957 was further mutagenized to obtain Glarea lozoyensis ATCC 74030, in which the pneumocandin B0 yield reached 241 mg / L, 13 times higher than that of the wild strain ATCC 20868. Qin T et al. used Glarea lozoyensis ATCC 74030 as the starting strain and obtained a stable high-yield strain G. lozoyensis Q1 by ARTP mutagenesis of protoplasts, with a pneumocandin B0 yield of 800 mg / L, no pneumocandin A0 content in the fermentation broth, and pneumocandin C0 content less than 3%. Through mutagenesis and breeding, the yield of Pneumocandin B0 was significantly improved, while the types and contents of structural analogues were reduced, laying the foundation for the development of subsequent fermentation processes.

[0005] The optimization of the culture medium is mainly carried out by adding precursors and optimizing the components in the culture medium by response surface method. Petersen et al. studied the effect of proline addition on the fermentation of Phoebe. The addition of proline increased the production of Phoebe B0 and Phoebe E0, reduced Phoebe C0 and Phoebe D0, and had a significant effect on the titer of Phoebe B0. When 10g / L proline was added to the culture medium, the titer of Phoebe B0 reached 494unit / L, which was 1.8 times that of the culture medium without proline. Yang Yuan et al. optimized the fermentation medium of the Phoebe B0 strain by response surface method. The results showed that mannitol, proline and glucose had the best effect on the study, and the production of Phoebe B0 increased by 42%.

[0006] Pneumocandin B0 is a hydrophobic secondary metabolite that accumulates in the mycelium. Due to feedback inhibition, the accumulation of high concentrations of Pneumocandin B0 in vivo becomes a challenge. Regulating and enhancing membrane permeability is one of the important means to alleviate feedback inhibition. Song et al. developed an osmotic pressure-controlled fed-batch strategy using mannitol as the sole carbon source together with an osmotic regulator. This strategy resulted in a maximum concentration of Pneumocandin B0 of 2711 mg / L and a productivity of 9.05 mg / L / h, which were 34.67% and 6.47% higher than the best results obtained in the first-stage fermentation, respectively.

[0007] At present, the construction of engineered bacteria for producing Pneumocandin B0 mainly focuses on the key genes in the Pneumocandin B0 production strain. "CRISPR / Cas9-Based Genome Editing in the Filamentous Fungus Glarealozoyensis and Its Application in Manipulating gloF" discloses a method for transforming the strain G.lozoyensis SIPI1208 using CRISPR / Cas9 gene editing technology, knocking out the GloF gene and replacing it with the Ap-HtyE gene, thereby obtaining 4 new strains for the production of Pneumocandin B0, which can well avoid the production of the impurity Pneumocandin C0. CN117586892A discloses that by overexpressing a gene encoding a non-ribosomal peptide synthetase, the fermentation yield of Pneumocandin B0 is increased by 16-37.4% compared with the wild-type strain.

[0008] For the production of Pneumocandin B0, there are problems with mutation direction and frequency in mutagenesis breeding, and fewer beneficial mutants; amino acids such as proline are expensive, and adding precursor amino acids to the fermentation medium will greatly increase production costs; there is a high off-target effect when using CRISPR / Cas9 gene editing technology to transform strains. Therefore, even based on the above multiple methods, the current maximum yield of Pneumocandin B0 produced by fermentation is still low, and it is difficult to meet the needs of industrial production. Summary of the invention

[0009] The purpose of the present invention is to overcome the problem of low yield of Pneumocandin B0 produced by fermentation in the prior art, and to provide an engineered bacterium, a construction method and application thereof, and a method for fermenting and producing Pneumocandin B0. The engineered bacterium has a high yield of Pneumocandin B0 produced by fermentation, providing a better potential choice for the microbial fermentation production of Pneumocandin B0.

[0010] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides an engineered bacterium, which is obtained by genetically modifying a starting strain. Compared with the starting strain, the engineered bacterium overexpresses a global transcriptional regulatory factor GLlaeA, and the amino acid sequence of the global transcriptional regulatory factor GLlaeA is shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence of the gene encoding the global transcriptional regulatory factor GLlaeA is shown in SEQ ID NO: 2.

[0012] Preferably, the starting strain is the filamentous fungus Glarea lozoyensis.

[0013] Preferably, the starting strain is a mutant strain Glarealozoyensis Q1 of Glarea lozoyensis ATCC74030.

[0014] The second aspect of the present invention provides a method for constructing an engineered bacterium, the method comprising: genetically modifying a starting strain so that the starting strain overexpresses a global transcriptional regulatory factor GLlaeA;

[0015] The method of causing the starting strain to overexpress the global transcriptional regulatory factor GLlaeA is to insert a gene encoding GLlaeA whose amino acid sequence is shown in SEQ ID NO: 1 into the genome of the starting strain.

[0016] Preferably, the starting strain is the filamentous fungus Glarea lozoyensis.

[0017] Preferably, the starting strain is a mutant strain Glarealozoyensis Q1 of Glarea lozoyensis ATCC74030.

[0018] Preferably, the method of making the starting strain overexpress the global transcriptional regulatory factor GLlaeA is to insert a GLlaeA expression gene having a nucleotide sequence as shown in SEQ ID NO: 2 into the genome of the starting strain.

[0019] Preferably, the promoter of the GLlaeA expression gene is PgpdA, and the nucleotide sequence of PgpdA is shown in SEQ ID NO:3.

[0020] The third aspect of the present invention provides the use of the above-mentioned engineered bacteria or the above-mentioned method in the production of Pneumocandin B0.

[0021] The fourth aspect of the present invention provides a method for producing pneumocandin B0 by fermentation, the method comprising: inoculating the above-mentioned engineered bacteria into a fermentation medium for fermentation;

[0022] Alternatively, the engineered bacteria are constructed according to the above method, and the obtained engineered bacteria are inoculated into a fermentation medium for fermentation.

[0023] Preferably, the fermentation conditions include: a temperature of 25-28° C., a rotation speed of 200-220 rpm, and a fermentation time of 12-18 days.

[0024] Preferably, the fermentation medium contains: glucose, mannitol, corn protein powder, cottonseed cake powder, K2HPO4, Fe3(PO4)2 and MnSO4.

[0025] Preferably, the fermentation medium contains: 10-20 g / L glucose, 60-80 g / L mannitol, 10-20 g / L corn gluten meal, 20-35 g / L cottonseed meal, 1.5-3.5 g / L K2HPO4, 0.2-0.8 g / L Fe3(PO4)2, 0.1-0.5 g / L MnSO4·H2O, and a pH of 6-7.5.

[0026] Preferably, the fermentation conditions include: temperature of 25-28°C, rotation speed of 300-500rpm, ventilation volume of 0.8-1.2vvm, and time of 8-15 days.

[0027] Preferably, the fermentation process comprises: starting from the 3rd to 4th day of fermentation, adding a mannitol solution with a concentration of 15-25 wt% at a rate of 0.1-0.3 g / L / h.

[0028] Preferably, the fermentation medium contains: glucose, mannitol, proline, cottonseed cake powder, K2HPO4, Fe3(PO4)2 and MnSO4.

[0029] Preferably, the fermentation medium contains: 10-20 g / L glucose, 60-80 g / L mannitol, 10-20 g / L proline, 20-35 g / L cottonseed meal, 1.5-3.5 g / L K2HPO4, 0.2-0.8 g / L Fe3(PO4)2, 0.1-0.5 g / L MnSO4·H2O, and a pH of 6-7.5.

[0030] Through the above technical solution, the beneficial effects of the present invention are:

[0031] The engineered bacteria provided by the present invention can promote the synthesis and accumulation of Pneumocandin B0 in microbial cells, effectively increase the yield of Pneumocandin B0, lay a foundation for metabolic engineering transformation to efficiently produce Pneumocandin B0, and provide a better potential choice for the microbial fermentation production of Pneumocandin B0; the engineering bacteria provided by the present invention are simple to construct, easy to use, and have good application prospects.

[0032] In the most preferred embodiment of the present invention, the content of Pneumocandin B0 in the fermentation broth obtained by fermentation with Glarea lozoyensis engineered bacteria can reach 7 g / L, which significantly improves the fermentation effect and the yield of Pneumocandin B0. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The amino acid sequence alignment of the global transcriptional regulatory factor LaeA of G. lozoyensis (XP_008077541.1) and A. nidulans (XP_658411.2) in Example 1;

[0034] Figure 2 is an amino acid sequence alignment of glyceraldehyde-3-phosphate dehydrogenase from G. lozoyensis (XP_008086288.1) and A. nidulans (P20445.2) in Example 1;

[0035] Figure 3 is a structural diagram of the recombinant plasmid pUC-HPH in Example 1, wherein PtrpC represents a promoter and TtrpC represents a terminator;

[0036] Figure 4 is a structural diagram of the recombinant plasmid pUC-GLlaeA in Example 1, wherein PgpdA represents a promoter and TgpdA represents a terminator;

[0037] Figure 5 The structure diagram of the recombinant plasmid pUC-HPH-GLlaeA in Example 1;

[0038] Figure 6 This is the detection spectrum of the pneumocandin B0 standard in Example 3, and the peak time is 6.91min;

[0039] Figure 7 This is the HPLC detection chart of the production of Pneumocandin B0 by the engineered bacteria shake flask in Example 3. DETAILED DESCRIPTION

[0040] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0041] The first aspect of the present invention provides an engineered bacterium, which is obtained by genetically modifying a starting strain. Compared with the starting strain, the engineered bacterium overexpresses a global transcriptional regulatory factor GLlaeA, and the amino acid sequence of the global transcriptional regulatory factor GLlaeA is shown in SEQ ID NO: 1.

[0042] The inventors found during the research process that there has been no research on randomly overexpressing the global transcriptional regulatory factor GLlaeA gene to increase the yield of pneumocandin B0 in the construction of engineered bacteria for producing pneumocandin B0. The present invention first discovered one of the global transcriptional regulatory factors GLlaeA in the filamentous fungus Glarea lozoyensis (abbreviated as G.lozoyensis) through homologous sequence alignment, and first discovered that randomly overexpressing the GLlaeA gene in Glarea lozoyensis can effectively increase the yield of pneumocandin B0.

[0043] In the present invention, the structural formula of Pneumocandin B0 is shown in formula (I),

[0044]

[0045] According to the present invention, preferably, the starting strain is the filamentous fungus Glarea lozoyensis. More preferably, the starting strain is a mutant strain Glarea lozoyensis Q1 of Glarea lozoyensis ATCC74030, which is described in the literature Tingting Qin, Ping Song, Xiaoting Wang, Xiaojun Ji, Lujing Ren & He Huang (2016). Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up, Bioscience, Biotechnology, and Biochemistry, 80: 11, 2241-2246.

[0046] In the present invention, in order to increase the yield of pneumocandin B0 of the engineered bacteria, preferably, the nucleotide sequence of the gene encoding the global transcriptional regulatory factor GLlaeA is as shown in SEQ ID NO: 2.

[0047] The inventors of the present invention have found that by using the mutant strain Glarealozoyensis Q1 of Glarea lozoyensis ATCC74030 as the starting strain and exogenously introducing a global transcriptional regulatory factor GLlaeA gene expression cassette into the engineered bacteria, the yield of the fermentation product Pneumocandin B0 can be more significantly increased.

[0048] The second aspect of the present invention provides a method for constructing an engineered bacterium, the method comprising: genetically modifying a starting strain so that the starting strain overexpresses a global transcriptional regulatory factor GLlaeA;

[0049] The method of causing the starting strain to overexpress the global transcriptional regulatory factor GLlaeA is to insert a gene encoding GLlaeA whose amino acid sequence is shown in SEQ ID NO: 1 into the genome of the starting strain.

[0050] According to the present invention, preferably, the starting strain is the filamentous fungus Glarea lozoyensis. More preferably, the starting strain is a mutant strain of Glarea lozoyensis ATCC74030, Glarea lozoyensis Q1.

[0051] According to the present invention, preferably, the method of making the starting strain overexpress the global transcriptional regulatory factor GLlaeA is to insert a GLlaeA expression gene having a nucleotide sequence as shown in SEQ ID NO: 2 into the genome of the starting strain.

[0052] In the present invention, the GLlaeA expression gene can use any suitable promoter. Preferably, the promoter of the GLlaeA expression gene is PgpdA, and the nucleotide sequence of PgpdA is shown in SEQ ID NO: 3, so that the expression amount of the global transcriptional regulatory factor GLlaeA gene is increased by regulating the promoter, which is conducive to the improvement of the yield of Pneumocandin B0.

[0053] In the present invention, overexpression refers to inserting a gene existing in the cell into the strain so that the gene is upregulated and the final gene expression product exceeds the normal level. In the present invention, overexpression is achieved by constructing an expression cassette (e.g., a recombinant vector) of the GLlaeA encoding gene and introducing the expression cassette into the starting strain.

[0054] Various methods known in the art for constructing recombinant vectors can be used to connect the target gene fragment to the vector to prepare the expression cassette, such as but not limited to the classic "enzyme cutting-ligation" method, the Gateway cloning system developed by Invitrogen, the Creator cloning system developed by Clontech, the Univector cloning system developed by Stephen Elledge's laboratory, and the GoldenGate cloning method based on type IIs restriction endonuclease.

[0055] Subsequently, the recombinant vector can be introduced into the starting strain (such as the filamentous fungus Glarealozoyensis) by conventional methods in the art, such as but not limited to microinjection, gene gun, transformation (such as electrotransformation), infection or transfection. The above-mentioned microinjection, gene gun, transformation, infection or transfection are all routine operations in the art. For example, transformation refers to treating cells by some known methods in molecular biology and genetic engineering, so that the treated cells are in a competent state, and thereby contact with exogenous DNA, so that the exogenous DNA enters the cells in the competent state. Commonly used transformation methods include protoplast transformation, chemical transformation and electroporation transformation. Infection refers to using an artificially modified phage live virus as a vector, recombining the vector with the target DNA sequence, and then packaging the recombinant DNA into a viable phage or virus in vitro using the coat protein of the phage or virus, so that the recombinant DNA enters the host cell in an infectious manner.

[0056] After the recombinant vector is introduced into the starting strain (such as the filamentous fungus Glarea lozoyensis), positive clones can be screened out by screening markers (such as resistance genes) and verified by genomic PCR or by genomic DNA sequencing to obtain the engineered bacteria that produce Pneumocandin B0.

[0057] Exemplarily, the engineered bacteria is obtained by using the filamentous fungus G. lozoyensis as a starting strain and randomly inserting a linearized recombinant plasmid pUC-HPH-GLlaeA into the genome of G. lozoyensis.

[0058] The recombinant plasmid pUC-HPH-GLlaeA is based on the recombinant plasmid pUC-HPH and the recombinant plasmid pUC-GLlaeA. The recombinant plasmid pUC-GLlaeA is obtained by inserting the PgpdA-GLlaeA-TgpdA expression cassette into the pUC57 vector. The recombinant plasmid pUC-HPH is obtained by inserting the PtrpC-HPH-TtrpC expression cassette into the pUC57 vector.

[0059] The recombinant plasmid pUC-HPH-GLlaeA is obtained by inserting the PgpdA-GLlaeA-TgpdA expression cassette into the pUC-HPH vector, and the insertion site is downstream of the PtrpC-HPH-TtrpC expression cassette.

[0060] Among them, the promoter of the PgpdA-GLlaeA-TgpdA expression cassette is the strong promoter of PgpdA of G. lozoyensis, and the terminator is the original terminator TgpdA of GLlaeA; the global transcriptional regulatory factor GLlaeA gene of the PgpdA-GLlaeA-TgpdA expression cassette is shown in SEQ ID NO: 2.

[0061] The promoter of the PtrpC-HPH-TtrpC expression cassette is the PtrpC promoter of Aspergillus nidulans; the terminator of the PtrpC-HPH-TtrpC is TtrpC of A. nidulans; the hygromycin resistance gene HPH of the PtrpC-HPH-TtrpC expression cassette is shown in SEQ ID NO: 23.

[0062] The strong promoter PgpdA gene is shown as SEQ ID NO:3, and the terminator TgpdA is shown as SEQ ID NO:4.

[0063] The promoter PtrpC gene is shown in SEQ ID NO:5, and the terminator TtrpC is shown in SEQ ID NO:6.

[0064] As a preferred embodiment, the method for constructing an engineered bacterium for efficiently producing Pneumocandin B0 of the present invention comprises the following steps:

[0065] (1) Construction of recombinant plasmid pUC-HPH:

[0066] The recombinant plasmid pUC-HPH is based on pUC57 and has a PtrpC-HPH-TtrpC expression cassette inserted into it. The three gene fragments of the promoter PtrpC, HPH and the terminator TtrpC are fused into one gene fragment by fusion PCR, namely the PtrpC-HPH-TtrpC expression cassette;

[0067] (2) Construction of recombinant plasmid pUC-GLlaeA:

[0068] The recombinant plasmid pUC-GLlaeA is based on pUC57 and has a PgpdA-GLlaeA-TgpdA expression cassette inserted into it. The three gene fragments of the strong promoter PgpdA, GLlaeA and the terminator TgpdA are fused into one gene fragment by fusion PCR, namely the PgpdA-GLlaeA-TgpdA expression cassette;

[0069] (3) Construction of recombinant plasmid pUC-HPH-GL1aeA:

[0070] The recombinant plasmid pUC-HPH-GLlaeA is based on the recombinant plasmid PUC-HPH, and the PgpdA-GLlaeA-TgpdA expression cassette is inserted downstream of the PtrpC-HPH-TtrpC expression cassette;

[0071] (4) The linearized recombinant plasmid pUC-HPH-GLlaeA was introduced into G. lozoyensis Q1 to obtain the engineered bacterium G. lozoyensis-OelaeA, which is an engineered bacterium for producing pneumocandin B0.

[0072] The third aspect of the present invention provides the use of the above-mentioned engineered bacteria or the above-mentioned method in the production of Pneumocandin B0.

[0073] The fourth aspect of the present invention provides a method for producing pneumocandin B0 by fermentation, the method comprising: inoculating the above-mentioned engineered bacteria into a fermentation medium for fermentation;

[0074] Alternatively, the engineered bacteria are constructed according to the above method, and the obtained engineered bacteria are inoculated into a fermentation medium for fermentation.

[0075] According to the present invention, preferably, the engineered bacteria are first prepared into seed liquid, and then the seed liquid is inoculated into a fermentation medium for fermentation to obtain a fermentation liquid.

[0076] According to the present invention, preferably, the method for preparing the seed solution comprises: picking a single colony of the engineered bacteria and inoculating it into a seed culture medium for seed culture to obtain the seed solution.

[0077] In the present invention, there is no particular limitation on the seed culture medium, and the seed culture medium conventionally used in the art can be used. Preferably, when the starting strain is the filamentous fungus Glarea lozoyensis, the seed culture medium contains: glucose, soybean powder, corn steep liquor powder, cottonseed cake powder, KH2PO4, FeSO4, ZnSO4, CaCl2, H3BO3 and (NH4)6Mo7O 24; Further preferably, the seed culture medium contains: glucose 30-50 g / L, soybean powder 15-25 g / L, corn steep liquor powder 5-15 g / L, cottonseed cake powder 5-15 g / L, KH2PO4 0.5-1.5 g / L, FeSO4·7H2O 0.01-0.02 g / L, ZnSO4·7H2O 0.001-0.003 g / L, CaCl2·2H2O 0.0007-0.0008 g / L, H3BO3 0.0005-0.0006 g / L and (NH4)6Mo7O 24 4H2O 0.001-0.003g / L.

[0078] In the present invention, there is no particular limitation on the conditions (including temperature, rotation speed, time, etc.) for the seed culture, which can be conventional culture conditions in the art. Preferably, when the starting strain is the filamentous fungus Glarea lozoyensis, the conditions for the seed culture include: a temperature of 25-28°C, a rotation speed of 200-220rpm, and a time of 4-6 days.

[0079] In the present invention, the single colony of the engineered bacteria can be selected from the freshly prepared engineered bacteria or the low-temperature frozen engineered bacteria (for example, the engineered bacteria for synthesizing Pneumocandin B0 frozen in a glycerol cryopreservation tube in a -80°C refrigerator).

[0080] In the present invention, there is no particular limitation on the fermentation method, and it can be a method for producing pneumocandin B0 by fermentation conventionally used in the art, for example, inoculating the seed liquid into the fermentation medium (for example, a shake flask or fermenter containing the fermentation medium) for fermentation to obtain a fermentation liquid.

[0081] In the present invention, in order to increase the yield of Pneumocandin B0, preferably, the inoculation amount of the seed liquid is 8-12 parts by volume relative to 100 parts by volume of the fermentation medium.

[0082] In the present invention, in order to further increase the yield of Pneumocandin B0 during shake flask fermentation, preferably, the fermentation conditions include: temperature of 25-28° C., rotation speed of 200-220 rpm, and time of 12-18 days.

[0083] In the present invention, there is no particular restriction on the components of the fermentation medium, and it is sufficient to provide nutrients for the growth and proliferation of the engineered bacteria. In order to further increase the yield of Pneumocandin B0 during the fermentation process, preferably, when the engineered bacteria are subjected to shake flask fermentation, the fermentation medium contains: glucose, mannitol, corn gluten powder, cottonseed cake powder, K2HPO4, Fe3(PO4)2 and MnSO4. Further preferably, the fermentation medium contains: glucose 10-20 g / L, mannitol 60-80 g / L, corn gluten powder 10-20 g / L, cottonseed cake powder 20-35 g / L, K2HPO4 1.5-3.5 g / L, Fe3(PO4)2 0.2-0.8 g / L, MnSO4·H2O 0.1-0.5 g / L, and pH is 6-7.5.

[0084] In the present invention, in order to further increase the yield of Pneumocandin B0 during fermentation in the fermenter, preferably, the fermentation conditions include: temperature of 25-28°C, rotation speed of 300-500rpm, ventilation volume of 0.8-1.2vvm, and time of 8-15 days. Further preferably, the fermentation process includes: starting from the 3rd to 4th day of fermentation, a mannitol solution with a concentration of 15-25wt% is added at a rate of 0.1-0.3g / L / h.

[0085] In order to further increase the yield of Pneumocandin B0 during the fermentation process, preferably, when the engineered bacteria are fermented in a fermenter, the fermentation medium contains: glucose, mannitol, proline, cottonseed cake powder, K2HPO4, Fe3(PO4)2 and MnSO4. Further preferably, the fermentation medium contains: glucose 10-20g / L, mannitol 60-80g / L, proline 10-20g / L, cottonseed cake powder 20-35g / L, K2HPO4 1.5-3.5g / L, Fe3(PO4)2 0.2-0.8g / L, MnSO4·H2O 0.1-0.5g / L, and pH is 6-7.5.

[0086] In the present invention, the pneumocandin B0 in the obtained fermentation broth can be separated by a known method, for example, the fermentation broth is mixed with an extraction solvent, pneumocandin B0 is extracted, and the supernatant is collected by centrifugation. The supernatant can also be further concentrated to crystallize the product, or ion exchange chromatography or the like can be performed to separate pneumocandin B0.

[0087] In the present invention, the pneumocandin B0 in the fermentation broth or the pneumocandin B0 separated from the fermentation broth can also be detected by known methods. For example, the content of pneumocandin B0 can be detected by high performance liquid chromatography and the like.

[0088] The present invention will be described in detail below through examples.

[0089] In the following examples, the starting strain used was a mutant strain of Glarea lozoyensis ATCC74030, Glarea lozoyensis Q1, provided by the research group of Professor Huang He of Nanjing Normal University, obtained by mutagenesis of Glarea lozoyensis ATCC74030. For details, see Tingting Qin, Ping Song, Xiaoting Wang, Xiaojun Ji, Lujing Ren & He Huang (2016). Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up, Bioscience, Biotechnology, and Biochemistry, 80: 11, 2241-2246.

[0090] The recombinant plasmid pUC-HPH is obtained by inserting the PtrpC-HPH-TtrpC expression cassette into the pUC57 vector (the construction method is shown in Example 1).

[0091] The recombinant plasmid pUC-GLlaeA is obtained by inserting the PgpdA-GLlaeA-TgpdA expression cassette into the pUC57 vector (the construction method is shown in Example 1).

[0092] The recombinant plasmid pUC-HPH-GLlaeA is obtained by inserting the PgpdA-GLlaeA-TgpdA expression cassette into the pUC-HPH vector (the construction method is shown in Example 1), and the insertion site is downstream of the PtrpC-HPH-TtrpC expression cassette.

[0093] Plasmid pUC57 and plasmid pUC were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.

[0094] The recombinant plasmid pUC57-HPH-gus-Ptrpc was constructed by our research group and contains the promoter sequence (PtrpC) and terminator sequence (TtrpC) of the fungal trpC gene and the screening gene hygromycin resistance gene HPH.

[0095] In the following examples, the seed culture medium formula is (g / L): glucose 40, soybean powder 20, corn steep liquor powder 10, cottonseed cake powder 10, KH2PO4 1, FeSO4·7H2O 0.01, ZnSO4·7H2O 0.002, CaCl2·2H2O 0.000756, H3BO3 0.00056,

[0096] (NH4)6Mo7O 24 4H2O 0.002, pH 5.0.

[0097] OM buffer is (g / L): MgSO4 144.48, Na2HPO4 0.4545, NaH2PO4 0.817, and Na2HPO4 is used to adjust the pH to 5.8.

[0098] STC buffer was (g / L): sorbitol 109.3, CaCl2 1.11, Tris 1.211, HCl adjusted to pH 7.5.

[0099] The solid culture medium formula is (g / L): glucose 30, yeast extract 5, tryptone 5, corn steep liquor powder 1.5, FeSO4·7H2O 0.01, ZnSO4·7H2O 0.002, CaCl2·2H2O 0.000756, H3BO3 0.00056, (NH4)6Mo7O 24 4H2O 0.002, pH 6.8, agar 22.

[0100] The formula of the regeneration medium is (g / L): NaCl 46.7, glucose 30, yeast extract 5, tryptone 5, corn steep liquor 1.5, FeSO4·7H2O 0.01, ZnSO4·7H2O 0.002, CaCl2·2H2O 0.000756, H3BO3 0.00056, (NH4)6Mo7O 24 4H2O 0.002, pH 6.8, agar 22.

[0101] The PEG solution is (g / L): PEG4000 600, CaCl2 5.55, Tris 6.055, and HCl adjusted to pH 7.5.

[0102] Example 1

[0103] 1. Amplification of genetic elements

[0104] 1. Preparation of target gene

[0105] According to the amino acid sequence of the global transcriptional regulatory factor LaeA from Aspergillus nidulans provided by NCBI (NCBI number XP_658411.2), NCBI BLAST ( BLAST:Basic Local AlignmentSearch Tool (nih.gov) ) were used for homologous sequence alignment. The amino acid sequence of LaeA from A. nidulans was input into NCBI BLAST, and the comparison species was set to G. lozoyensis. The sequence with the highest score was selected and the amino acid sequence of LaeA from A. nidulans was located in G. lozoyensis.

[0106] S-adenosyl-L-methionine-dependent methyltransferase (NCBI number XP_008077541.1), which has a sequence homology of 43% with LaeA from A. nidulans, see Figure 1 shown.

[0107] The gene encoding S-adenosyl-L-methionine-dependent methyltransferase was named GLlaeA, and the genomic DNA sequence of GLlaeA was located at GLAREA_11161. Using the G. lozoyensis genome as a template, GLlaeA-F (nucleotide sequence as shown in SEQ ID NO: 7) and GLlaeA-R (nucleotide sequence as shown in SEQ ID NO: 8) were used as primers to amplify the GLlaeA gene with PgpdA promoter homology arms and TgpdA terminator homology arms, and the nucleotide sequence of the GLlaeA gene is shown in SEQ ID NO: 1.

[0108] In order to overexpress laeA in G. lozoyensis, a strong natural promoter was found in the G. lozoyensis genome. According to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase (GPD) from A. nidulans provided by NCBI (NCBI number P20445.2), the amino acid sequence of GPD from A. nidulans was input into NCBI BLAST, and the comparison species was set to G. lozoyensis. The sequence with the highest score was selected, and GPD was located in G. lozoyensis (NCBI number XP_008086288.1), which has a sequence homology of about 82% with A. nidulans GPD, see Figure 2The gene encoding GPD was named GLgpdA. The genomic DNA sequence of GLgpdA was located at GLAREA_03012. The sequence of about 2 kb upstream was used as the promoter PgpdA. The G. lozoyensis genome was used as a template, and PgpdA-F (nucleotide sequence as shown in SEQ ID NO: 9) and PgpdA-R (nucleotide sequence as shown in SEQ ID NO: 10) were used as primers to amplify the promoter gene PgpdA with the homology arms of the pUC57 vector and the homology arms of the GLlaeA gene. The nucleotide sequence of PgpdA is shown in SEQ ID NO: 3.

[0109] A sequence of about 1 kb downstream of GLlaeA was used as the terminator TgpdA. The G. lozoyensis genome was used as a template and TgpdA-F (the nucleotide sequence is shown in SEQ ID NO: 11) and TgpdA-R (the nucleotide sequence is shown in SEQ ID NO: 12) were used as primers to amplify the terminator gene TgpdA with the GLlaeA gene homologous arms and the pUC57 vector homologous arms. The nucleotide sequence of TgpdA is shown in SEQ ID NO: 4.

[0110] Using pUC57-HPH-gus-Ptrpc plasmid as a template and HPH-F (nucleotide sequence as shown in SEQ ID NO: 13) and HPH-R (nucleotide sequence as shown in SEQ ID NO: 14) as primers, the hygromycin resistance gene HPH with a PtrpC promoter homology arm and a TtrpC terminator homology arm was amplified. The nucleotide sequence of HPH is shown in SEQ ID NO: 23.

[0111] Using the pUC57-HPH-gus-Ptrpc plasmid as a template, the promoter gene PtrpC with pUC57 vector homology arms and HPH homology arms was amplified using primers PtrpC-F (nucleotide sequence as shown in SEQ ID NO: 15) and PtrpC-R (nucleotide sequence as shown in SEQ ID NO: 16). The nucleotide sequence of PtrpC is shown in SEQ ID NO: 5.

[0112] Using pUC57-HPH-gus-Ptrpc plasmid as a template and TtrpC-F (nucleotide sequence as shown in SEQ ID NO: 17) and TtrpC-R (nucleotide sequence as shown in SEQ ID NO: 18) as primers, the terminator gene TtrpC with HPH homology arms and pUC57 vector homology arms was amplified. The nucleotide sequence of TtrpC is shown in SEQ ID NO: 6.

[0113] 2. Construction of recombinant plasmid

[0114] 1. Construction of recombinant plasmid pUC-HPH

[0115] The recombinant plasmid pUC-HPH is based on pUC57 and inserted with the PtrpC-HPH-TtrpC expression cassette. The PtrpC-HPH-TtrpC expression cassette is constructed by fusion PCR.

[0116] Using PtrpC and HPH as templates, PtrpC-F and HPH-R as primers, PtrpC and HPH were fused to obtain a PtrpC-HPH gene fragment with a pUC57 vector homology arm and a TtrpC terminator homology arm; then using PtrpC-HPH and TtrpC as templates, PtrpC-F and TtrpC-R as primers, PtrpC-HPH and TtrpC were fused to obtain a PtrpC-HPH-TtrpC expression cassette. Universal CloneMix homologously recombined the PtrpC-HPH-TtrpC expression cassette with the linearized pUC57 vector to obtain the recombinant plasmid pUC-HPH; the circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened by ampicillin resistance plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HPH (structure diagram as shown in Figure 3 The build is successful.

[0117] 2. Construction of recombinant plasmid pUC-GLlaeA

[0118] The recombinant plasmid pUC-GLlaeA is based on PUC57 and has the PgpdA-GLlaeA-TgpdA expression cassette inserted into it.

[0119] The PgpdA-GLlaeA-TgpdA expression cassette was constructed using fusion PCR.

[0120] Using PgpdA and GLaeA as templates, PgpdA-F and GLaeA-R as primers, PgpdA and GLaeA were fused to obtain a PgpdA-GLlaeA gene fragment with a pUC57 vector homology arm and a PgpdA terminator homology arm. Then, using PgpdA-GLlaeA and TgpdA as templates, PgpdA-F and TgpdA-R as primers, PgpdA-GLlaeA and TgpdA were fused to obtain a PgpdA-GLlaeA-TgpdA expression cassette fragment; using the Tolo Harbor Biotechnology Co., Ltd. Universal CloneMix homologously recombined the PgpdA-GLlaeA-TgpdA expression cassette fragment with the linearized pUC57 vector to obtain the recombinant plasmid pUC-GLlaeA; the circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened by ampicillin resistance plate and verified by colony PCR and sequencing, and the positive recombinant plasmid pUC-GLlaeA (structure diagram as shown in Figure 4 The build is successful.

[0121] 3. Construction of recombinant plasmid pUC-HPH-GLlaeA

[0122] The recombinant plasmid pUC-HPH-GLlaeA is based on PUC-HPH as a skeleton, and a PgpdA-GLlaeA-TgpdA expression cassette is inserted downstream of the PtrpC-HPH-TtrpC expression cassette. Using the PUC-HPH plasmid as a template and pUCZT-F (nucleotide sequence as shown in SEQ ID NO: 19) and TtrpC-R1 (nucleotide sequence as shown in SEQ ID NO: 20) as primers, a linearized pUC-HPH plasmid with PgpdA and TgpdA homology arms is obtained as a linearized vector. Using pUC-GLlaeA as a template, PgpdA-F1 (nucleotide sequence as shown in SEQ ID NO: 21) and TgpdA-R1 (nucleotide sequence as shown in SEQ ID NO: 22) as primers, a linearized PgpdA-GLlaeA-TgpdA expression cassette fragment with PUC-HPH plasmid vector homology arms and TtrpC homology arms was amplified. Universal CloneMix homologously recombined the PgpdA-GLlaeA-TgpdA expression cassette fragment with the linearized PUC-HPH plasmid vector to obtain the recombinant plasmid pUC-HPH-GLlaeA. The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by ampicillin resistance plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HPH-GLlaeA (structure diagram as shown in Figure 5 The build is successful.

[0123] The PCR enzyme used in the above PCR reaction is 2×PhantaFlash Master Mix (Dye Plus) high-fidelity enzyme produced by Nanjing Novozymes Biotech Co., Ltd.; the above PCR amplification system is shown in Table 1 below.

[0124] Table 1

[0125] Components volume 2×Phanta Flash Master Mix(Dye Plus) 25μL Primer 1 (10 μM) 2μL Primer 2 (10 μM) 2μL Template <20ng Sterile distilled water up to 50μL

[0126] The PCR procedure was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C (extension time = 5 s / kb), repeated 35 cycles, and extension at 72°C for another 1 min.

[0127] Each fragment was purified and recovered using FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.).

[0128] The homologous recombination system is shown in Table 2.

[0129] Table 2

[0130]

[0131] The recombinant plasmids constructed in this example are shown in Table 3, and the primer sequences used are shown in Table 4.

[0132] Table 3 Insertion sequences in each recombinant plasmid

[0133] Plasmid name Insert sequence pUC-HPH PtrpC-HPH-TtrpC pUC-GLlaeA PgpdA-GLlaeA-TgpdA pUC-HPH-GLlaeA PtrpC-HPH-TtrpC-PgpdA-GLlaeA-TgpdA

[0134] Table 4 Primer sequences

[0135]

[0136]

[0137] Example 2 Construction of engineering bacteria

[0138] 1. Preparation of G. lozoyensis protoplasts

[0139] The mutant strain Glarea lozoyensis Q1 was inoculated into 50 mL seed culture medium, cultured at 25°C and 220 rpm for 3-5 days, then 4 mL of the shaken bacterial solution was taken with a 5 mL sterile pipette tip into a 50 mL sterile centrifuge tube, centrifuged at 8000 rpm for 5 minutes, the upper culture medium was discarded, the bacterial precipitate was washed with sterile water, and centrifuged at 8000 rpm for 5 minutes, and repeated twice. Subsequently, the mycelium was drawn into a 50 mL conical flask containing 10 mL of enzymatic solution (2% Yatalase + 3% Lysing Enzymes dissolved in 10 mL OM buffer, filtered through a 0.22 μm sterile filter membrane) with a 5 mL sterile pipette tip, cultured on a shaker at 25°C and 80 rpm for 3.5-4 hours, and then examined under a microscope, and 20 μL of the count reached 10 7 / mL is the best. Use sterile cotton balls to filter the culture solution into a new 50mL sterile centrifuge tube, centrifuge at 5000rpm, 4℃ for 15 minutes, collect the precipitate is the protoplasts, rinse the protoplasts with 0.8mol / L STC buffer, centrifuge at 5000rpm, 4℃ for 15 minutes, repeat twice. Take 150μL 0.8mol / L STC to resuspend the G.lozoyensis protoplasts.

[0140] 2. Protoplast transformation

[0141] The recombinant plasmid pUC-HPH-GLlaeA was used as a template, and PtrpC-F (nucleotide sequence as shown in SEQ ID NO: 15) and TgpdA-R1 (nucleotide sequence as shown in SEQ ID NO: 22) were used as primers for amplification to obtain the linearized plasmid pUC-HPH-GLlaeA. 50 μL of the linearized plasmid pUC-HPH-GLlaeA was added to 150 μL of G. lozoyensis protoplasts, and the mixture was placed on ice for 40 minutes. Then, 500 μL of PEG solution was added and the mixture was allowed to stand at room temperature for 20 minutes. 100 μL of the mixture was spread on a regeneration medium containing hygromycin resistance, and the mixture was cultured at 25°C for 14 days to observe the growth of colonies. The colonies were inoculated on a solid medium containing hygromycin resistance, and a single colony was picked to extract the genome for PCR verification and sent to Qingke Company for sequencing. The positive clones that were correctly identified by PCR and sequenced were named as the engineered bacteria G. lozoyensis-OElaeA.

[0142] Example 3 Application of engineered bacteria G. lozoyensis-OElaeA in shake flask production of Pneumocandin B0

[0143] 1. Cultivation of engineered bacteria and product extraction

[0144] First, the engineered bacteria obtained in Example 2 were activated, and the colonies that grew well on the solid culture medium were picked and cultured in a seed culture medium at a temperature of 25° C. and a rotation speed of 220 rpm for 5 days to obtain a seed solution. The seed solution was inoculated into 50 mL of a fermentation medium at an inoculum volume ratio of 10%, and cultured at a temperature of 25° C. and a rotation speed of 220 rpm for 15 days. After the fermentation was completed, 1 mL of the fermentation liquid and 4 mL of anhydrous ethanol were added to a 15 mL EP tube, and then shaken with a vortex mixer for 10 min, and centrifuged at 25° C. and 8000 g for 10 min, and the supernatant was collected for use.

[0145] The fermentation medium formula is (g / L): glucose 20, mannitol 80, corn protein powder 10, cottonseed cake powder 28, K2HPO4 2.5, Fe3(PO4)2 0.5, MnSO4·H2O 0.3, pH 6.8.

[0146] 2. Qualitative and quantitative analysis of Pneumocandin B0

[0147] 1 mL of the supernatant was passed through a 0.22 μm organic nylon membrane for liquid phase detection. Mobile phase B was 100% acetonitrile, and mobile phase A was ultrapure water (filtered through a membrane to remove impurities and ultrasonic bubbles before use). The gradient elution process is shown in Table 5. A Venusil MP C18 column (250 mm × 4.6 mm) and a UV detector were used. The flow rate was set to 1 mL / min, the column oven was set to 35°C, the detection wavelength was set to 210 nm, the injection volume was set to 20 μL, and the standard substance of Pneumocandin B0 was used for qualitative and quantitative analysis. Figure 6 This is the detection spectrum of the standard substance of Pneumocandin B0. Figure 7 This is the HPLC detection chart of Pneumocandin B0 produced by the engineered bacteria G.lozoyensis-OElaeA. According to the test, after 15 days of shake flask fermentation, the production of Pneumocandin B0 of the engineered bacteria G.lozoyensis-OElaeA reached 6.15g / L.

[0148] Table 5

[0149] Time (min) Organic phase (%) 0 50 10 75 15 95 20 95 25 50 30 50

[0150] Example 4

[0151] A method for producing Pneumocandin B0 using the engineered bacteria G. lozoyensis-OElaeA in a 5L fermenter.

[0152] The fermentation tank culture medium was added to a 5L fermentation tank, the liquid volume was 3L, and sterilized at 121°C for 30 minutes; the seed solution was obtained according to the method in Example 3, and 300mL of the cultured shake flask seed solution was inoculated into the fermentation tank, and cultured at 400rpm, 1vvm, and 25°C for 11 days. On the third day of fermentation, 0.2g -1 ·L -1 ·h -1 20% mannitol was added at a rate of ; judgment of the fermentation end point: the mycelium is dark in color, with more vacuoles, and the mycelium is broken and autolyzed.

[0153] The fermentation tank culture medium formula is (g / L): glucose 20, mannitol 80, proline 10, cottonseed cake powder 28, K2HPO4 2.5, Fe3(PO4)2 0.5, MnSO4·H2O 0.3, pH 6.8.

[0154] According to the above fermentation conditions and process, three batches of fermentation experiments were carried out in the tank. After the fermentation was completed, 1 mL of fermentation liquid and 4 mL of anhydrous ethanol were added to a 15 mL EP tube, and then vortexed for 10 min; after centrifugation at 25 ° C and 8000 g for 10 min, the supernatant was collected for use; the yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The yield of Pneumocandin B0 in three batches of fermentation of the engineered bacteria G. lozoyensis-OElaeA in a 5L fermenter is shown in Table 6.

[0155] Table 6

[0156] Fermentation batch <![CDATA[Output of pneumocandin B0 (g / L)]]> 1 6.75 2 7.01 3 6.89

[0157] Example 5

[0158] The process provided in Example 3 was used to carry out shake flask fermentation of the engineered bacteria G. lozoyensis-OelaeA, except that the shake flask fermentation medium and the fermentation culture process were replaced by:

[0159] The fermentation medium formula was (g / L): glucose 10, mannitol 60, corn gluten meal 20, cottonseed meal 35, K2HPO4 3.5, Fe3(PO4)2 0.2, MnSO4·H2O 0.5, pH 7.5;

[0160] The seed solution was inoculated into 50 mL of fermentation medium at an inoculum volume ratio of 12%, and cultured at 28° C. and 200 rpm with shaking for 12 days.

[0161] The yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The results are shown in Table 7.

[0162] Example 6

[0163] The process provided in Example 3 was used to carry out shake flask fermentation of the engineered bacteria G. lozoyensis-OelaeA, except that the shake flask fermentation medium and the fermentation culture process were replaced by:

[0164] The fermentation medium formula was (g / L): glucose 15, mannitol 70, corn gluten meal 15, cottonseed meal 25, K2HPO4 1.5, Fe3(PO4)2 0.8, MnSO4·H2O 0.1, pH 6.5;

[0165] The seed solution was inoculated into 50 mL of fermentation medium at an inoculum volume ratio of 8%, and cultured at 26° C. and 210 rpm with shaking for 18 days.

[0166] The yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The results are shown in Table 7.

[0167] Example 7

[0168] The process provided in Example 4 was used to carry out fermentation of the engineered bacteria G. lozoyensis-OelaeA in a fermenter, except that the fermenter medium and the fermenter culture process were replaced by:

[0169] The fermentation tank medium formula was (g / L): glucose 15, mannitol 70, proline 15, cottonseed meal 20, K2HPO4 3.5, Fe3(PO4)2 0.8, MnSO4·H2O 0.1, pH 7.5;

[0170] 240 mL of the cultured shake flask seed solution was inoculated into the fermenter and cultured at 500 rpm, 0.8 vvm, and 28 °C for 8 days. On the second day of fermentation, 0.1 g -1 ·L -1 ·h -1 25% mannitol was added at a rate of ; judgment of the fermentation end point: the mycelium is dark in color, with more vacuoles, and the mycelium is broken and autolyzed.

[0171] The yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The results are shown in Table 7.

[0172] Example 8

[0173] The process provided in Example 4 was used to carry out fermentation of the engineered bacteria G. lozoyensis-OelaeA in a fermenter, except that the fermenter medium and the fermenter culture process were replaced by:

[0174] The fermentation tank medium formula was (g / L): glucose 10, mannitol 60, proline 20, cottonseed meal 35, K2HPO4 1.5, Fe3(PO4)2 0.2, MnSO4·H2O 0.1, pH 6.2;

[0175] 360 mL of the cultured shake flask seed solution was inoculated into the fermenter and cultured at 300 rpm, 1.2 vvm, and 26 °C for 13 days. On the fourth day of fermentation, 0.3 g -1 ·L -1 ·h -1 15% mannitol was added at a rate of 100%. The fermentation endpoint was judged as follows: the mycelium was dark in color, with more vacuoles, and the mycelium was broken and autolyzed.

[0176] The yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The results are shown in Table 7.

[0177] Example 9

[0178] The process provided in Example 4 was used to carry out fermentation of the engineered bacteria G. lozoyensis-OelaeA in a fermenter, except that the formula of the fermenter culture medium was replaced with: glucose 20 g / L, mannitol 80 g / L, corn gluten meal 10 g / L, cottonseed cake meal 28 g / L, K2HPO4 2.5 g / L, Fe3(PO4)2 0.5 g / L, MnSO4·H2O 0.3 g / L, pH 6.8.

[0179] The yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The results are shown in Table 7.

[0180] Comparative Example 1

[0181] The method of Example 4 was used to produce pneumocandin B0 by fermentation, except that the engineered bacteria G. lozoyensis-OelaeA was replaced by the starting strain Glarea lozoyensis Q1.

[0182] The yield of Pneumocandin B0 was detected according to the liquid phase detection method in Example 3. The results are shown in Table 7.

[0183] Table 7

[0184] serial number <![CDATA[Output of pneumocandin B0 (g / L)]]> Example 5 6.08 Example 6 6.17 Example 7 6.78 Example 8 6.90 Example 9 6.36 Comparative Example 1 4.09

[0185] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An engineered bacterium, characterized in that: The engineered bacteria is obtained by genetically modifying a starting strain. Compared with the starting strain, the engineered bacteria overexpresses a global transcriptional regulatory factor GLlaeA, and the amino acid sequence of the global transcriptional regulatory factor GLlaeA is shown in SEQ ID NO:

1.

2. The engineered bacteria according to claim 1, characterized in that The nucleotide sequence of the gene encoding the global transcriptional regulatory factor GLlaeA is shown in SEQ ID NO:

2.

3. The engineered bacteria according to claim 1 or 2, characterized in that: The starting strain is the filamentous fungus Glarealozoyensis; Preferably, the starting strain is a mutant strain Glarealozoyensis Q1 of Glarea lozoyensis ATCC74030.

4. A method for constructing an engineered bacterium, characterized in that: The method comprises: genetically modifying a starting strain so that the starting strain overexpresses a global transcriptional regulatory factor GLlaeA; The method of causing the starting strain to overexpress the global transcriptional regulatory factor GLlaeA is to insert a gene encoding GLlaeA whose amino acid sequence is shown in SEQ ID NO: 1 into the genome of the starting strain.

5. The method according to claim 4, characterized in that The starting strain is the filamentous fungus Glarealozoyensis; Preferably, the starting strain is a mutant strain Glarealozoyensis Q1 of Glarea lozoyensis ATCC74030.

6. The method according to claim 4 or 5, characterized in that: The method of causing the starting strain to overexpress the global transcriptional regulatory factor GLlaeA is to insert a GLlaeA expression gene with a nucleotide sequence as shown in SEQ ID NO: 2 into the genome of the starting strain; Preferably, the promoter of the GLlaeA expression gene is PgpdA, and the nucleotide sequence of PgpdA is shown in SEQ ID NO:

3.

7. Use of the engineered bacteria described in any one of claims 1 to 3 or the method described in any one of claims 4 to 6 in the production of Pneumocandin B0.

8. A method for producing pneumocandin B0 by fermentation, characterized in that: The method comprises: inoculating the engineered bacteria according to any one of claims 1 to 3 into a fermentation medium for fermentation; Alternatively, the engineered bacteria are constructed according to the method described in any one of claims 4 to 6, and the obtained engineered bacteria are inoculated into a fermentation medium for fermentation.

9. The method according to claim 8, characterized in that The fermentation conditions include: temperature of 25-28°C, rotation speed of 200-220rpm, and time of 12-18 days; Preferably, the fermentation medium contains: glucose, mannitol, corn protein powder, cottonseed cake powder, K2HPO4, Fe3(PO4)2 and MnSO4; Preferably, the fermentation medium contains: 10-20 g / L glucose, 60-80 g / L mannitol, 10-20 g / L corn gluten meal, 20-35 g / L cottonseed meal, 1.5-3.5 g / L K2HPO4, 0.2-0.8 g / L Fe3(PO4)2, 0.1-0.5 g / L MnSO4·H2O, and a pH of 6-7.

5.

10. The method according to claim 8, characterized in that The fermentation conditions include: temperature of 25-28°C, rotation speed of 300-500rpm, ventilation volume of 0.8-1.2vvm, and time of 8-15 days; Preferably, the fermentation process comprises: starting from the 3rd to 4th day of fermentation, adding a mannitol solution with a concentration of 15-25 wt% at a rate of 0.1-0.3 g / L / h; Preferably, the fermentation medium contains: glucose, mannitol, proline, cottonseed meal, K2HPO4, Fe3(PO4)2 and MnSO4; Preferably, the fermentation medium contains: 10-20 g / L glucose, 60-80 g / L mannitol, 10-20 g / L proline, 20-35 g / L cottonseed meal, 1.5-3.5 g / L K2HPO4, 0.2-0.8 g / L Fe3(PO4)2, 0.1-0.5 g / L MnSO4·H2O, and a pH of 6-7.5.

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