CN113403242B

CN113403242B - Mutant Aspergillus oryzae strains

Info

Publication number: CN113403242B
Application number: CN202010187442.7A
Authority: CN
Inventors: 吴伟; 戴小军; 曹海生; 牛其文
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2024-05-24
Anticipated expiration: 2040-03-17
Also published as: CN113403242A

Abstract

The present invention relates to mutant Aspergillus oryzae strains. In particular, the present invention relates to mutant Aspergillus oryzae (Aspergillus oryzae) strains having an increased productivity of endogenous enzymes, e.g. amylases and/or exogenous proteins, such as food lipases, preferably food lipases selected from Rhizomucor miehei lipase, thermomyces lanuginosus lipase and Fusarium oxysporum lipase, relative to the unmutated strains. The invention also relates to various uses of the strain.

Description

Mutant Aspergillus oryzae strains

Technical Field

The invention relates to the field of microorganisms, in particular to a mutant aspergillus oryzae strain, a recombinant strain obtained by adopting the strain, a biocatalyst and the like, and a method for producing exogenous proteins by using the strain.

Background

Aspergillus oryzae (Aspergillus oryzae) is a strain commonly used in the fermentation industry and is a aerobic fungus belonging to the genus Aspergillus, the phylum Deuteromycotina. The history of aspergillus oryzae production of many fermented foods such as soy sauce, bean flour paste, sake and the like has been traced to a grade GRAS (Generally Regards As Safe) by the U.S. Food and Drug Administration (FDA) and has been approved by the world health organization (machidam Progress ofAspergillus oryzae genemics AdvAppl Microbio, l 2002, 51:81, 106), which is a very common species for use in filamentous fungi, and is widely used in the food brewing industry and industrial enzyme preparation industry.

Aspergillus oryzae is a strain capable of producing complex enzymes including amylase, protease, lipase, pectinase, phytase, etc., and has high-efficiency protein secretion and expression capability. The Aspergillus oryzae expresses the exogenous protein and has the characteristics of large expression quantity, high extracellular secretion rate, close to higher eukaryotic cells of a protein molecular folding and modifying system and the like, and the expressed exogenous protein has natural activity. In addition, aspergillus oryzae can also undergo various post-translational processes such as glycosylation modified protease cleavage and disulfide bond formation （Meyer V. Genetic engineering of filamentous fungi-progress, obstacle and future trends. Biotechnol Adv, 2008, 26(2):177-185）., and thus, the use of Aspergillus oryzae as an expression strain for expressing homologous and heterologous proteins is increasingly gaining attention.

In industrial processes, aspergillus oryzae expresses heterologous proteins at lower levels than homologous proteins (Zhang Jianjun, cai Ronghua, li Jiang, etc., strategies to increase the amount of protein expressed in Aspergillus oryzae journal of Chinese bioengineering, 2009, 29 (1): 111-115). The expression of the foreign protein in Aspergillus oryzae can be enhanced by constructing a self protease deficient strain, using a strong promoter, fusion expression and the like. However, protease deficient strains generally affect the growth rate of the strain, which is disadvantageous for industrial production, and fusion expression may present regulatory risks in the production process of enzymes for food, so strategies using strong promoters are generally more common.

Further suitable selectable markers are an important component of the expression system. The most commonly used auxotroph selection markers in Aspergillus oryzae are the nitrate reductase auxotroph (niaD ^-) conversion system and the orotidine-5' phosphate decarboxylase auxotroph (pyrG ^-) conversion system (Dairy, ma Shiliang. Aspergillus oryzae exogenesis expression systems research progress. J. China bioengineering, 2016, 36 (9): 94-100). However, because the hypha of Aspergillus oryzae is polynuclear and most of conidia are polynuclear, the efficiency of mutagenesis or genetic modification is greatly reduced, and the difficulty of establishing a transformation system is increased （Jun-ichi MARUYAMA., Visualization of Nuclei in Aspergillus oryzaewith EGFP and Analysis of the Number of Nuclei in Each Conidium by FACS.Biosci.Biotechnol.Biochem,65(7),1504-1510,2001）.

In China, the enzyme preparation applied to food must meet the national standard GB2760 of the people's republic of China-national food standard\food additive use standard. Wherein the usual heterologously expressed food lipase according to regulations: lipase from rhizopus oryzae (Rhizomucor miehei), thermomyces gossypii (Thermomyces lanuginosus) and fusarium oxysporum (Fusarium oxysporum) can be heterologously expressed only by aspergillus oryzae, which reflects the importance of the aspergillus oryzae expression system in the production of enzymes for food. However, according to the prior art, a lipase for food is heterologously expressed from Aspergillus oryzae, and the expression level is often very low, for example: wang expressing the Rhizopus oryzae (Rhizomucor miehei) -derived lipase RML in Aspergillus oryzae auxotroph host bacteria (Aspergillus oryzae niaD, niaD-) and taking the culture supernatant of 7 days of culture, and determining that the enzyme activity is only 2.5U/mL by adopting an alkali titration method (Wang, pan Li, guo Yong. Construction of a filamentous fungus Aspergillus oryzae exogenous gene expression system. Proc. Natl. Acad. Sci. Huanan, 2009, 37 (6): 84-90), thus failing to meet the industry requirements.

Thus, there remains a need in the art for Aspergillus oryzae strains capable of efficiently expressing various proteins, especially food lipases.

Disclosure of Invention

The invention constructs a orotidine-5' -phosphate decarboxylase auxotroph (pyrG ^-) strain by taking aspergillus oryzae CICC2012 as an initial strain, and improves the protein expression capability by ARTP mutagenesis, so that the strain becomes an effective heterologous expression host, and can efficiently and heterologously express various proteins, in particular to lipase for food.

In particular, the present invention relates to the following aspects.

In one aspect, the invention relates to a mutant Aspergillus oryzae (Aspergillus oryzae) strain having increased productivity of an endogenous enzyme, e.g., an amylase and/or an exogenous protein, e.g., a food lipase, e.g., a lipase selected from Rhizomucor miehei lipase, thermomyces lanuginosus lipase, and Fusarium oxysporum lipase, relative to an unmutated strain. In the present invention, the term "endogenous enzyme" refers to an enzyme expressed by the mutant Aspergillus oryzae (Aspergillus oryzae) strain itself, for example, including but not limited to an amylase expressed by the mutant Aspergillus oryzae (Aspergillus oryzae) strain itself.

In a specific embodiment, the mutant Aspergillus oryzae strain of the present invention has a preservation number of CGMCC No. 18825.

The novel Aspergillus oryzae strain of the present invention is capable of efficiently expressing heterologous proteins. The aspergillus oryzae strain is a orotidine-5' -phosphate decarboxylase auxotroph strain, and the endogenous amylase expression level can be improved by more than 40% compared with the original strain, for example, 40% -100%, particularly 40% -60%, especially 45% -55%. When the RML is expressed in a heterologous way, the protein expression level of the strain is improved by 50 to 100 percent, particularly 50 to 80 percent, especially 55 to 70 percent compared with that of the original strain. Furthermore, when TLL and sol are expressed, the protein expression level is increased by more than 40%, in particular 40-50%, compared to the original strain cic 2012. Therefore, the strain has obvious practicability of efficiently expressing the heterologous protein.

In another aspect, the present invention relates to a recombinant A.oryzae strain obtained by introducing a gene encoding a foreign protein into the mutated A.oryzae strain described above. In one embodiment, the exogenous protein is an enzyme, for example a food lipase, such as a food lipase selected from rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase.

In another aspect, the present invention relates to a method for producing a protein of interest, comprising introducing a gene encoding said protein of interest into a mutant strain of Aspergillus oryzae as described above, and culturing said strain to produce the protein of interest. Or comprises culturing the recombinant A.oryzae strain described above to produce the protein of interest. In one embodiment, the exogenous protein is an enzyme, for example a food lipase, such as a food lipase selected from rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase.

In another aspect, the invention relates to a biocatalyst comprising the above mutant Aspergillus oryzae strain into which a gene encoding an exogenous enzyme, such as a food lipase, e.g. a food lipase selected from Rhizomucor miehei lipase, thermomyces lanuginosus lipase and Fusarium oxysporum lipase, has been introduced.

In another aspect, the invention relates to a foreign protein produced by the strain described above. The exogenous protein is an enzyme, such as a food lipase, e.g., a food lipase selected from rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase.

The invention also relates to recombinant microbial cells into which the intra-bacterial components derived from the mutated A.oryzae strains described above have been introduced. After the strain of the present invention is obtained, its intracellular components may be isolated by conventional techniques and introduced into other microorganisms. Recombinant microbial cells incorporating the components have excellent properties of the strains of the invention. In the present invention, the term "intra-bacterial component" refers to the sum of all genetic material of an organism, including, in particular, but not limited to: coding DNA and non-coding DNA, mitochondrial DNA.

In particular, the mutant Aspergillus oryzae strain of the present invention can be used to express exogenous proteins. After the mutant Aspergillus oryzae of the present invention is obtained, a conventional expression vector may be introduced therein for expressing the foreign protein. For example, the expression vector may include a promoter and a terminator, and a gene encoding a foreign protein may be inserted between the promoter and the terminator with multiple cloning sites. One or more copies of the enhancer may be included in the promoter. A number of commercial vectors can be used for expression of the foreign proteins in the mutant Aspergillus oryzae strains of the invention.

When expressing exogenous proteins that are not of Aspergillus oryzae origin, the codons of certain species may be rare codons in Aspergillus oryzae. Therefore, when the expression vector is introduced, the codon optimization of the Aspergillus oryzae suitable for the invention can be performed on the gene encoding the exogenous protein, thereby increasing the expression quantity.

The Aspergillus oryzae of the present invention can express a variety of foreign proteins including food enzymes such as food lipases, pharmaceutical proteins, various enzymes derived from plants, animals and bacteria, membrane receptor proteins, prosthetic group-containing proteins, proteins useful for studying crystal structures, and the like. The aspergillus oryzae expressing the exogenous enzyme component of the present invention can also use whole cells as biocatalysts.

Drawings

FIG. 1 is a schematic diagram of an expression vector for the RML gene constructed in the present invention.

FIG. 2 is a comparison of the enzymatic activities of the endogenous amylases expressed by the strain BC4 of the invention and the starting strain CICC2012 and the strain pyrG L4.

FIG. 3 is a protein electrophoretogram of the endogenous amylase expressed by the strain BC4 of the invention and the starting strain CICC2012 and the strain pyrG L4.

FIG. 4 is an enzyme activity comparison of strain BC4 of the invention with the Rhizomucor miehei lipase RML expressed by the original strain CICC2012 and the strain pyrG L4.

FIG. 5 is a protein electrophoretogram of the RML expressed by the strain BC 4of the present invention and the starting strain CICC2012 and the strain pyrG L4.

FIG. 6 is a graph comparing the enzymatic activities of the amylase expressed by the strain BC4 of the invention and the original strain CICC2012 and the strain pyrG L4 in a 500L fermenter.

FIG. 7 is a graph comparing the enzymatic activities of the strain BC4 of the invention with the Rhizomucor miehei lipase RML expressed by the starting strain CICC2012 and the strain pyrG L4 in a 500L fermenter.

Detailed Description

The initial strain Aspergillus oryzae of the invention is purchased from China industry microbiological culture collection center (CICC for short), and the strain deposit number CICC2012. The initial strain is firstly enriched and grown on an inositol plate, then subjected to ultraviolet mutagenesis, and finally screened by adding 5-fluoroorotic acid and uracil on a screening plate, so that the orotidine-5' phosphate decarboxylase auxotroph strain pyrG L4 is obtained. And screening a strain BC4 by utilizing an ARTP mutagenesis mode, wherein the amylase activity of the strain BC4 on a 500L fermentation tank reaches 12264U/mL, and compared with the original strains CICC2012 and pyrG L4, the amylase expression quantity is improved by 52.2 percent and 51.4 percent. In the present invention, the term "ARTP" is an abbreviation for atmospheric pressure room temperature plasma (Atmospheric and Room Temperature Plasma), and specifically refers to a plasma jet capable of generating a plasma jet having a high concentration of active particles (including helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc. in an excited state) at a temperature of 25 to 40 ℃ at atmospheric pressure. The term ARTP mutagenesis, namely strain mutagenesis by utilizing the normal pressure room temperature plasma technology, specifically, a normal pressure room temperature plasma source adopting helium as working gas contains various chemical active particle components such as OH, nitrogen molecule di-positive system, nitrogen molecule-negative system, excited helium atom, hydrogen atom, oxygen atom and the like. The active energy particles enriched in ARTP cause damage to genetic material of strains/plants/cells, etc., and induce biological cells to initiate SOS repair mechanisms. SOS repair is a repair with high fault tolerance, so that various mismatch sites can be generated in the repair process, and finally stable inheritance is realized to form mutant strains. The intensity of SOS repair is strongly related to the extent to which DNA is damaged.

When heterologous expression of Rhizomucor miehei lipase RML for food was attempted using an enolase promoter with 12 copies of the enhancer sequence added to Aspergillus oryzae strain BC4, it was found that the activity of the RML lipase expressed by BC4 on a 500L fermenter reached 6488U/mL, which was 68.1% and 58.0% higher than that of the original strains CICC2012 and pyrG L4. Compared with the prior art, for example: wang expressing Aspergillus oryzae auxotroph host bacteria (Aspergillus oryzae niaD, niaD-) Lipase RML derived from Rhizopus oryzae (Rhizomucor miehei) with an enzyme activity of only 2.5U/mL (Wang, pan Li, guo Yong. Construction of exogenous gene expression System for filamentous fungi Aspergillus oryzae. University of North America, 2009, 37 (6): 84-90), the advantages of the present invention are very obvious when Aspergillus oryzae is used for heterologous expression of Lipase RML for food.

The inventor also carries out the expression of thermomyces lanuginosus (Thermomyces lanuginosus) lipase TLL and fusarium oxysporum (Fusarium oxysporum) lipase FOL in the aspergillus oryzae strain BC4, and can carry out high-efficiency expression, and the result shows that when TLL and FOL obtained by BC4 expression are used, the protein expression level is improved by 45.6 percent and 40.5 percent compared with that of the original strain CICC2012, thereby proving that the expression system has obvious advantages when being used as a lipase expression system for food. Furthermore, the inventors have found that the use of the enzymes of interest expressed by Aspergillus oryzae BC4, such as RML, TLL and FOL, has significant utility in specific applications, such as:

1. The immobilized RML enzyme preparation and the immobilized TLL enzyme preparation obtained can be immobilized using RML and TLL obtained by BC4 expression of aspergillus oryzae. When the immobilized RML enzyme preparation and the immobilized TLL enzyme preparation are used for OPO production, under the requirements that the two-position palmitic acid content in the OPO product is more than 52 percent and the 1, 3-dioleoyl-2-palmitic acid triglyceride content (calculated by C52) is more than 40 percent, the use batch of the immobilized RML is not less than 40 times, the use batch of the immobilized TLL is not less than 60 times, which is obviously superior to the use batch of the immobilized enzyme obtained by using the commercial enzyme Novozym ^® 40086, and the RML and the TLL expressed by the Aspergillus oryzae BC4 are applied to OPO production, not only can meet the production index requirement, but also can be repeatedly used for a plurality of batches, thereby greatly reducing the production cost and having obvious economic value.

2. The FOL obtained by using the expression of the aspergillus oryzae BC4 is used for enzymatic degumming of crude oil, the degumming effect is remarkable, and specifically, the phosphorus content of the oil can be reduced to below 10ppm through one-step degumming, so that the subsequent physical refining is directly carried out, a chemical refining section is omitted, refining procedures and refining consumption are saved, the production cost is reduced, and the environment-friendly effect is achieved.

Example 1: acquisition of orotidine-5' phosphate decarboxylase auxotrophic Aspergillus oryzae Strain

Referring to patent CN201310288060.3, aspergillus oryzae CICC2012 strain spores were inoculated and spread onto MM solid medium (1% glucose, 0.15% KH ₂PO₄,0.6% NaNO₃,0.05% KCl,0.05% MgSO₄, 2% agar powder) with 0.04% inositol added, and were left to stand at 28 ℃ for 5 days to obtain enriched aspergillus oryzae spores. Fresh aspergillus oryzae CICC2012 spores were eluted with spore wash (0.9% nacl,0.05% tween 80), prepared as spore suspension by Miracloth (Calbiochem, cat# 475885) filtration, and the cells were washed 2 times with sterile water and adjusted to 1 x 10 ⁷/mL. 2mL of spore suspension is evenly dispersed on the surface layer of a culture dish, the culture dish is placed under an ultra-clean workbench ultraviolet lamp to be irradiated for 90s, 100 mu L of the spore suspension is coated on an MM solid culture medium added with 0.3% uracil (Uracil) and 1mg/mL 5-fluoroorotic acid (5-FOA), and the spore suspension is cultivated at 28 ℃ in a dark place (the whole process is operated under red light to prevent reverse mutation) for 7 days. And transferring the single colony growing on the MM solid culture medium in the last step to the MM solid culture medium and the MM-Uracil solid culture medium, and picking the strain which can only grow on the MM-Uracil solid culture medium, thereby obtaining the orotidine-5' phosphate decarboxylase auxotroph Aspergillus oryzae pyrG L4 strain. By sequencing the pyrG gene of the strain Aspergillus oryzae pyrG L4, it was found that the 34 th nucleotide was changed from C to T and the corresponding amino acid change was from arginine (CGC) to cysteine (TGC), resulting in inactivation of the pyrG gene.

Example 2: construction of RML expression vector

The exogenous Rhizomucor miehei (Rhizomucor miehei) lipase (RML) gene sequence is as follows.

Nucleic acid sequence:

gtgccaatcaagagacaatcaaacagcacggtggatagtctgccacccctcatcccctctcgaacctcggcaccttcatcatcaccaagcacaaccgaccctgaagctccagccatgagtcgcaatggaccgctgccctcggatgtagagactaaatatggcatggctttgaatgctacttcctatccggattctgtggtccaagcaatgagcattgatggtggtatccgcgctgcgacctcgcaagaaatcaatgaattgacttattacactacactatctgccaactcgtactgccgcactgtcattcctggagctacctgggactgtatccactgtgatgcaacggaggatctcaagattatcaagacttggagcacgctcatctatgatacaaatgcaatggttgcacgtggtgacagcgaaaaaactatctatatcgttttccgaggttcgagctctatccgcaactggattgctgatctcacctttgtgccagtttcatatcctccggtcagtggtacaaaagtacacaagggattcctggacagttacggggaagttcaaaacgagcttgttgctactgttcttgatcaattcaagcaatatccaagctacaaggttgctgttacaggtcactcactcggtggtgctactgcgttgctttgcgccctgggtctctatcaacgagaagaaggactctcatccagcaacttgttcctttacactcaaggtcaaccacgggtaggcgaccctgcctttgccaactacgttgttagcaccggcattccttacaggcgcacggtcaatgaacgagatatcgttcctcatcttccacctgctgcttttggttttctccacgctggcgaggagtattggattactgacaatagcccagagactgttcaggtctgcacaagcgatctggaaacctctgattgctctaacagcattgttcccttcacaagtgttcttgaccatctctcgtactttggtatcaacacaggcctctgtacttaa (SEQ ID NO:1).

Amino acid sequence:

VPIKRQSNSTVDSLPPLIPSRTSAPSSSPSTTDPEAPAMSRNGPLPSDVETKYGMALNATSYPDSVVQAMSIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT(SEQ ID NO:2).

For specific procedures, reference is made to the method of molecular cloning Experimental guidelines (third edition, new York, cold spring harbor laboratory Press, new York: cold Spring Harbor Laboratory Press, 1989), which constructs the RML gene expression vector pAOP-Eno-E, as shown in FIG. 1, by the following procedure:

The RML gene obtained from the total gene synthesis of the biological engineering (Shanghai) Co., ltd (SEQ ID NO:1, with the Aspergillus oryzae alpha-amylase signal peptide (NCBI SEQ ID NO: XM_001821384.2,1-63bp sequence)) was inserted into the expression cassette with the Aspergillus oryzae enolase promoter (NCBI SEQ ID NO: D63941.1, 215-734bp; containing 12 copies of the enhancer sequence (GTCGTGTCGGGCATTTATCGGGGGATGGACCAATCAGCGTAGG, SEQ ID NO: 3)) and the Aspergillus niger glucoamylase terminator (NCBI SEQ ID NO: AF214480.1, wherein the terminator sequence portion), the entire expression cassette was inserted into the multiple cloning site of cloning vector pSP72 with BglII and XhoI, and finally the Aspergillus oryzae-derived pyrG expression gene (NCBI SEQ ID NO: AB 017705.1) was inserted into the vector with the XhoI cleavage site, thereby constructing the RML gene expression vector pAOP-Eno-E.

Example 3: aspergillus oryzae pyrG L4 anaplerotic experiment

Fresh Aspergillus oryzae PyrG L4 spores were eluted with spore wash (0.9% NaCl,0.05% Tween 80), prepared as spore suspension by Miracloth filtration, and adjusted to 1X 10 ⁷/mL. 1mL of spore suspension was inoculated into mycelium culture medium (2% tryptone, 1% yeast extract, 2% glucose, 0.3% uracil), cultured at 28℃and 180rpm for 40 hours, and the grown mycelium was collected by filtration with a sterile Miracloth.

The collected mycelia were washed three times with sterilized osmostat (0.6M MgSO ₄,10mM NaH₂PO₄, ph=5.8) and pressed dry. Hyphae were transferred to 100mL Erlenmeyer flasks. Every 0.8g mycelium is resuspended in 20mL of enzymolysis liquid (1% of lyase, 1% of cellulase and 0.1% of snailase enzymolysis liquid prepared by osmotic stabilizer, and filtered and sterilized by a microporous filter membrane with the size of 0.22 μm), and the enzymolysis is carried out for 60-90min at 30 ℃ and 90 rpm. The hydrolyzed protoplast mixture was filtered with Miracloth, the filtrate was collected, centrifuged at 4℃for 10min at 1000g, the protoplast pellet was resuspended in 5mL of pre-chilled 1.0mol/L sorbitol solution, centrifuged at 800g at 4℃for 10min and the supernatant was discarded. The protoplasts were then adjusted to 1X 10 ⁷ per mL with pre-chilled STC solution (1.0M sorbitol, 50mM CaCl ₂, 50mM Tris-HCl, pH=7.5) and ice-bath ready for use.

To 200. Mu.L of the protoplast suspension, 10. Mu.L of the RML expression vector pAOP-Eno-E at a concentration of 1. Mu.g/. Mu.L was added, followed by addition of 50. Mu.L of PTC solution (40% PEG4000, 50mM CaCl ₂, 50mM Tris-HCl, pH=7.5), and after mixing, ice bath was carried out for 30 minutes. Adding 0.2mL of PTC solution, uniformly mixing, adding 0.8mL of PTC solution, uniformly mixing, and keeping at room temperature for 30min.

The mixture was spread on a regeneration medium (1% glucose ,0.6%NaNO₃,0.15% KH₂PO₄,0.05% KCl,0.05% MgSO₄,0.001% FeSO₄,1M sucrose, 2% agar powder) and incubated at 28℃for 7 days until colonies developed.

Colonies growing on the plates were transferred to PDA-Rhodamine B screening medium and incubated at 28℃for 3-6 days.

The preparation method of the PDA-Rhodamine B screening culture medium is as follows:

PDA medium: 200g of potato (peeled), 20g of glucose, 1000mL of agar 20g of water and natural pH value;

-olive oil emulsion: adding olive oil and 4% polyvinyl alcohol (PVA) solution in a volume ratio of 1:3, mixing, homogenizing for 15min at 8000rpm with a high-speed homogenizer, and stirring for 3min after 5 min;

PDA culture medium and olive oil emulsion were autoclaved for 15min at 115 ℃ respectively; after cooling to about 60 ℃, PDA culture medium and olive oil emulsion are added according to the ratio of 9:1, and then 1/100 volume of filter sterilized 0.1% rhodamine B (Rhodamine B) solution is added, and after uniform mixing, the mixture is immediately poured into a flat plate.

The transformation discovers that the aspergillus oryzae pyrG L4 can well realize pyrG gene anaplerotic experiments, and the transformant shows lipase activity in a PDA-Rhodamine B screening culture medium, and further proves that an aspergillus oryzae expression system taking orotidine-5' phosphate decarboxylase auxotroph aspergillus oryzae strain pyrG L4 as a host is successfully established.

Example 4: aspergillus oryzae pyrG L4 ARTP mutagenesis experiment

Fresh Aspergillus oryzae PyrG L4 spores were eluted with spore wash (0.9% NaCl,0.05% Tween 80), prepared as spore suspension by Miracloth filtration, and adjusted to 2X 10 ⁷/mL. After mixing the spore suspension with 10% glycerol 1:1, 10. Mu.L of the mixture was applied to an iron sheet and treated with an ARTP instrument (model: ARTP-M, instrument model: qingshan Biotechnology Co., ltd.) for 100s, instrument parameters were set: the radio frequency power range is 120W, the helium quantity is 10 SLM (99.999 percent of high-purity helium), and the irradiation distance is 2 mm.

After the treatment, the small iron sheet is taken down and put into a centrifuge tube filled with 1mL of sterile water, and then repeatedly sucked by a gun head, so that thalli on the iron sheet are washed off, diluted to a proper concentration, coated on an amylase screening plate added with uracil (1% of soluble starch and 0.3% of uracil are added into a PDA culture medium), and placed in a 30 ℃ incubator for 3d, and colonies with larger hydrolysis circles are selected.

Example 5: screening of amylase high-producing strains

The colonies selected in example 4 with larger amylase hydrolysis circles were inoculated into 24-well plates, and 3mL of fermentation medium (4% corn steep liquor dry powder, 3.5% corn dextrin, 0.15% potassium dihydrogen phosphate, 0.6% disodium hydrogen phosphate dodecahydrate, 0.05% magnesium sulfate heptahydrate, 0.3% uracil) was added to the plates at 180rpm for 8 days, and preliminary screening was performed.

And then carrying out shake flask fermentation on the strain with higher amylase activity obtained by the primary screening to carry out secondary screening, wherein the secondary screening fermentation medium is subjected to primary screening, and the liquid loading amount is 50mL/250mL of triangular shake flask, 150rpm and 28 ℃ for 8 days.

Amylase assay method:

Taking a clean sterile centrifuge tube (2 mL or 1.5 mL), sequentially adding 200uL of starch substrate (20 mL of soluble starch (20 g/L) is absorbed into a test tube, adding phosphate buffer (45.23 g of disodium hydrogen phosphate dodecahydrate and 8.07g of citric acid monohydrate, dissolving with water and fixing the volume to 1000mL, and pH value of 6.0) to 5mL, shaking uniformly) and 100uL of enzyme solution, fully mixing the starch substrate and the enzyme solution upside down, and rapidly placing into a water bath kettle at 60 ℃ for enzymatic reaction for 10 min. Immediately after the reaction, 450uL of DNS (10 g of 3, 5-dinitrosalicylic acid is weighed in water, 20g of NaOH and 200g of sodium tartrate are added after dissolution, water is added to a constant volume of 500mL, 2g phenol and 0.5g of anhydrous sodium sulfite are added after heating dissolution, heating dissolution and cooling are carried out, water is used to a constant volume of 1000mL, the mixture is stored in a brown bottle and used after 1 week), and the mixture is fully mixed to terminate the reaction rapidly, and the mixture is transferred into an ice water bath for cooling rapidly after 10 minutes in a boiling water bath. And (3) sequentially adding 190uL of distilled water into the clean ELISA plate, uniformly mixing, and measuring the absorbance value of each tube of reaction liquid at 540nm by using an ELISA instrument.

Definition of amylase activity (U) units: in the above specific assay mode, the unit time (min) catalyzes the hydrolysis of the soluble starch and produces reducing sugar with a reducing power corresponding to the amount of enzyme required for 1umol of glucose.

Calculation formula of enzyme activity

Enzyme activity (U/mL) =

Wherein:

n: dilution factor;

ODt: absorbance at 540nm for the experimental group;

ODc: absorbance at 540nm for the control group;

1000: unit conversion multiple;

0.106: slope of glucose standard curve;

10: reaction time at 60 DEG C

The final results are shown in FIG. 2 for amylase enzyme activity and FIG. 3 for protein electrophoresis. The amylase high-yield strain BC4 is obtained through screening, the amylase activity of the amylase high-yield strain BC4 reaches 6415U/mL, and compared with the original strains CICC2012 and pyrG L4, the amylase expression quantity is respectively improved by 40.4% and 45.1%.

The strain BC4 is preserved in China general microbiological culture collection center (CGMCC) for 10 months and 30 days in 2019, and the preservation number is CGMCC No.18825, and is named as Aspergillus oryzae (Aspergillus oryzae) in the China national academy of sciences of China, the West-Lu No.1 and the 3 of the university of China, north Star, which are the areas of Korean of Beijing.

Example 6: investigation of the expression Capacity of the amylase high-producing Strain BC4

Transformation procedure for A.oryzae BC4 the RML expression vector pAOP-Eno-E was transformed into the strain as in example 3.

Transformation screening was performed using the Aspergillus oryzae pyrG L4 transformant of example 3 as a control, and pAOP-Eno-E was additionally transformed into Aspergillus oryzae CICC2012, using p3SR2 ((BCCM/LMBP: accession number: 2363), containing the acetamidase (amdS) gene, since Aspergillus oryzae CICC2012 failed to use pyrG as a screening marker. Regeneration medium requires removal of sodium nitrate and addition of 15mM acetamide and 20mM cesium chloride.

40 Transformants were selected for each of the three transformants and subjected to shaking fermentation, and the fermentation medium (4% corn steep liquor dry powder, 3.5% corn dextrin, 0.15% potassium dihydrogen phosphate, 0.6% disodium hydrogen phosphate dodecahydrate, 0.05% magnesium sulfate heptahydrate) was subjected to 150rpm,28℃for 8 days, and the RML lipase activity was measured.

The RML lipase activity assay is as follows: olive oil titration lipase assay

(1) Preparation of 0.05M NaOH: firstly, preparing 5M NaOH stock solution by using CO ₂ -free water; accurately diluting 50 times, weighing 0.38g of potassium hydrogen phthalate which is dried to constant weight at 100 ℃, dissolving in 80mL of CO ₂ -free water, calibrating the accurate concentration, and calculating the stock solution concentration; the NaOH solution of 0.05M NaOH is prepared in the form of CO ₂ -free water stock solution;

(2) Mixing 100mL of olive oil and 300mL of 4% PVA1750 (polyvinyl alcohol), heating for melting, emulsifying with ultrasonic wave with power of 300W, ultrasonic wave for 3s, intermittent for 4s,99 times, and circulating for 2 times;

(3) Adding 4mL of emulsion substrate, 2mL of 0.2M Tris-HCl buffer solution with pH of 8.0 and 3 mL sterile water into a 100mL triangular flask, and placing the triangular flask in a constant-temperature water bath shaking table at 40 ℃ for incubation at 150rpm for 5min;

(4) Adding 1mL of properly diluted enzyme solution into the substrate and the buffer solution, reacting at 40 ℃ and 150rpm for 15min, and adding 10mL of absolute ethyl alcohol to terminate the reaction;

(5) 2 drops of phenolphthalein are dripped as an indicator, and 0.05M NaOH is used for titrating fatty acid generated by enzymolysis until the reaction liquid turns pink;

blank procedure was identical to that described above except that after the reaction was stopped with absolute ethanol when the substrate and buffer were in a water bath for 15min, 1mL of an appropriately diluted enzyme solution was added.

(6) Definition and calculation formula of enzyme activity

The lipase enzyme activity units are defined as: the amount of enzyme catalyzing the substrate to release 1. Mu. Mol fatty acid per minute was 1 lipase activity unit (U).

Enzyme activity calculation formula:

Enzyme activity (U/mL) =

Wherein: v: volume of NaOH solution consumed by titration of sample solution (ml)

V ₀: volume of NaOH solution (ml) consumed by titration of blank

T: reaction time (min)

N: enzyme liquid volume (ml)

M: concentration of NaOH solution for titration (mmol/L)

The results of the enzyme activity measurement are shown in FIG. 4. The result shows that the enzyme activity of the BC4 expressed RML lipase reaches 5560U/mL, which is improved by 66.9 percent and 60.6 percent compared with the original strains CICC2012 and pyrG L4.

Polyacrylamide gel electrophoresis analysis: the supernatant was filtered through a 0.22 μm filter membrane, and an equal amount of the supernatant was concentrated to the same volume using a Millipore 10kDa ultrafiltration concentration tank, and then the same volume of concentrated enzyme solution was taken for polyacrylamide gel electrophoresis analysis. The result of electrophoresis is shown in FIG. 5. The results of the protein electrophoretogram showed that the RML protein band concentration of BC4 transformants was significantly higher than the control.

The specific reason for the increased levels of protein expression in the BC4 strain is presumably due to the increased intracellular transcription, translation or secretion efficiency, which can be exploited in the expression of RML, and thus the level of heterologous expression of RML.

Example 7: BC4 and BC4-RML Strain 500L fermenter experiment

The 500L fermentation tank culture medium is prepared according to the total volume of 300L for each nutrient component concentration, and comprises the initial constant volume of 245L, the tank elimination condensed water of about 25L, and the dextrin solution of 30L is added in 0 hour (0 h).

Basal medium: 5610g of corn steep liquor dry powder, 207g of potassium dihydrogen phosphate, 994g of disodium hydrogen phosphate dodecahydrate, 41.53 magnesium sulfate heptahydrate and 245L of constant volume (0.3% uracil is needed for fermenting pyrG L4 and BC 4); the fed-batch culture medium is consistent with the basic culture medium, and the volume is fixed by 38L;

defoaming agent: 10% silicone emulsion, 200g/2000mL;

Dextrin solution: the concentration of the preparation is 200g/L, 150L is prepared in total, and 30kg is required.

Fermentation parameters: the fermentation temperature is 28 ℃, the ventilation is 0.6vvm, the defoamer is added as required, the rotating speed is 135-280rpm, the adjustment is carried out according to the actual situation in the fermentation process, the feeding of the fed-batch culture medium and the dextrin solution is started from 24 hours, and the fermentation is carried out for about 184 hours.

The activities of amylase and RML lipase obtained from a specific 500L fermenter are shown in fig. 6 and 7.

The results in FIG. 6 show that amylase activity of amylase high-producing strain BC4 on a 500L fermenter reached 12264U/mL, and amylase expression levels were increased by 52.2% and 51.4% as compared to the original strains CICC2012 and pyrG L4.

The results in FIG. 7 show that the BC4 expressed RML lipase on 500L fermentors reached 6488U/mL, 68.1% and 58.0% improvement over the starting strain CICC2012 and pyrG L4.

Example 8: expression of TLL and FOL in A.oryzae strain BC4

The construction procedure of the expression vectors for TLL and FOL was the same as in example 2, and the TLL (NCBI sequence No. AF054513.1 and FOL (NCBI sequence No. EF 613329.1) gene sequences were all synthesized by the division of Biotechnology (Shanghai).

Transformation procedure for A.oryzae BC4 the TLL and FOL expression vectors were transformed into the strain as in example 3.

40 Transformants were selected for each of the two transformants, subjected to shaking fermentation, and the fermentation medium (4% corn steep liquor dry powder, 3.5% corn dextrin, 0.15% potassium dihydrogen phosphate, 0.6% disodium hydrogen phosphate dodecahydrate, 0.05% magnesium sulfate heptahydrate) was subjected to 150rpm,28℃for 8 days, and the lipase activity was measured.

The results showed that TLL and sol expressed by BC4 increased protein expression levels by 45.6% and 40.5% compared to the starting strain cic 2012.

Example 9: application of RML and TLL expressed in Aspergillus oryzae strain BC4 in the production of 1, 3-dioleate-2-palmitic acid triglyceride (OPO)

Macroporous adsorption resin D101 (available from Hebei Baohen chemical Co., ltd.) is soaked in deionized water for about 12h to remove impurities, then soaked in 95% ethanol overnight to remove organic impurities and other insoluble substances, rinsed with deionized water until no alcohol smell is present, and placed in deionized water for use.

40G of the treated D101 resin was weighed into a 250mL triangular flask, 40mL of an RML or TLL enzyme solution having a protein concentration of 20g/L and a pH of 5 was added, and the mixture was shaken in a 25℃shaker at 150rpm for 2 hours, then the resin was taken out and placed into a dry petri dish, and dried in a freeze-dryer to obtain immobilized RML and immobilized TLL, respectively, and Novozym ^® 40086, a commercial RML enzyme of Norwestine, was used as a control (the immobilized enzyme was designated as immobilized 40086).

According to GB30604-2015 the OPO product has a two-position palmitic acid content of more than 52% and a1, 3-dioleoyl-2-palmitic acid triglyceride content (calculated as C52) of more than 40%.

800G of palm oil stearin (IV 6) and 200g of high oleic sunflower oil were mixed, heated to 105℃and dehydrated under vacuum with stirring for 30min. 1g of sodium methoxide was added thereto, the reaction was stirred at 105℃under vacuum for 30 minutes, 20g of aqueous citric acid solution (15% by weight) was added thereto, and the reaction was terminated by stirring for 20 minutes. The reaction was repeatedly washed with hot water and desapodized. Heating to 105deg.C, vacuum stirring for 30min, and dehydrating and drying to obtain ester-exchanged oil.

Mixing 500g of the ester exchange grease and 1000g of oleic acid, and carrying out gas bath reaction for 6 hours at 60 ℃ and 250rpm by using the addition amount of the immobilized enzyme. The product was removed and replaced with a new substrate for repeated reactions (examination of the number of enzyme reusability).

Removing free fatty acid from the product by molecular distillation, heating deacidified oil to 105deg.C under vacuum, adding activated clay 1.5% of oil weight, stirring under vacuum for 30min, and filtering to obtain hump-colored oil. Introducing nitrogen (serving as stirring and deodorizing medium) into the decolorized oil, maintaining the vacuum degree at about 10-20mBar, deodorizing at 240 ℃ for 1h to obtain refined OPO product, and determining the content of two-bit palmitic acid in all palmitic acid and the content of 1, 3-dioleoyl-2-palmitic acid triglyceride (calculated by C52).

The repeated experiment results show that under the condition that the requirement that the content of two-bit palmitic acid in an OPO product is more than 52 percent and the content of 1, 3-dioleoyl-2-palmitic acid triglyceride (calculated by C52) is more than 40 percent is met, the use batch of immobilized RML is not less than 40 times, the use batch of immobilized TLL is not less than 60 times, and the use batch of immobilized 40086 is not more than 20 times under most conditions, so that the application of the RML and the TLL expressed by the Aspergillus oryzae BC4 in OPO production can be proved, the production index requirement can be met, the use of multiple batches can be repeated, the production cost is greatly reduced, and the economic value is obvious.

Example 10: FOL expressed in aspergillus oryzae strain BC4 applied to soybean oil enzymatic degumming

Weighing 30g of crude soybean oil, and heating to 55 ℃; adding citric acid monohydrate aqueous solution with an addition amount of 0.065% (w/w based on anhydrous citric acid weight), shearing at 10000rpm for 1.5min (first shearing); stirring and reacting for 1h at 55 ℃; adding NaOH aqueous solution, wherein the addition amount is 1.5 times of the molar ratio of anhydrous citric acid (calculated by NaOH weight), and shearing at 10000rpm for 30s (second shearing); adding FOL enzyme solution diluted with water, wherein the water addition amount is 2.5%, FOL enzyme (product concentration is 10 g/L) addition amount is 50ppm, shearing is performed at 10000rpm for 1.5min (third shearing); stirring and reacting for 4 hours at the temperature of 55 ℃; heating to 85 ℃, and reacting for 8min;10000g, centrifuging for 10min, taking out supernatant to obtain degummed oil, detecting phosphorus content of degummed oil, and judging degummed effect.

	Soy oil 1	Soy oil 2
			Initial phosphorus content (ppm) of crude oil	703.21	806.67
Phosphorus content (ppm) after FOL degumming	5.21	6.26

The results show that the FOL enzyme method expressed by the aspergillus oryzae BC4 is used for degumming, the crude oil can realize one-step degumming to reduce the phosphorus content to below 10ppm, so that the subsequent physical refining is directly carried out, a chemical refining section is omitted, refining procedures and refining consumption are saved, the production cost is reduced, and the environment-friendly effect is achieved.

Sequence listing

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Claims

1. The collection number of the mutant Aspergillus oryzae strain is CGMCC No.18825.

2. A recombinant aspergillus oryzae strain obtained by introducing a gene encoding a foreign protein selected from rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase into the strain of claim 1.

3. A method of producing a protein of interest comprising introducing a gene encoding the protein of interest into the strain of claim 1 and culturing the strain to produce the protein of interest, or culturing the recombinant aspergillus oryzae strain of claim 2 to produce the protein of interest, wherein the protein of interest is selected from the group consisting of rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase.

4. A biocatalyst comprising the mutated aspergillus oryzae strain of claim 1 or the recombinant aspergillus oryzae strain of claim 2.