Genetically engineered bacterium for producing pneumocandin B0 as well as construction and application of genetically engineered bacterium
By genetically engineering and overexpressing the cytochrome P450 enzyme gene of the Numocanding-producing strain, the difficulty and high cost of Numocanding-producing B0 were solved, the PB5/PB0 ratio was reduced, and the yield and purification of Numocanding-producing B0 was improved. efficiency.
Patent Information
- Application Number
- CN202310773781.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-27
- Publication Date
- 2024-12-27
AI Technical Summary
In the prior art, separation and purification of Numconding B0 is difficult, and high-cost forward chromatography columns are required, resulting in high separation cost of impurity PB5 and difficult separation of PB5 and PB0.
Genetically engineered the Nümoconding-producing strain G.lozoyensis ATCC 74030 to reduce the production of impurity PB5, and constructed for overexpression in the strain by overexpressing GloO or its homologous protein, combining G418 resistant plasmid and molecular cloning methods. The cytochrome P450 enzyme gene was introduced into the plasmid using Agrobacterium-mediated transformation technology to obtain genetically engineered bacteria and reduce the PB5/PB0 ratio.
The PB5/PB0 ratio in the fermentation product was successfully reduced from 2.9% to 0.3%, which increased the content of Newmocontin B0 and reduced production costs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for producing pneumocandin B 0 Genetically engineered bacteria and their construction and application. Background Art
[0002] Pneumocandins belong to the class of echinocandins, which can non-competitively inhibit β-(1,3)-D-glucan synthase involved in the synthesis of fungal cell walls. Due to their mechanism of action, these antibiotics have the advantage of low toxicity and side effects. Currently, the echinocandins on the market include caspofungin, anidulafungin and micafungin, which are mainly used to treat deep fungal infections.
[0003] Pneumocandin is a series of structurally similar compounds produced by the filamentous fungus G. lozoyensis, which differ in the first amino acid residue on the ring, PB 0 is 4R,5R-dihydroxy-L-Orn, and PB 5 It is 5R-dihydroxy-L-Orn. Due to the similar structure, separation and purification are difficult. Summary of the invention
[0004] In order to solve the problem that it is difficult to reduce impurities such as PB in the existing technology 5 , and PB 5 and PB 0 The separation of Pneumocandin B is difficult, and a forward chromatographic column is required for production, resulting in a high separation cost. 0 Genetically engineered bacteria and their construction and application.
[0005] The present invention is through PB 0 The strain G.lozoyensis ATCC 74030 was genetically modified to reduce the impurity PB 5 The production of PB 5 / PB 0 ratio.
[0006] The present invention analyzes the biosynthesis pathway of Pneumocandins in the strain Glarea lozoyensis ATCC 74030, and reduces PB by overexpressing GloO or its homologous protein. 5Production. A plasmid pAgG containing a G418 resistance gene was constructed based on pAg1-H3. Based on the plasmid pAgG, plasmids for overexpressing GLoO, CEP450-2, ECDH and GloP in strain G.lozoyensis ATCC 74030 were constructed using molecular cloning methods. The constructed plasmids were introduced into G.lozoyensis by Agrobacterium tumefaciens-mediated transformation (AMT), and finally genetically engineered bacteria Gl-pAgG, Gl-gloO, Gl-CEp450-2, Gl-ecdH, and Gl-gloP were obtained.
[0007] The first aspect of the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium overexpresses a cytochrome P450 enzyme gene, wherein the starting strain of the genetically engineered bacterium is the filamentous fungus Glarea lozoyensis.
[0008] In certain embodiments, the cytochrome P450 enzyme gene is present in a plasmid or randomly integrated into the genome of the filamentous fungus by homologous recombination.
[0009] In certain embodiments, the cytochrome P450 enzyme gene is selected from one or more of the genes encoding the following proteins: GLoO, CEP450-2, and ECDH.
[0010] In certain embodiments,
[0011] The amino acid sequence of GLoO is shown in SEQ ID NO: 16,
[0012] The amino acid sequence of CEP450-2 is shown in SEQ ID NO: 18,
[0013] The amino acid sequence of the ECDH is shown in SEQ ID NO:20.
[0014] In certain embodiments, the starting strain is the filamentous fungus Glarea lozoyensis ATCC 74030.
[0015] In certain embodiments, the cytochrome P450 enzyme gene is in a gene expression cassette, and has a promoter and a terminator upstream and downstream thereof, respectively; preferably, the promoter and the terminator are trpC promoter and trpC terminator, respectively.
[0016] In certain embodiments, the gene expression cassette is in an expression vector, and the backbone of the expression vector is preferably plasmid pAgG.
[0017] The second aspect of the present invention provides a method for preparing the genetically engineered bacteria according to the first aspect of the present invention, the preparation method comprising the following steps:
[0018] 1) constructing an expression vector containing the cytochrome P450 enzyme gene;
[0019] 2) Using the filamentous fungus Glarea lozoyensis as a host bacterium, transforming the expression vector obtained in step 1) to obtain a genetically engineered bacterium that overexpresses cytochrome P450 enzyme.
[0020] Preferably, in 1), the method for constructing the expression vector is:
[0021] A. constructing an expression vector containing a gene expression cassette containing a gpd promoter fragment and a gpd terminator fragment;
[0022] B. Inserting the cytochrome P450 enzyme gene into the gene expression cassette.
[0023] In certain embodiments, the transformation utilizes AMT technology or electroporation, such as AMT technology.
[0024] The third aspect of the present invention provides a method for preparing pneumocandin B by using the genetically engineered bacteria as described in the first aspect of the present invention. 0 On use.
[0025] The fourth aspect of the present invention provides a method for preparing pneumocandin B 0 The method comprises using a fermentation medium to culture the genetically engineered bacteria as described in the first aspect of the present invention, so that the genetically engineered bacteria are fermented and produce pneumocandin B 0 .
[0026] The method meets one or more of the following conditions:
[0027] (1) The formula of the fermentation medium includes: mannitol, cottonseed cake powder, soybean cake powder, K 2 HPO 4 、CaCO 3 and L-Pro;
[0028] (2) The pH value of the fermentation medium is 6 to 7, for example 6.5;
[0029] (3) The culture temperature is 20-30°C, for example, 25°C;
[0030] (4) The culture is a shaking culture, preferably, the shaking speed is 200-300 rpm, for example, 220 rpm;
[0031] (5) The culture period is 5 to 15 days, for example 10 days.
[0032] In certain embodiments, the fermentation medium comprises mannitol 60-140 g / L, for example, 100 g / L, cottonseed meal 3-8 g / L, for example, 5 g / L, soybean meal 5-15 g / L, for example, 10 g / L, K 2 HPO 4 3-6g / L, for example 4g / L, CaCO 3 0.5-2g / L, for example, 1g / L, L-Pro 10-30g / L, for example, 20g / L.
[0033] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0034] The reagents and raw materials used in the present invention are commercially available.
[0035] The positive and progressive effects of the present invention are:
[0036] The present invention can reduce the PB in the fermentation product by expressing the cytochrome P450 enzyme gene in the starting strain. 5 / PB 0 ratio, in reducing PB 5 The content of PB 0 PB 5 / PB 0 The ratio of PB can be reduced from 2.9% to at least 1%. In a preferred embodiment, PB 5 / PB 0 The ratio decreased from 2.9%±0.1% to 0.3%±0.1%. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 PB 0 and PB 5 Schematic diagram of the chemical structure of .
[0038] Figure 2 This is a liquid phase diagram of the plasmid and strain construction in Example 1.
[0039] Figure 3 This is a liquid phase diagram of the plasmid and strain construction in Example 2.
[0040] Figure 4 This is a liquid phase diagram of the plasmid and strain construction in Example 3.
[0041] Figure 5 This is a liquid phase diagram of the plasmid and strain construction in Example 4.
[0042] Figure 6 This is a liquid phase diagram of the plasmid and strain construction in Example 5.
[0043] Figure 7 This is the map of plasmid pAgG.
[0044] Figure 8 This is the map of plasmid pAgG-gloO.
[0045] Fig. 9 This is a map of plasmid pAgG-CEp450-2.
[0046] Fig.10 This is a map of plasmid pAgG-ecdH.
[0047] Fig.11 This is the map of plasmid pAgG-gloP. DETAILED DESCRIPTION
[0048] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0049] PB 0 and PH 5 The schematic diagram of the chemical structure is as follows Figure 1 As shown. Visible PB 0 and PB 5 The structural difference lies in the first amino acid residue in the loop, PB 0 is 4R,5R-dihydroxy-L-Orn, and PB 5 It is 5R-dihydroxy-L-Orn, which is difficult to separate and purify in the prior art.
[0050] Culture medium used in the present invention
[0051] 1. Seed culture medium formula (1L):
[0052] Glucose 20g, soybean cake powder 10g, KH2PO4. pH value is 5.5; sterilize at 121℃ for 20 minutes.
[0053] 2. Fermentation medium formula (1L):
[0054] Mannitol 100g, cottonseed cake powder 5g, soybean cake powder 10g, K2HPO 4 4g, CaCO 3 1g, L-Pro 20g. pH value is 6.5; sterilize at 121℃ for 20 minutes.
[0055] The primer sequences used in the present invention are shown in Table 1.
[0056] Table 1 Specific primer sequences
[0057]
[0058] Example 1 Construction of plasmid pAgG and strain G1-pAgG
[0059] Using pAg1-H3 as a template, PCR was performed with primers Ptrpc-F / Ptrpc-R to obtain the trpC promoter fragment. Using plasmid pEGFP-N2 as a template, PCR was performed with primers NeoR-F / NeoR-R to obtain the G418 resistance gene Neo fragment. Using pAg1-H3 as a template, PCR was performed with primers Ttrpc-F / Ttrpc-R to obtain the trpC terminator fragment. Using the trpC promoter fragment, the G418 resistance gene Neo fragment, and the trpC terminator fragment as templates, overlapping PCR was performed with primers Ptrpc-F and Ttrpc-R to obtain the G418 resistance gene expression cassette, and the expression cassette was connected to the HindIII / SpeI linearized pAg1-H3 vector to finally obtain the plasmid pAg1-HG. pAg1-HG was self-ligated after single cutting with EcoRI to obtain pAgG (such as Figure 7 shown).
[0060] The plasmid pAgG was introduced into the G. lozoyensis ATCC 74030 strain by Agrobacterium tumefaciens-mediated transformation (AMT) to obtain the engineered strain Gl-pAgG.
[0061] In order to detect the PB of the control strain Gl-pAgG 5 The yield was determined by inoculating it into 20 mL of seed culture medium, culturing it at 25°C, 220 rpm for 4 days, and then inoculating it into 30 mL of fermentation medium at 10% of the inoculum amount, culturing it at 25°C, 220 rpm for 10 days.
[0062] Detection of PB in fermentation broth by HPLC 5 Content and PB 5 / PB 0 The detection method is: take 2mL of fermentation liquid, add 8ml of anhydrous ethanol and mix thoroughly, ultrasonically vibrate for 30min, filter and obtain the filtrate for detection. Mobile phase: Phase A: H 2 O (0.1% phosphoric acid) Phase B: acetonitrile (0.1% phosphoric acid). Column: 4.6*250mm C18 ODS, Dalian Yilite, detection wavelength: 210nm, flow rate: 1.5mL / min.
[0063] The results showed that the PB in the fermentation product of the control strain G. lozoyensis ATCC 74030 0The yield is 2200±10mg / L, PB 5 / PB 0 2.9%±0.1%. PB in the fermentation product of strain Gl-pAgG 0 The yield was 2180±10mg / L, PB 5 / PB 0 The liquid phase diagram results are shown in Figure 2 .
[0064] Example 2 Construction of plasmid pAgG-gloO and strain Gl-gloO
[0065] First, construct an expression plasmid pAgG-gloO containing the gloO gene. Using the genome of G. lozoyensis ATCC 74030 as a template and gloO-F / R as primers, PCR was performed to obtain the gloO gene. The gloO gene was connected to the pAgG linearized vector cut with EcoRI / BglII using a homologous recombination kit (ClonExpress II one step cloning kit, catalog number: Vazyme C112-02) to obtain the plasmid pAgG-gloO (eg Figure 8 shown).
[0066] The plasmid pAgG-gloO was introduced into the G. lozoyensis ATCC 74030 strain using AMT to obtain the engineered strain Gl-gloO.
[0067] To confirm the PB of Gl-gloO strain 5 The yield was determined by inoculating it into 20 mL of seed culture medium, culturing it at 25°C, 220 rpm for 4 days, and then inoculating it into 30 mL of fermentation medium at 10% of the inoculum amount, culturing it at 25°C, 220 rpm for 10 days.
[0068] Detection of PB in fermentation broth by HPLC 5 Content and PB 5 / PB 0 The detection method is: take 2mL of fermentation liquid, add 8ml of anhydrous ethanol and mix thoroughly, ultrasonically vibrate for 30min, and filter the filtrate for detection. Mobile phase: Phase A: H 2 O (0.1% phosphoric acid) Phase B: acetonitrile (0.1% phosphoric acid). Column: 4.6*250mm C18 ODS, Dalian Yilite, detection wavelength: 210nm, flow rate: 1.5mL / min.
[0069] The results showed that the PB 0 The yield was 2290±10mg / L, PB 5 / PB 0 The liquid phase diagram is shown in Figure 3 The results show that the engineered strain Gl-gloO not only reduces PB 5 content, while increasing the PB 0 The content.
[0070] Example 3 Construction of plasmid pAgG-CEp450-2 and strain G1-CEp450-2
[0071] CEP450-2 is a homologous protein of GloO, derived from the strain Coleophoma empetri F-11899, and is a P450 enzyme in the FR901379 synthesis pathway.
[0072] First, an expression plasmid pAgG-CEp450-2 containing the CEp450-2 gene was constructed. The CEp450-2 gene was obtained by PCR using the genome of Coleophoma empetriF-11899 as a template and CEP450-2-F / R as primers. The CEp450-2 gene was connected to the pAgG linearized vector cut with EcoRI / BglII using a homologous recombination kit (ClonExpress II one step cloning kit, catalog number: Vazyme C112-02) to obtain the plasmid pAgG-CEp450-2 (such as Fig. 9 shown).
[0073] The plasmid pAgG-CEp450-2 was introduced into the G. lozoyensis ATCC 74030 strain using AMT to obtain the engineered strain Gl-CEp450-2.
[0074] In order to confirm the PB of Gl-CEp450-2 strain 5 The yield was determined by inoculating it into 20 mL of seed culture medium, culturing it at 25°C, 220 rpm for 4 days, and then inoculating it into 30 mL of fermentation medium at 10% of the inoculum amount, culturing it at 25°C, 220 rpm for 10 days.
[0075] Detection of PB in fermentation broth by HPLC 5 Content and PB 5 / PB 0 The detection method is: take 2mL of fermentation liquid, add 8ml of anhydrous ethanol and mix thoroughly, ultrasonically vibrate for 30min, filter and obtain the filtrate for detection. Mobile phase: Phase A: H 2 O (0.1% phosphoric acid) Phase B: acetonitrile (0.1% phosphoric acid). Column: 4.6*250mm C18 ODS, Dalian Yilite, detection wavelength: 210nm, flow rate: 1.5mL / min.
[0076] The results showed that the PB 0 The yield was 2240±10mg / L, PB 5 / PB 0 The liquid phase diagram results are shown in Figure 4 The results show that the engineered strain Gl-CEp450-2 not only reduces PB 5 content, while increasing the PB 0 The content.
[0077] Example 4 Construction of plasmid pAgG-ecdH and strain G1-ecdH
[0078] EcdH is a homologous protein of GloO, derived from the strain Aspergillus pachycristatus NRRL11440, and is a P450 enzyme in the echinocandin B biosynthesis pathway.
[0079] First, an expression plasmid pAgG-ecdH containing the ecdH gene was constructed. The ecdH gene was obtained by PCR using the genome of Aspergillus pachycristatusNRRL11440 as a template and ecdH-F / R as primers. The ecdH gene was connected to the pAgG linearized vector cut with EcoRI / BglII using a homologous recombination kit (ClonExpress II one step cloning kit, catalog number: Vazyme C112-02) to obtain the plasmid pAgG-ecdH (as Fig.10 shown).
[0080] The plasmid pAgG-ecdH was introduced into the G. lozoyensis ATCC 74030 strain using AMT to obtain the engineered strain Gl-ecdH.
[0081] To confirm the PB of Gl-ecdH strain 5 The yield was determined by inoculating it into 20 mL of seed culture medium, culturing it at 25°C, 220 rpm for 4 days, and then inoculating it into 30 mL of fermentation medium at 10% of the inoculum amount, culturing it at 25°C, 220 rpm for 10 days.
[0082] Detection of PB in fermentation broth by HPLC 5 Content and PB 5 / PB 0 The detection method is: take 2mL of fermentation liquid, add 8ml of anhydrous ethanol and mix thoroughly, ultrasonically vibrate for 30min, and filter the filtrate for detection. Mobile phase: Phase A: H 2O (0.1% phosphoric acid) Phase B: acetonitrile (0.1% phosphoric acid). Column: 4.6*250mm C18 ODS, Dalian Yilite, detection wavelength: 210nm, flow rate: 1.5mL / min.
[0083] The results showed that the PB 0 The yield was 2220±10mg / L, and the PB in the fermentation product was 5 / PB 0 1% ± 0.1%. Liquid phase diagram results are shown in Figure 5 The results show that the engineered strain Gl-ecdH not only reduces PB 5 content, while increasing the PB 0 The content.
[0084] Example 5 Construction of plasmid pAgG-gloP and strain Gl-gloP
[0085] GloP is another P450 enzyme in the biosynthetic pathway of Pneumocandins, responsible for the hydroxylation of the fourth amino acid residue. First, an expression plasmid pAgG-gloP containing the gloP gene was constructed. The gloP gene was obtained by PCR using the genome of G. lozoyensis ATCC 74030 as a template and gloP-F / R as primers. The gloP gene was connected to the pAgG linearized vector cut with EcoRI / BglII using a homologous recombination kit (ClonExpress II one step cloning kit, catalog number: Vazyme C112-02) to obtain the plasmid pAgG-gloP (such as Fig.11 shown).
[0086] The plasmid pAgG-gloP was introduced into the G. lozoyensis ATCC 74030 strain using AMT to obtain the engineered strain Gl-gloP.
[0087] To confirm the PB of Gl-gloO strain 5 The yield was determined by inoculating it into 20 mL of seed culture medium, culturing it at 25°C, 220 rpm for 4 days, and then inoculating it into 30 mL of fermentation medium at 10% of the inoculum amount, culturing it at 25°C, 220 rpm for 10 days.
[0088] Detection of PB in fermentation broth by HPLC 5 Content and PB 5 / PB 0 The detection method is: take 2mL of fermentation liquid, add 8ml of anhydrous ethanol and mix thoroughly, ultrasonically vibrate for 30min, filter and obtain the filtrate for detection. Mobile phase: Phase A: H 2O (0.1% phosphoric acid) Phase B: acetonitrile (0.1% phosphoric acid). Column: 4.6*250mm C18 ODS, Dalian Yilite, detection wavelength: 210nm, flow rate: 1.5mL / min.
[0089] The results showed that the PB in the fermentation product of the engineered strain Gl-gloP 0 The yield was 2190±10mg / L, PB 5 / PB 0 The liquid phase diagram results are shown in Figure 6 .
[0090] gloO gene sequence (including introns) (SEQ ID NO: 15)
[0091]
[0092] GloO amino acid sequence (SEQ ID NO:16)
[0093] miaalfttnlqlgavgvfifallafafnklttweysipkevqwvdrrtqpfsylrakaralarskentleayfrfnklgkaaalavpfgrpllllpqtfvrwivdqpesiisldpihddfhvfvggdltgdhtvqellrreltlnldklisvindeivcalddvlgnspewkstsladdlktivartsnrvfvgkdlcrnkhyistvkglalvimpetvlqdlipqflkgplsritkafnniygmkkfsslllgvvrqryidvkdvlegsgdktrlpddlltwmvqksirkgessanidkllvariamanlaaietttaamtrsvldlvtqgseggflkavqeetlavvegcnyepskkdvlklvltenaikealrlqvafpglmrqvvapngvtlenglhvpcgtrlgvsaagihvdesiyedpttynpgrflvrdldprgdpspmwkgnenylafslgrrscpgrwyvtdqlkltlahifskyeirfekaaetasalrkilpgapqdrvmirrrsvgkr
[0094] CEp450-2 gene sequence (including introns) (SEQ ID NO:17)
[0095]
[0096] CEP450-2 Amino Acid Sequence (SEQ ID NO:18)
[0097] misavvpiitstnlvfygatglvlfailayslnrlttweysipnevqwidrrkesfsylrakarslvrnkenvleayfkfnklgnaaacavafgrpllllpptfirwivdqpestisldpihddfhafvgdgligdhtvqellrreltlnldkltpmineeivsalddvlgnstewkttsladdlktiiartsnrvfmgkdlsqnedyintakglamvvmpetvlqdlvpqilkgplskitrvfnniyamkritshllpvvrqryidvknvfdgsgdkeqlpdnlltwmvqksirrgestanidkvlvarigmvnlaaietttaamtksildlvtvgveggfleavqeealavlegcnyqpeksdvsklnftenaikeslrlqvafpglmrqvvspkgvtldnglhvpygtrvgvsaagihvddsvyenattynpgrfmvidldargkpnplwkgtenylafglgrrscpgrwyvtdqlkltlahifskyeikfekaakktsalrkilpgapqdhvmirrrskval
[0098] ecdH Gene Sequence (including introns) (SEQ ID NO:19)
[0099]
[0100] EcdH amino acid sequence (SEQ ID NO:20)
[0101] MEFPLYTTTLLCGVISSTLLLLLLNKLTTWEHIVPSKVPWIDRRSEPFGYLRAKCRSFFNMKENITEAYYAFNKEGRAAALAIAFGRPQIILPPKYIRWIIDQPESVLSIDPIHNEFHAFVRDGLVGDHLVQEVLRRELAGHLAHLTREMNEEIADSVESVLGVSKEWATVPLRDSMRIIIARISNRLFVGKELCRNEDYIRNAVGLGMAVMPQTLVQDLLPQLLKGPLSFATKLFTRITLAGLSGHLSPVVRQRIQDVQTAEKDQLPLELLTWMAQRALQRGESATSIEEKLIARIAMANLASIETTTNTITKCMEDMTALSNETGGYLELMRQEAHTVLEACNYSPIKADLEKLVHIENALKESLRLAVVFPGLIRQVTSRTGVTLDDGTHLPHGARISVAAYAIHRDDANWTDAARYDPSRHEKASLPMSRGSEQLLSFGLGKRACPGRFFVTDELKLLFAHILTKYEFKVIKPPVTKVGLLKELTMRGPQEQLVIRRVK*
[0102] gloP gene sequence (including introns) (SEQ ID NO:21)
[0103]
[0104] GloP amino acid sequence (SEQ ID NO: 22)
[0105] Mlseviarvelligeqtlsggiltflfivviahfvltrftvhsrfwnsqawtgvreewfpkmrakvrtignirqmlsdgyegfskqnkafalpviaekpwlvlphscipellaksdseidmkiiheeqlmheyttg slgrhvvdvpiqydillrqvnrklpllisafneeldksfchywgtdtsysevnlsetcekivtqalnrifagkeicrdegflehsrlysegvgrnaimvrmlppllrpllapfitysnrkhrdiclrvclpvirerv qhtaakradaehkweppldvlqwiieesfarndpkeldprmitqrllalnfvaidtthmsmahtildlyrspnsddflvglreecervlqanggqwtksglddlvcvdstiresmrysdlgyisltrmvvdpkgtq fnangtnsssplsvppgiricvpahaihrdaalypspyefqafrfskarekyrgtqtelsepkvsivtttdkflpfghgrhacpgrffaaqqmklmlaylvqnydveklstkiqnkimvgttkpdaslkikvkrrkv
[0106] Finally, the above specific implementation method is only used to illustrate the technical solution of the present invention, but not to limit it.
Claims
1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria overexpresses cytochrome P450 enzyme genes, wherein the starting strain of the genetically engineered bacteria is the filamentous fungus Glarea lozoyensis.
2. The genetically engineered bacterium according to claim 1, characterized in that The cytochrome P450 enzyme gene is present in a plasmid or randomly integrated into the genome of the filamentous fungus through homologous recombination.
3. The genetically engineered bacterium according to claim 1 or 2, characterized in that: The cytochrome P450 enzyme gene is selected from one or more genes encoding the following proteins: GLoO, CEP450-2 and ECDH; preferably, The amino acid sequence of GLoO is shown in SEQ ID NO: 16, The amino acid sequence of CEP450-2 is shown in SEQ ID NO: 18, The amino acid sequence of the ECDH is shown in SEQ ID NO:
20.
4. The genetically engineered bacterium according to any one of claims 1 to 3, characterized in that The starting strain is the filamentous fungus Glarea lozoyensis ATCC 74030.
5. The genetically engineered bacterium according to any one of claims 1 to 4, characterized in that The cytochrome P450 enzyme gene is in a gene expression cassette, and has a promoter and a terminator upstream and downstream thereof, respectively; preferably, the promoter and the terminator are trpC promoter and trpC terminator, respectively.
6. The genetically engineered bacterium according to any one of claims 1 to 5, characterized in that The gene expression cassette is in an expression vector, and the backbone of the expression vector is preferably plasmid pAgG.
7. A method for preparing a genetically engineered bacterium according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: 1) constructing an expression vector containing the cytochrome P450 enzyme gene; 2) using the filamentous fungus Glarea lozoyensis as a host bacterium, transforming the expression vector obtained in step 1) to obtain a genetically engineered bacterium that overexpresses cytochrome P450 enzyme; Preferably, in 1), the method for constructing the expression vector is: A. constructing an expression vector containing a gene expression cassette containing a gpd promoter fragment and a gpd terminator fragment; B. Inserting the cytochrome P450 enzyme gene into the gene expression cassette.
8. The method for preparing a genetically engineered bacterium according to claim 7, characterized in that: The transformation utilizes AMT technology or electrotransduction, such as AMT technology.
9. Use of the genetically engineered bacteria according to any one of claims 1 to 6 in the preparation of Pneumocandin B0.
10. A method for preparing pneumocandin B0, characterized in that: The method comprises using a fermentation medium to culture the genetically engineered bacteria as described in any one of claims 1 to 6, so as to ferment and produce Pneumocandin B0.
11. The method according to claim 10, characterized in that It meets one or more of the following conditions: (1) The formula of the fermentation medium includes: mannitol, cottonseed cake powder, soybean cake powder, K2HPO4, CaCO3 and L-Pro; (2) The pH value of the fermentation medium is 6 to 7, for example 6.5; (3) The culture temperature is 20-30°C, for example, 25°C; (4) The culture is a shaking culture, and the shaking speed is 200-300 rpm, for example, 220 rpm; (5) The culture period is 5 to 15 days, for example 10 days.