Mutant aspergillus oryzae strains
1. Mutant Aspergillus oryzae (Aspergillus oryzae) A strain which is an orotidine-5' phosphate decarboxylase auxotrophic strain and which has an increased production capacity of an endogenous enzyme, e.g. an amylase and/or an exogenous protein, such as a food lipase, preferably a food lipase selected from the group consisting of rhizomucor miehei lipase, thermomyces lanuginosus lipase and fusarium oxysporum lipase, relative to an unmutated strain.
2. The mutant aspergillus oryzae strain has the preservation number of CGMCC No. 18825.
3. A recombinant Aspergillus oryzae strain obtained by introducing a gene encoding a foreign protein into the strain of claim 1 or 2.
4. The recombinant Aspergillus oryzae strain of claim 3, wherein the exogenous protein is an enzyme, preferably a food lipase, such as a food lipase selected from the group consisting of Rhizomucor miehei lipase, Thermomyces lanuginosus lipase and Fusarium oxysporum lipase.
5. A method for producing a protein of interest, comprising introducing a gene encoding the protein of interest into the strain of claim 1 or 2, and culturing the strain to produce the protein of interest, or culturing the recombinant aspergillus oryzae strain of claim 3 or 4 to produce the protein of interest.
6. The process of claim 5, wherein the protein of interest is an enzyme, preferably a food lipase, such as a food lipase selected from the group consisting of Rhizomucor miehei lipase, Thermomyces lanuginosus lipase and Fusarium oxysporum lipase.
7. A biocatalyst comprising a recombinant aspergillus oryzae strain of claim 1 or 2 into which has been introduced a gene encoding an enzyme, preferably a food lipase, such as a food lipase selected from the group consisting of rhizomucor miehei lipase, thermomyces lanuginosus lipase and fusarium oxysporum lipase.
8. A foreign protein produced by the strain of any one of claims 1 to 4.
9. The foreign protein of claim 8, wherein the foreign protein is an enzyme, preferably a food lipase, such as a food lipase selected from the group consisting of Rhizomucor miehei lipase, Thermomyces lanuginosus lipase and Fusarium oxysporum lipase.
10. A recombinant microbial cell into which an intrabacterial component derived from the strain of claim 1 or 2 has been introduced.
Background
Aspergillus oryzae (Aspergillus oryzae) Is a commonly used strain in fermentation industry, belongs to aspergillus and deuteromycotina, and is aerobic fungus. Production of soy sauce and bean flour sauce by aspergillus oryzaeThe history of many fermented foods such As sake can be traced back to over 1000 years, and is classified As GRAS (generally Regards As safe) by the U.S. Food and Drug Administration (FDA) and recognized by the world health organization (Machidam. Progress of Aspergillus oryzae genes. AdvAppl Microbio, l 2002, 51: 81, 106), which is a very common species used in filamentous fungi, and is widely used in food brewing industry and industrial enzyme preparation industry.
Aspergillus oryzae is a strain capable of producing complex enzyme, including amylase, protease, lipase, pectinase, phytase, etc., and has high-efficiency protein secretion and expression capacity. The aspergillus oryzae expressed exogenous protein has the characteristics of large expression quantity, high extracellular secretion rate, protein molecule folding and modifying system close to higher eukaryotic cells and the like, and the expressed exogenous protein has natural activity. In addition, Aspergillus oryzae can also be subjected to various post-translational processes such as glycosylation modification protease cleavage and disulfide bond formation (Meyer V. Genetic engineering of fibrous fungi-progress, obstacle and future trands. Biotechnol Adv, 2008, 26(2): 177-185). Thus, the use of Aspergillus oryzae as an expression strain for the expression of homologous and heterologous proteins is gaining increasing attention.
In the industrial production process, when aspergillus oryzae expresses heterologous proteins, the expression level is lower than that of homologous proteins (Zhang Jianjun, Chuai Wai Hua, Li Qiang, etc.. strategy for improving the expression level of proteins in aspergillus oryzae, China journal of bioengineering, 2009, 29 (1): 111-. Generally, 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 and are not conducive to industrial production, and fusion expression may present regulatory risks during the production of enzymes for food use, so strategies using strong promoters are generally used.
Suitable selection markers are furthermore important components of expression systems. Auxotrophic selection marker in Aspergillus oryzae the most used at present is nitrate reductase auxotrophy (niaD) - ) Transformation system and orotidine-5' phosphate decarboxylase auxotrophy: ( pyrG - ) Transformation system (natural, Ma Shi, progress on the exogenous expression system of Aspergillus oryzae, J.Bioengineering, China, 2016, 36 (9): 94-100). However, since Aspergillus oryzae hyphae are multinucleated and most of conidia are multinucleated, the efficiency of mutagenesis or genetic engineering is greatly reduced, and the difficulty of establishing transformation systems is increased (Jun-ichi MARUYAMA., Visualization of nucleic acids inAspergillus oryzaewith EGFP and Analysis of the Number of Nuclei in Each Conidium by FACS.Biosci.Biotechnol.Biochem,65(7),1504-1510,2001)。
In China, an enzyme preparation applied to food must meet the national standard GB2760 of the people's republic of China-the national standard of food \ the use standard of food additives. Wherein the commonly used heterologous expression food lipase complying with the regulations: rhizopus oryzae (A. rhizopus oryzae) ((A. rhizopus oryzae))Rhizomucor miehei) Thermomyces lanuginosus (Thermomyces lanuginosus) (II)Thermomyces lanuginosus) And Fusarium oxysporum (F.), (Fusarium oxysporum) The derived lipases can be heterogeneously expressed only by Aspergillus oryzae, which reflects the importance of Aspergillus oryzae expression systems in food enzyme production. However, according to the prior art, the expression level of a lipase for food products by Aspergillus oryzae is often very low, for example: an auxotrophic host bacterium for Aspergillus oryzae (c)Aspergillus oryzae Expression of Rhizopus oryzae in niaD300, niaD-)Rhizomucor miehei) The source lipase RML is obtained by taking culture solution supernatant cultured for 7 days, and the enzyme activity of the culture solution is only 2.5U/mL (han and panli, guo and brave) determined by an alkaline titration method, and the enzyme activity is only 2.5U/mL (2009, 37 (6): 84-90) in the statement of university of south China university of science and engineering).
Therefore, there remains a need in the art for a strain of Aspergillus oryzae that is capable of efficiently expressing various proteins, particularly lipases for food use.
Disclosure of Invention
The invention constructs orotidine-5' phosphate decarboxylase auxotroph (pyrG) by taking aspergillus oryzae CICC2012 as an original strain - ) The bacterial strain, and the protein expression capacity is improved by ARTP mutagenesis, so that the bacterial strain becomes an effective heterologous expression host,it can high-effectively heterologously express various proteins, in particular to lipase for food.
Specifically, the present invention relates to the following aspects.
In one aspect, the invention relates to a mutant Aspergillus oryzae (Aspergillus oryzae) A strain having an increased production capacity of an endogenous enzyme, e.g. an amylase, and/or an exogenous protein, e.g. a food lipase selected from the group consisting of 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 a mutant Aspergillus oryzae (Aspergillus oryzae) Enzymes expressed by the strain itself, for example, including but not limited to, mutated Aspergillus oryzae (A. oryzae) ((B. oryzae))Aspergillus oryzae) Amylase expressed by the strain itself.
In a specific embodiment, the mutant A.oryzae strain of the present invention has a accession number of CGMCC No. 18825.
The novel aspergillus oryzae strain can efficiently express heterologous protein. The aspergillus oryzae strain is an orotidine-5' phosphate decarboxylase auxotrophic strain, and the expression level of endogenous amylase can be improved by more than 40 percent compared with the original strain, such as 40 to 100 percent, particularly 40 to 60 percent, and particularly 45 to 55 percent. When the RML is expressed in a heterologous way, the protein expression level is improved by 50 to 100 percent, particularly 50 to 80 percent, and particularly 55 to 70 percent compared with the protein expression level of the original strain. In addition, when TLL and FOL are expressed, the protein expression level is improved by more than 40%, particularly 40-50% compared with that of the starting strain CICC 2012. Therefore, the strain has obvious practicability of efficiently expressing heterologous proteins.
In another aspect, the present invention relates to a recombinant A.oryzae strain obtained by introducing a gene encoding a foreign protein into the above-mentioned mutant A.oryzae strain. In one embodiment, the exogenous protein is an enzyme, e.g., a food lipase, such as a food lipase selected from the group consisting of rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase.
In another aspect, the present invention relates to a method for producing a target protein, comprising introducing a gene encoding the target protein into the above-mentioned mutant A.oryzae strain, and culturing the strain to produce the target protein. Alternatively, it comprises culturing the above recombinant Aspergillus oryzae strain to produce the protein of interest. In one embodiment, the exogenous protein is an enzyme, e.g., a food lipase, such as a food lipase selected from the group consisting of rhizomucor miehei lipase, thermomyces lanuginosus lipase, and fusarium oxysporum lipase.
In another aspect, the present invention relates to a biocatalyst comprising a mutant Aspergillus oryzae strain as described above, into which a gene encoding a foreign enzyme, such as a food lipase, for example, a food lipase selected from the group consisting of Rhizomucor miehei lipase, Thermomyces lanuginosus lipase and Fusarium oxysporum lipase, has been introduced.
In another aspect, the present invention relates to a foreign protein produced by the strain described above. The foreign protein is an enzyme, such as a food lipase, for example a food lipase selected from the group consisting of Rhizomucor miehei lipase, Thermomyces lanuginosus lipase and Fusarium oxysporum lipase.
The invention also relates to a recombinant microbial cell into which an intrabacterial component derived from the mutated Aspergillus oryzae strain described above has been introduced. After obtaining the strain of the present invention, its intra-bacterial components can be isolated by conventional techniques and introduced into other microorganisms. The recombinant microbial cells into which the components are introduced have excellent properties of the strain of the present invention. In the present invention, the term "in-bacterial composition" refers to the sum of all genetic material of an organism, specifically including, but not limited to: coding and non-coding DNA, mitochondrial DNA.
In particular, the mutant A.oryzae strains of the invention can be used to express foreign proteins. After obtaining the mutant Aspergillus oryzae of the present invention, a commonly used expression vector can be introduced therein for expression of a foreign protein. For example, the expression vector may contain a promoter and a terminator, and a gene encoding a foreign protein may be inserted with a multiple cloning site between the promoter and the terminator. One or more copies of the enhancer may be included in the promoter. Many commercial vectors can be used for the expression of the foreign protein in the mutant A.oryzae strain of the present invention.
In expressing exogenous proteins from sources other than A.oryzae, codons for certain species may be rare codons in A.oryzae. Therefore, when introducing an expression vector, the gene encoding a foreign protein can be first subjected to codon optimization suitable for Aspergillus oryzae of the present invention, thereby increasing the expression amount.
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, and proteins useful for studying crystal structures, etc. The Aspergillus oryzae expressing the exogenous enzyme component of the present invention may also have whole cells as biocatalysts.
Drawings
FIG. 1 is a schematic diagram of an RML gene expression vector constructed according to the present invention.
Fig. 2 is a graph comparing the enzyme activity of the endogenous amylase expressed by the strain BC4 of the present invention with the original strain cic 2012 and the strain PyrG L4.
Fig. 3 is a protein electrophoresis diagram of the endogenous amylase expressed by the strain BC4 of the present invention and the starting strain cic 2012 and the strain PyrG L4.
FIG. 4 is a comparison graph of the enzyme activities of the Rhizomucor miehei lipase RML expressed by the strain BC4 and the original strain CICC2012 and the strain PyrG L4.
Fig. 5 is a protein electrophoresis diagram of RML expressed by the strain BC4 of the present invention and the starting strain cic 2012 and the strain PyrG L4.
FIG. 6 is a graph comparing the enzyme activities of the amylases expressed by the strain BC4 of the present invention and the starting strain CICC2012 and the strain PyrG L4 in a 500L fermenter.
FIG. 7 is a graph comparing the enzyme activities of the Rhizomucor miehei lipase RML expressed by the strain BC4 of the present invention and the starting strain CICC2012 and the strain PyrG L4 in a 500L fermenter.
Detailed Description
The initial strain Aspergillus oryzae is purchased from China industrial microorganism culture Collection management center (CICC for short), and the strain number is CICC 2012. The initial strain is subjected to ultraviolet mutagenesis after the initial strain is enriched and grows the mononucleated spores on an inositol plate, and finally, the screening is carried out 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 then, an ARTP mutagenesis mode is utilized to screen a strain BC4, the amylase activity of the strain BC4 in 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% and 51.4%. In the present invention, the term "ARTP" is an abbreviation of 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 Temperature of 25 to 40 ℃ at Atmospheric pressure and having a high concentration of reactive species (including helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc. in an excited state). The term "ARTP mutagenesis" means that strain mutagenesis is performed by using an atmospheric pressure room temperature plasma technique, and specifically, an atmospheric pressure room temperature plasma source using helium gas as a working gas contains a plurality of chemically active particle components such as OH, a nitrogen molecule two positive system, a nitrogen molecule one negative system, an excited state helium atom, a hydrogen atom, an oxygen atom and the like. ARTP-rich active energy particles cause damage to genetic materials of strains/plants/cells and the like and induce biological cells to initiate SOS repair mechanism. The SOS repair process is a high fault tolerance repair, so that abundant types of mismatching sites can be generated in the repair process, and finally, the mismatching sites are stably inherited to form mutant strains. The strength of SOS repair, and the extent to which DNA is damaged, are strongly correlated.
An attempt is made to heterologously express the rhizomucor miehei lipase RML for food by using an enolase promoter added with 12 copies of an enhancer sequence in an Aspergillus oryzae strain BC4, and the result shows that the enzyme activity of the RML lipase expressed by BC4 in a 500L fermentation tank reaches 6488U/mL, and is improved by 68.1% and 58.0% compared with the starting strains CICC2012 and PyrG L4. Compared with the prior art, for example: an auxotrophic host bacterium for Aspergillus oryzae (c)Aspergillus oryzae Expression of Rhizopus oryzae in niaD300, niaD-)Rhizomucor miehei) The lipase RML from the source has the enzyme activity of only 2.5U/mL (Wang and bin, Panli and Guo and courage) and the construction of an exogenous gene expression system of filamentous fungus Aspergillus oryzae, university of south China college of academic Press, 2009, 37 (6): 84-90)Is very obvious.
The inventor also expresses Thermomyces lanuginosus lipase TLL and Fusarium oxysporum lipase FOL in aspergillus oryzae strain BC4, and the results show that when TLL and FOL obtained by BC4 expression are used, the protein expression level is improved by 45.6% and 40.5% compared with that of original strain CICC2012, so that the inventor proves that the lipase expression system has obvious advantages as a lipase expression system for food. Furthermore, the inventors have found that the use of the enzyme of interest expressed by aspergillus oryzae BC4, such as RML, TLL, FOL, etc., has significant utility in specific applications, such as:
1. the immobilized RML and TLL obtained by expressing aspergillus oryzae BC4 can be used for immobilization, and the obtained immobilized RML enzyme preparation and immobilized TLL enzyme preparation. When an immobilized RML enzyme preparation and an immobilized TLL enzyme preparation are used for producing OPO, under the condition of meeting the requirements of GB30604-2015 that the content of dipalmitic acid in an OPO product is more than 52 percent and the content of 1, 3-dioleate-2-palmitic acid triglyceride (calculated by C52) is more than 40 percent, the using batch of the immobilized RML is not less than 40 times, the using batch of the immobilized TLL is not less than 60 times, and the production method is obviously superior to the production method using a commercial enzyme Novozym®40086 the immobilized enzyme obtained by the method is used in batches, and RML and TLL expressed by Aspergillus oryzae BC4 are applied to the production of OPO, so that the method not only can meet the requirement of production indexes, but also can be used repeatedly in multiple batches, greatly reduces the production cost, and has obvious economic value.
2. FOL obtained by Aspergillus oryzae BC4 expression is used for carrying out enzymatic degumming on crude oil, the degumming effect is obvious, specifically, the phosphorus content of oil can be reduced to below 10ppm through one-step degumming, so that physical refining is directly carried out subsequently, a chemical refining section is omitted, the refining process and refining consumption are saved, the production cost is reduced, and the environment friendliness is achieved.
Example 1: acquisition of an orotidine-5' phosphate decarboxylase auxotrophic Aspergillus oryzae Strain
Referring to patent CN201310288060.3, spores of aspergillus oryzae CICC2012 strain were inoculated and spread on MM solid medium (1% glucose, 0.15% KH) supplemented with 0.04% inositol2PO4,0.6% NaNO3,0.05% KCl,0.05% MgSO42% agar powder) at 28 deg.C, standing for 5 days to obtain enriched spores of Aspergillus oryzae. Fresh Aspergillus oryzae CICC2012 spores were eluted with spore wash (0.9% NaCl, 0.05% Tween 80), prepared as a spore suspension by filtration through Miracloth (Calbiochem, Cat # 475885), and the cells were washed 2 times with sterile water and adjusted to 1X 107one/mL. 2mL of spore suspension was uniformly dispersed on the surface layer of a petri dish, and the petri dish was irradiated with ultraviolet light on an ultraclean bench for 90 seconds, and 100. mu.L of the spore suspension was applied to MM solid medium supplemented with 0.3% Uracil (Uracil) and 1mg/mL of 5-fluoroorotic acid (5-FOA) and cultured in the dark at 28 ℃ for 7 days (the whole process was performed under red light to prevent reversion). And (3) transferring the single colony grown 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 a strain which only can grow on the MM-Uracil solid culture medium, thereby obtaining the orotidine-5' phosphate decarboxylase auxotrophic Aspergillus oryzae PyrG L4 strain. Sequencing the pyrG gene from A.oryzae pyrG L4 strain revealed 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 the RML expression vector
Exogenous Rhizomucor miehei (A), (B), (C), (B), (C)Rhizomucor miehei) The lipase (RML) gene sequence is as follows.
Nucleic acid sequence:
gtgccaatcaagagacaatcaaacagcacggtggatagtctgccacccctcatcccctctcgaacctcggcaccttcatcatcaccaagcacaaccgaccctgaagctccagccatgagtcgcaatggaccgctgccctcggatgtagagactaaatatggcatggctttgaatgctacttcctatccggattctgtggtccaagcaatgagcattgatggtggtatccgcgctgcgacctcgcaagaaatcaatgaattgacttattacactacactatctgccaactcgtactgccgcactgtcattcctggagctacctgggactgtatccactgtgatgcaacggaggatctcaagattatcaagacttggagcacgctcatctatgatacaaatgcaatggttgcacgtggtgacagcgaaaaaactatctatatcgttttccgaggttcgagctctatccgcaactggattgctgatctcacctttgtgccagtttcatatcctccggtcagtggtacaaaagtacacaagggattcctggacagttacggggaagttcaaaacgagcttgttgctactgttcttgatcaattcaagcaatatccaagctacaaggttgctgttacaggtcactcactcggtggtgctactgcgttgctttgcgccctgggtctctatcaacgagaagaaggactctcatccagcaacttgttcctttacactcaaggtcaaccacgggtaggcgaccctgcctttgccaactacgttgttagcaccggcattccttacaggcgcacggtcaatgaacgagatatcgttcctcatcttccacctgctgcttttggttttctccacgctggcgaggagtattggattactgacaatagcccagagactgttcaggtctgcacaagcgatctggaaacctctgattgctctaacagcattgttcccttcacaagtgttcttgaccatctctcgtactttggtatcaacacaggcctctgtacttaa (SEQ ID NO:1)。
amino acid sequence:
VPIKRQSNSTVDSLPPLIPSRTSAPSSSPSTTDPEAPAMSRNGPLPSDVETKYGMALNATSYPDSVVQAMSIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT(SEQ ID NO:2)。
specifically, the RML gene expression vector pAOP-Eno-E was constructed by referring to the method of molecular cloning Laboratory Manual (third edition, New York, Cold Spring Harbor Laboratory Press, 1989), as shown in FIG. 1, and the procedure was as follows:
the RML gene (SEQ ID NO:1, with Aspergillus oryzae. alpha. -amylase signal peptide (NCBI SEQ ID NO: XM-001821384.2, sequence 1-63 bp)) obtained by whole gene synthesis from Biotechnology engineering (Shanghai) GmbH was inserted into the expression cassette of the Aspergillus oryzae enolase promoter (NCBI SEQ ID NO: D63941.1, 215-734 bp; containing 12 copies of the enhancer sequence (gtcgtgtcgggcatttatcgggggatggaccaatcagcgtagg, SEQ ID NO: 3)) and the glucoamylase terminator (NCBI SEQ ID NO: AF214480.1, part of the terminator sequence) using SphI and HindIII enzyme sites, the entire expression cassette was inserted into the multicloning site of the cloning vector pSP72 as BglII and XhoI, and finally the Aspergillus oryzae-derived pyrG expression gene (NCBI sequence No.: AB 017705.1) was inserted into the vector as XhoI cleavage site, thereby constructing the RML gene expression vector pAOP-Eno-E.
Example 3: aspergillus oryzae pyrG L4 anaplerosis experiment
Washing with spore lotion (0.9% NaCl, 0.05% spit)Weak 80) fresh Aspergillus oryzae PyrG L4 spores were eluted, prepared into a spore suspension by Miracloth filtration, and adjusted to 1X 107one/mL. 1mL of spore suspension was inoculated into a hyphal medium (2% tryptone, 1% yeast extract, 2% glucose, 0.3% uracil), cultured at 28 ℃ and 180rpm for 40 hours, and the grown hyphae were collected by filtration using sterilized Miracloth.
The collected mycelia were treated with sterilized osmotic pressure stabilizer (0.6M MgSO4,10mM NaH2PO4pH = 5.8) was washed three times and press dried. The mycelia were transferred to a 100mL Erlenmeyer flask. Every 0.8g hypha is suspended in 20mL of enzymolysis solution (prepared with osmotic pressure stabilizer to obtain 1% lyase, 1% cellulase, and 0.1% helicase, and sterilized by filtration with 0.22 μm microporous membrane) and is subjected to enzymolysis at 30 deg.C and 90 rpm for 60-90 min. Filtering the zymolyzed protoplast mixed solution by Miracloth, collecting the filtrate, centrifuging at 4 ℃ for 10min at 1000g, suspending the protoplast precipitate by 5mL of precooled 1.0mol/L sorbitol solution, centrifuging at 800g at 4 ℃ for 10min, and discarding the supernatant. Then precooled STC solution (1.0M sorbitol, 50mM CaCl)250mM Tris-HCl, pH = 7.5) adjusting protoplasts to 1X 107Per mL, ice bath 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, and 50. mu.L of PTC solution (40% PEG4000, 50mM CaCl)250mM Tris-HCl, pH = 7.5), mixed well and ice-cooled for 30 min. Adding 0.2mL of PTC solution, mixing, adding 0.8mL of PTC solution, mixing, and keeping at room temperature for 30 min.
The mixture was applied to a regeneration medium (1% glucose, 0.6% NaNO)3,0.15% KH2PO4,0.05% KCl,0.05% MgSO4,0.001% FeSO41M sucrose, 2% agar powder) at 28 ℃ for 7 days until colonies grow out.
The colonies grown on the plate were transferred to PDA-Rhodamine B screening medium and cultured at 28 ℃ for 3-6 days.
The preparation method of the PDA-Rhodamine B screening culture medium comprises the following steps:
PDA medium: 200g of potato (peeled), 20g of glucose, 20g of agar and 1000mL of water, and the pH value is natural;
-olive oil emulsion: adding olive oil and 4% polyvinyl alcohol (PVA) solution at a volume ratio of 1:3, mixing, homogenizing with a high speed homogenizer at 8000rpm for 15min, and stirring for 3min after 5min;
respectively sterilizing PDA culture medium and olive oil emulsion at 115 deg.C under high pressure for 15 min; cooling to about 60 deg.C, adding PDA culture medium and olive oil emulsion at a ratio of 9:1, adding 1/100 volume of 0.1% rhodamine B (Rhodamine B) solution, mixing, and pouring into plate.
The transformation finds that the Aspergillus oryzae PyrG L4 can well realize the pyrG gene complementation experiment, and the transformant shows lipase activity in a PDA-Rhodamine B screening culture medium, and further proves that an Aspergillus oryzae expression system taking the 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 a spore suspension by Miracloth filtration, and adjusted to 2X 107one/mL. After mixing the spore suspension with 10% glycerol 1:1, 10. mu.L of the mixed solution was put on an iron plate and treated for 100s with an ARTP instrument (No-Sn source Qingtian Wood Biotech Co., Ltd., instrument model: ARTP-M) with the instrument parameters set: the radio frequency power range is 120W, the helium amount is 10 SLM (99.999 percent of high-purity helium), and the irradiation distance is 2 mm).
After the treatment is finished, the small iron sheets are taken down and placed into a centrifuge tube filled with 1mL of sterile water, then the centrifuge tube is repeatedly sucked by a gun head, so that thalli on the iron sheets are washed off, the iron sheets are diluted to a proper concentration and then coated on a amylase screening plate added with uracil (a PDA culture medium is added with 1% of soluble starch and 0.3% of uracil), the amylase screening plate is placed in an incubator at the temperature of 30 ℃ for culture for 3d, and colonies with larger hydrolysis circles are selected.
Example 5: screening of amylase high-producing strains
The colonies with the larger amylase hydrolysis loop selected in example 4 were inoculated into a 24-well plate, and 3mL of a 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 plate at 180rpm, 28 ℃ and 8 days for primary screening.
And then, carrying out shake flask fermentation on the strains with higher amylase activity obtained by primary screening for re-screening, wherein the re-screening fermentation medium is the same as the primary screening, the liquid loading amount is 50mL/250mL of triangular shake flask, 150rpm, 28 ℃, and 8 days.
The amylase determination method comprises the following steps:
taking a clean sterile centrifuge tube (2 mL or 1.5 mL), sequentially adding a starch substrate with the concentration of 200uL (absorbing 20mL of soluble starch (20 g/L), adding 5mL of a phosphate buffer solution (45.23 g of disodium hydrogen phosphate dodecahydrate and 8.07g of citric acid monohydrate are weighed, dissolved with water and added to 1000mL of constant volume, the pH value is 6.0) and 100uL of an enzyme solution which is properly diluted), fully mixing the starch substrate and the enzyme solution by turning upside down, and quickly putting the mixture into a water bath kettle at 60 ℃ for enzymatic reaction for 10 min. After the reaction is finished, 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 the sodium tartrate is dissolved, water is added to a constant volume of 500mL, 2 g of phenol and 0.5g of anhydrous sodium sulfite are added after the sodium tartrate is heated and dissolved, the mixture is heated and dissolved, cooled, water is added to a constant volume of 1000mL, the mixture is stored in a brown bottle and used after 1 week) is added into each tube, the mixture is fully mixed to stop the reaction quickly, and the mixture is transferred into an ice water bath to be cooled quickly after being boiled in a water bath for 10 min. And (3) sequentially adding 190uL of distilled water into a clean enzyme label plate, uniformly mixing, and measuring the light absorption value of each tube of reaction liquid at 540nm by using an enzyme label instrument.
Definition of amylase activity (U) units: in the above specific assay mode, the unit time (min) catalyzes the hydrolysis of soluble starch and produces reducing sugars, the reducing power of which corresponds to the amount of enzyme required for 1umol of glucose.
Formula for calculating enzyme activity
Enzyme activity (U/mL) =
Wherein:
n: dilution times;
ODt: absorbance at 540nm for the experimental group;
ODc: absorbance at 540nm for the control;
1000: unit conversion multiple;
0.106: the slope of the glucose standard curve;
10: reaction time at 60 DEG C
The final results are shown in FIG. 2 amylase activity diagram and FIG. 3 protein electrophoresis diagram. Screening to obtain the amylase high-yield strain BC4, wherein the amylase activity of the strain BC4 reaches 6415U/mL, and compared with the original strains CICC2012 and PyrG L4, the amylase expression levels are respectively improved by 40.4% and 45.1%.
The strain BC4 is preserved in China general microbiological culture Collection center (CGMCC) 10 months and 30 days 2019, the microbial research institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Kyoho, Beijing City, with the preservation number of CGMCC No.18825, and is classified and named as Aspergillus oryzae (A), (B), and (BAspergillus oryzae) 。
Example 6: examination of expression ability of amylase highly producing Strain BC4
Transformation of Aspergillus oryzae BC4 in the same manner as in example 3, RML expression vector pAOP-Eno-E was transformed into the strain.
The A.oryzae PyrG L4 transformant of example 3 was used as a control, and pAOP-Eno-E was additionally transformed into A.oryzae CICC2012, and since A.oryzae CICC2012 could not use pyrG as a selection marker, p3SR2 ((BCCM/LMBP: Accession number: 2363), acetamidase (amdS) -containing gene) was used for transformation screening. Regeneration medium requires removal of sodium nitrate and further supplementation with 15mM acetamide and 20mM cesium chloride.
For each of the three transformants, 40 transformants were selected for shake flask fermentation in a 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) at 150rpm, 28 ℃ for 8 days, and the RML lipase activity was measured.
RML lipase activity assay as follows: olive oil titration method lipase determination method
(1) Preparation of 0.05M NaOH: first use without CO2Preparing 5M NaOH stock solution by using water; then, the product is processedAccurately diluting 50 times, weighing 0.38g of potassium hydrogen phthalate, drying at 100 deg.C to constant weight, and dissolving in 80mL of solution without CO2In water, calibrating the accurate concentration of the stock solution, and then calculating the concentration of the stock solution; 0.05M NaOH solution in NaOH to be free of CO2Preparing water in situ after using a stock solution;
(2) preparing PVA-olive oil emulsion substrate, mixing 100mL olive oil and 300mL 4% PVA1750 (polyvinyl alcohol), heating for melting, emulsifying with ultrasonic wave at power of 300W for 3s, intermittently for 4s, 99 times, and circulating for 2 times;
(3) adding 4mL of emulsion substrate, 2mL of 0.2M Tris-HCl buffer solution with pH8.0 and 3mL of sterile water into a 100mL triangular flask, placing the triangular flask in a constant-temperature water bath shaker at 40 ℃, and incubating for 5min at 150 rpm;
(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) dripping 2 drops of phenolphthalein as an indicator, and titrating the fatty acid generated by enzymolysis by using 0.05M NaOH until the reaction solution turns pink;
the blank run was identical to that described above except that 1mL of the appropriately diluted enzyme solution was added after the substrate and buffer were allowed to warm up in a water bath for 15min and the reaction was stopped with absolute ethanol.
(6) Enzyme activity definition and calculation formula
The lipase activity unit is defined as: the amount of enzyme catalyzing the release of 1. mu. mol of fatty acid from the substrate per minute is 1 lipase activity unit (U).
The enzyme activity calculation formula is as follows:
enzyme activity (U/mL) =
In the formula: v: the volume (ml) of NaOH solution consumed for titration of the sample liquid
V0: titration of NaOH solution volume (ml) consumed for blank
t: reaction time (min)
n: volume of enzyme solution (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 RML lipase expressed by BC4 reaches 5560U/mL, and is improved by 66.9% and 60.6% compared with the original strains CICC2012 and PyrG L4.
Polyacrylamide gel electrophoresis analysis: the supernatant was filtered through a 0.22 μm filter, and an equal amount of the supernatant was concentrated to the same volume using a Milipore 10KDa ultrafiltration concentration tank, and the same volume of the concentrated enzyme solution was subjected to polyacrylamide gel electrophoresis analysis. The electrophoresis results are shown in FIG. 5. The results of the protein electrophorogram show that the RML protein band concentration of the BC4 transformant is significantly higher than that of the control.
The specific reason for the increased protein expression level of the strain BC4 is presumably due to the increased intracellular transcription, translation or secretion efficiency, and the increased utilization of the strain during the expression of RML, which in turn increases 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 by mixing each nutrient concentration according to the total volume of 300L, and comprises the initial constant volume of 245L, the tank condensate water consumption of about 25L, and dextrin solution addition of 30L within 0 hour (0 h).
Basic culture medium: 5610g of corn steep liquor dry powder, 207g of potassium dihydrogen phosphate, 994g of disodium hydrogen phosphate dodecahydrate, 41.53 g of magnesium sulfate heptahydrate, and keeping the volume to 245L (0.3% of uracil needs to be supplemented for fermentation of PyrG L4 and BC 4); the feeding culture medium is consistent with the basic culture medium, and the volume is fixed to 38L;
defoaming agent: 10% emulsified silicone oil, 200g/2000mL;
dextrin solution: the preparation concentration is 200g/L, the total preparation concentration is 150L, and the total preparation concentration is 30 kg.
The fermentation parameters are as follows: the fermentation temperature is 28 ℃, the aeration is 0.6vvm, the antifoaming agent is added according to the requirement, the rotating speed is 135-280rpm, the adjustment is carried out according to the actual situation in the fermentation process, the fed-batch culture medium and the dextrin solution are fed-batch from 24 hours, and the fermentation is about 184 hours.
Specific activities of amylase and RML lipase obtained from 500L fermentors are shown in fig. 6 and 7.
The results in fig. 6 show that the amylase activity of the amylase high-producing strain BC4 in the 500L fermenter reaches 12264U/mL, and the amylase expression level is increased by 52.2% and 51.4% compared with the original strains cic 2012 and PyrG L4.
The results in fig. 7 show that the RML lipase enzyme activity expressed by BC4 in a 500L fermentor reached 6488U/mL, which was 68.1% and 58.0% higher than the starting strains cic 2012 and PyrG L4.
Example 8: expression of TLL and FOL in A.oryzae Strain BC4
The construction of expression vectors for expression of TLL and FOL was carried out in the same manner as in example 2, and the gene sequences of TLL (NCBI SEQ ID NO: AF054513.1 and FOL (NCBI SEQ ID NO: EF 613329.1) were synthesized in the whole gene of Biotechnology engineering (Shanghai) Ltd.
Transformation of Aspergillus oryzae BC4 the TLL and FOL expression vectors were transformed into strains as in example 3.
40 transformants were selected for each of the two transformants for shake flask fermentation in a 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) at 150rpm, 28 ℃ for 8 days, and lipase activity was measured.
The results show that the protein expression levels of TLL and FOL expressed by BC4 are improved by 45.6% and 40.5% compared with the original strain CICC 2012.
Example 9: application of RML and TLL expressed in Aspergillus oryzae strain BC4 in production of 1, 3-dioleoyl-2-palmitic acid triglyceride (OPO)
The macroporous adsorption resin D101 (purchased from Hebei Cang, Baoyen 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, washed with deionized water until no alcohol smell exists, and placed in deionized water for later use.
Weighing 40g of the treated D101 resin into a 250mL triangular flask, adding 40mL of RML or TLL enzyme solution with the protein concentration of 20g/L and the pH of 5, shaking for 2h at 150rpm in a shaking table at 25 ℃, taking out the resin, putting the resin into a dried culture dish, putting the culture dish into a freeze dryer for drying to obtain immobilized RML and immobilized TLL respectively, and using Novaxin RML commercial enzyme Novozym®40086 as a control (the enzyme obtained by immobilization was designated as immobilization 40086).
According to GB30604-2015, the content of dipalmitic acid in OPO products is more than 52%, and the content of 1, 3-dioleoyl-2-palmitic acid triglyceride (calculated as C52) is 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 for 30min with stirring. 1g of sodium methoxide was added thereto, and the mixture was reacted at 105 ℃ under vacuum with stirring for 30 minutes, and 20g of an aqueous citric acid solution (15% by weight) was added thereto and stirred for 20 minutes to terminate the reaction. The reactant was washed repeatedly with hot water to remove soap. Heating to 105 deg.C, vacuum stirring for 30min, and dehydrating to obtain ester-exchanged oil.
500g of the above-mentioned transesterified oil and fat and 1000g of oleic acid were mixed, and the mixture was reacted at 60 ℃ for 6 hours in a 250rpm gas bath with 6% of immobilized enzyme added. The product was removed and replaced with new substrate for repeat reaction (see the number of reusability of the enzyme).
Removing free fatty acid from the product by molecular distillation, heating deacidified oil to 105 deg.C under vacuum, adding activated clay 1.5% of the weight of oil, stirring under vacuum for 30min, and filtering to obtain camel oil. Introducing nitrogen (as stirring and deodorizing medium) into the decolorized oil, maintaining the vacuum degree at about 10-20mBar, deodorizing at 240 deg.C for 1h to obtain refined OPO product, and measuring the content of dipalmitic acid in all palmitic acid and the content of 1, 3-dioleate-2-palmitic acid triglyceride (calculated as C52).
Repeated experiments show that under the condition that the requirements of GB30604-2015 are met, the content of dipalmitic acid in an OPO product is more than 52%, and the content of 1, 3-dioleate-2-palmitic acid triglyceride (calculated by C52) is more than 40%, 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 in most cases, so that the application of the RML and TLL expressed by Aspergillus oryzae BC4 in the production of OPO is proved, the requirements of production indexes can be met, the multiple batches can be used repeatedly, the production cost is greatly reduced, and the economic value is obvious.
Example 10: application of FOL expressed in Aspergillus oryzae strain BC4 in enzymatic degumming of soybean oil
Weighing 30g of crude soybean oil, and heating to 55 ℃; adding citric acid monohydrate aqueous solution in an amount of 0.065% (based on anhydrous citric acid weight, w/w), and shearing at 10000rpm for 1.5min (first shearing); stirring and reacting for 1h at the temperature of 55 ℃; adding NaOH aqueous solution, wherein the adding amount of the NaOH aqueous solution is 1.5 times of the molar ratio of the anhydrous citric acid (calculated by weight of NaOH), and shearing the citric acid at 10000rpm for 30s (second shearing); adding FOL enzyme solution diluted with water, wherein the water addition amount is 2.5%, the FOL enzyme (finished product concentration is 10 g/L) addition amount is 50ppm, and shearing at 10000rpm for 1.5min (third shearing); stirring and reacting for 4 hours at the temperature of 55 ℃; heating to 85 deg.C, and reacting for 8 min; 10000g, centrifuging for 10min, taking out supernatant fluid to obtain the degummed oil, detecting the phosphorus content of the degummed oil, and judging the degumming effect.
Soybean oil 1
Soybean 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 crude oil can be degummed by using the FOL enzyme method expressed by Aspergillus oryzae BC4 to reduce the phosphorus content to below 10ppm by one-step degumming, so that the subsequent physical refining is directly carried out, a chemical refining section is omitted, the refining process and refining consumption are saved, the production cost is reduced, and the environmental protection is realized.
Sequence listing
<110> Fengyi (Shanghai) Biotechnology research and development center, Inc
<120> mutant A.oryzae strain
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accggcattc cttacaggcg cacggtcaat gaacgagata tcgttcctca tcttccacct 840
gctgcttttg gttttctcca cgctggcgag gagtattgga ttactgacaa tagcccagag 900
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Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro
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Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr
20 25 30
Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp
35 40 45
Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp
50 55 60
Ser Val Val Gln Ala Met Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr
65 70 75 80
Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn
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Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala Thr Trp Asp Cys Ile His
100 105 110
Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu
115 120 125
Ile Tyr Asp Thr Asn Ala Met Val Ala Arg Gly Asp Ser Glu Lys Thr
130 135 140
Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala
145 150 155 160
Asp Leu Thr Phe Val Pro Val Ser Tyr Pro Pro Val Ser Gly Thr Lys
165 170 175
Val His Lys Gly Phe Leu Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu
180 185 190
Val Ala Thr Val Leu Asp Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val
195 200 205
Ala Val Thr Gly His Ser Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala
210 215 220
Leu Asp Leu Tyr Gln Arg Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe
225 230 235 240
Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly Asp Pro Ala Phe Ala Asn
245 250 255
Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg
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Asp Ile Val Pro His Leu Pro Pro Ala Ala Phe Gly Phe Leu His Ala
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Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser Pro Glu Thr Val Gln Val
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Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys Ser Asn Ser Ile Val Pro
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Phe Thr Ser Val Leu Asp His Leu Ser Tyr Phe Gly Ile Asn Thr Gly
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Leu Cys
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gtcgtgtcgg gcatttatcg ggggatggac caatcagcgt agg 43