24-dehydrocholesterol reductase mutant and engineering strain and application

Abstract

The present application relates to the technical field of protein engineering, in particular to 24-dehydrocholesterol reductase mutant and engineering strain and application. The present application carries out point mutation on the 24-dehydrocholesterol reductase (Gallus gallus (chicken) DHCR24) of chicken origin obtained by gene synthesis through protein engineering technology, and obtains a mutant sequence with higher 7-DHC production capacity than the natural 24-dehydrocholesterol reductase. The optimal mutant sequence is integrated into a high-yield cholesta-5,7,24-triene-3beta alcohol strain, and a series of strain modification is carried out, and an engineering strain F-6B with high 7-DHC production capacity is obtained. The yield in 50L fermenter for 35h fermentation can reach 56.2mg / g. The existing strain can greatly shorten the fermentation time, simultaneously reduce the cost of high 7-DHC production, and meet the demand of large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, and particularly to 24-dehydrocholesterol reductase mutants, engineered strains, and their applications. Background Technology

[0002] 7-Dehydrocholesterol (7-DHC) is a sterol and a key precursor to vitamin D3. 7-DHC undergoes photoisomerization to form vitamin D3. Vitamin D3 is a fat-soluble steroid hormone that plays a crucial role in balancing metabolism and maintaining normal growth and health. Current methods for microbial synthesis of 7-DHC suffer from problems such as slow cell growth, metabolic imbalance, and accumulation of toxic intermediate metabolites like terpenes. Chassis strains for modifying microorganisms to produce 7-DHC primarily include *Saccharomyces cerevisiae* and *Yersinia lipolytica*. *Saccharomyces cerevisiae*, as a highly robust eukaryote, is an ideal host for sterol natural products. *Saccharomyces cerevisiae* possesses advantages such as a short growth cycle, ease of genetic manipulation, and the presence of its own sterol biosynthetic module, making it suitable as a chassis cell for 7-DHC synthesis. Saccharomyces cerevisiae itself does not synthesize 7-DHC. It is necessary to introduce the exogenous enzyme DHCR24 (3β-hydroxysterol-D24 reductase) to construct the 7-DHC synthesis pathway intracellularly, which generates 7-DHC by reducing cholesterol-5,7,24-trienol.

[0003] Naturally derived 24-dehydrocholesterol reductase (DHCR24) has weak enzyme activity and a weak ability to synthesize 7-DHC. By mutating the genetically synthesized chicken-derived 24-dehydrocholesterol reductase, a beneficial mutant sequence was obtained and applied to industrial yeast to increase the yield of 7-DHC.

[0004] Currently known strains for producing 7-DHC have long fermentation times, with fermentation tank times generally around 120 hours. This invention optimizes the fermentation process of genetically engineered strains, shortening the fermentation time to any integer number of fermentation times from 2 to 30, thereby increasing fermentation yield. Summary of the Invention

[0005] In view of this, this invention utilizes protein engineering technology to perform point mutations on genetically synthesized chicken-derived 24-dehydrocholesterol reductase (Gallus gallus (chicken) DHCR24), obtaining a mutant sequence with a higher 7-DHC production capacity than the natural 24-dehydrocholesterol reductase. The optimal mutant sequence was integrated into a high-cholesterol-5,7,24-trien-3β-ol-producing strain, and a series of strain modifications were performed to obtain an engineered strain F-6B with high 7-DHC production capacity. Currently, the yield in a 50L fermenter after 35 hours of fermentation reaches 56.2 mg / g. This existing strain can significantly shorten fermentation time and achieve high 7-DHC production while reducing costs, meeting the needs of large-scale industrial production.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a mutant of 24-dehydrocholesterol reductase, wherein the mutation sites of the mutant include: S324L, R58F, A25Y, A25F, T431D, M60L, S324V, M40L, Q51Y, T431E, S324I, Y145T, S265E, K310G, S126T, A25H, S265D, and V403. One or more of the following: E, M497L, I238V, S265Q, M231I, M497Y, N406E, L405I, S324T, R295A, N298T, M115H, Q496K, T445A, R295S, N406Q, M115Q, R295C, R58L, V403N, V403Q, M188V, or V403S.

[0008] In some specific embodiments of the present invention, the accession number of the 24-dehydrocholesterol reductase is NCBI Reference Sequence: NP_001026459; the gene of the 24-dehydrocholesterol reductase is the chicken-derived DHCR24 gene.

[0009] The 24-dehydrocholesterol reductase has the amino acid sequence shown in SEQ ID No. 90.

[0010] In some specific embodiments of the present invention, the mutant has:

[0011] (I) An amino acid sequence as shown in any of SEQ ID No. 91~130;

[0012] (II) An amino acid sequence obtained by substituting, deleting or adding one or more residues as shown in (I), and whose function is the same or similar to that of (I);

[0013] (III) An amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (I) or (II).

[0014] The plurality can be any integer from 2 to 30.

[0015] In some specific embodiments of the present invention, the mutant has an amino acid sequence as shown in SEQ ID No. 103.

[0016] Secondly, the present invention also provides a nucleic acid molecule encoding the mutant.

[0017] Thirdly, the present invention also provides an expression frame including the nucleic acid molecule.

[0018] Fourthly, the present invention also provides an expression vector, comprising the nucleic acid molecule or the expression cassette.

[0019] Fifthly, the present invention also provides a bacterial strain, said strain:

[0020] (A) Expressing the mutant;

[0021] (B) Transform or transdye the expression box;

[0022] (C) Transform or transfect the expression vector.

[0023] In some specific embodiments of the present invention, the strain includes Saccharomyces cerevisiae.

[0024] In some specific embodiments of the present invention, the chassis strain of the strain is taken as the starting strain F-3, and any of the following are performed:

[0025] (I) Integration of two ERG2 and / or ERG3 genes;

[0026] (II) Knock out oxygen-binding protein vhb and / or transcription factor ROX1;

[0027] (III) Integrating glucose transport-related genes HXT1, HXT7 and / or GCR1;

[0028] The F-3 strain is a knockout Saccharomyces cerevisiae FX-2 (… Saccharomyces cerevisiae Saccharomyces cerevisiae obtained from the ERG4, ERG5, and ERG6 genes in FX-2;

[0029] The preservation number of the brewing yeast FX-2 is CCTCC No: M 2016418.

[0030] Sixthly, the present invention also provides a method for constructing the strain, wherein in the chassis strain:

[0031] (a) Transform or transdye the expression box;

[0032] (b) Transformation or transfection of the expression vector.

[0033] In some specific embodiments of the present invention, the strain includes Saccharomyces cerevisiae.

[0034] In some specific embodiments of the present invention, the chassis strain uses strain F-3 as the starting strain and performs any of the following:

[0035] (I) Integration of two ERG2 and / or ERG3 genes;

[0036] (II) Knock out oxygen-binding protein vhb and / or transcription factor ROX1;

[0037] (III) Integrating glucose transport-related genes HXT1, HXT7 and / or GCR1;

[0038] The F-3 strain is a knockout Saccharomyces cerevisiae FX-2 (… Saccharomyces cerevisiae Saccharomyces cerevisiae obtained from the ERG4, ERG5, and ERG6 genes in FX-2;

[0039] The preservation number of the brewing yeast FX-2 is CCTCC No: M 2016418.

[0041] In a seventh aspect, the present invention also provides any of the following applications in increasing 7-DHC yield, shortening 7-DHC fermentation time, and / or reducing 7-DHC production costs:

[0042] (I) The expression vector;

[0043] (II) The strain described above;

[0044] (III) The strains obtained by the construction method.

[0045] Eighthly, the present invention also provides the use of any of the following in the preparation of 7-DHC:

[0046] (i) The mutant;

[0047] (ii) The nucleic acid molecule;

[0048] (iii) The expression box;

[0049] (iv) The expression vector;

[0050] (v) The strain described;

[0051] (vi) The strain obtained by the construction method.

[0052] In a ninth aspect, the present invention also provides a method for preparing 7-DHC, comprising:

[0053] (a) Cultivate the strain, collect the fermentation broth, purify it, and obtain the 7-DHC;

[0054] (b) Cultivate the strain obtained by the construction method, collect the fermentation broth, purify it, and obtain the 7-DHC.

[0055] This invention mutates chicken-derived 24-dehydrocholesterol reductase (DHCR24) obtained through gene synthesis to solve the problem of the weak ability of natural 24-dehydrocholesterol reductase to produce 7-DHC, thereby increasing the yield of 7-DHC, optimizing the fermentation process of 7-DHC genetically engineered strains, shortening fermentation time, increasing fermentation yield, achieving cost reduction and efficiency improvement, and thus realizing industrial production.

[0056] Biological Preservation Instructions

[0057] Biological material: Saccharomyces cerevisiae FX-2, deposited on August 1, 2016 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M 2016418. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0059] Figure 1 This shows the implementation flowchart of the technical solution;

[0060] Figure 2 Plasmid map;

[0061] Figure 3 This shows positive clones selected from transformed E. coli;

[0062] Figure 4 Screening for positive transformants by transforming Saccharomyces cerevisiae;

[0063] Figure 5 Fermentation results showing the 7-DHC content of DHCR24 enzyme point mutation (wild type and corresponding mutants 1-20).

[0064] Figure 6 Fermentation results showing the 7-DHC content of DHCR24 enzyme point mutation (wild type and corresponding mutants 21-40).

[0065] Figure 7 Gel image showing ERG2-ERG3 gene integration PCR;

[0066] Figure 8 A gel image showing PCR integration of the vhb gene;

[0067] Figure 9 Showing a gel image of ROX1 gene integration PCR;

[0068] Figure 10 Gel image showing GCR1 gene integration PCR;

[0069] Figure 11 Showing HXT1 gene integration PCR gel image;

[0070] Figure 12 This shows a gel image of HXT7 gene integration PCR.

[0071] Figure 13 This shows a gel image of PCR integration of the DHCR24(M40L) gene.

[0072] Figure 14 The 7-DHC content and cell wet weight of strain F-6B at different time points during fermentation in a 50L bioreactor are shown.

[0073] Figure 15 The 7-DHC content of strains F-6A and F-6B at different time points during the fermentation process in a 50L bioreactor is shown. Detailed Implementation

[0074] This invention discloses a 24-dehydrocholesterol reductase mutant, its engineered strain, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0075] pSCm-N20-G418-GAPp-DHCR24-adh1t plasmid sequence (as shown in SEQ ID No. 81):

[0076]

[0077] Anshengda Biotechnology Co., Ltd. (Suzhou) synthesized chicken-derived DHCR24 sequence (as shown in SEQ ID No. 82):

[0078]

[0079] ERG2 gene sequence (as shown in SEQ ID No.83):

[0080] ATGAAGTTTTTCCCACTCCTTTTGTTGATTGGTGTTGTAGGCTACATTATGAACGTATTGTTCACTACCTGGTTGCCAACCAATTACATGTTCGATCCAAAAACTTTGAACGAAATATGTAACTCGGTGATTAGCAAACACAACGCAGCAGAAGGTTTATCCACTGAAGACCTGTTACAGGATGTCAGAGACGCACTTGCCTCTCATTACGGGGACGAATACATCAACAAGTACGTCAAAGAAGAATGGGTCTTCAACAATGCTGGTGGTGCGATGGGCCAAATGATCATCCTACACGCTTCCGTATCCGAGTACTTAATTCTATTCGGAACCGCTGTTGGTACTGAAGGGCACACAGGTGTTCACTTTGCTGACGACTATTTTACCATCTTACATGGTACGCAAATCGCAGCATTGCCATATGCCACTGAAGCCGAAGTTTACACTCCTGGTATGACTCATCACTTGAAGAAGGGATACGCCAAGCAATACAGCATGCCAGGTGGTTCCTTTGCCCTTGAATTGGCTCAAGGCTGGATTCCATGTATGTTGCCATTCGGGTTTTTGGACACTTTCTCCAGTACTCTTGATTTATACACTCTATATAGAACTGTCTACCTGACTGCCAGGGACATGGGTAAGAACTTGTTGCAAAACAAAAAGTTCTAA

[0081] ERG3 gene sequence (as shown in SEQ ID No.84):

[0082]

[0083] HXT1 gene sequence (as shown in SEQ ID No. 85):

[0084]

[0085] HXT7 gene sequence (as shown in SEQ ID No. 86):

[0086]

[0087] GCR1 gene sequence (as shown in SEQ ID No. 87):

[0088]

[0089] VHB (Vitreoscilla hemoglobin) gene sequence (as shown in SEQ ID No.88):

[0090] ATGTTGGATCAACAAACTATTAATATTATTAAAGCTACTGTTCCTGTTTTAAAAGAACACGGTGTTACAATTACAACTACATTTTATAAGAACTTGTTTGCTAAACATCCTGAAGTTAGACCATTGTTTGATATGGGTAGACAAGAATCTTTGGAACAACCAAAAGCTTTGGCTATGACTGTTTTGGCTGCAGCTCAAAATATTGAAAATTTGCCTGCTATTTTGCCTGCTGTTAAAAAAATTGCTGTTAAACATTGTCAAGCTGGTGTTGCTGCAGCTCATTATCCAATTGTTGGTCAAGAATTGTTGGGTGCTATTAAAGAAGTTTTGGGTGATGCTGCTACTGATGATATTTTGGATGCTTGGGGTAAAGCTTATGGTGTTATTGCTGATGTTTTTATTCAAGTTGAAGCTGATTTGTATGCTCAAGCTGTTGAATAA

[0091] ROX1 (transcriptional repressors Rox1p) sequence (as shown in SEQ ID No.89):

[0092]

[0093] The original sequence of the 24-dehydrocholesterol reductase (as shown in SEQ ID No. 90):

[0094] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQ VVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEE SKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDM LVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0095] The following mutants were all obtained by mutation based on the original sequence of the 24-dehydrocholesterol reductase.

[0096] S324L mutant (as shown in SEQ ID No. 91):

[0097] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPLRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0098] S324V mutant (as shown in SEQ ID No.92):

[0099] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPVRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0100] S324I mutant (as shown in SEQ ID No.93):

[0101] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPIRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0102] S324T mutant (as shown in SEQ ID No.94):

[0103] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPTRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0104] R58F mutant (as shown in SEQ ID No.95):

[0105] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVFRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0106] R58L mutant (as shown in SEQ ID No.96):

[0107] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVLRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0108] A25Y mutant (as shown in SEQ ID No.97):

[0109] MSAVWSLGAGLLLLLLWVRHRGLEYVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0110] A25F mutant (as shown in SEQ ID No.98):

[0111] MSAVWSLGAGLLLLLLWVRHRGLEFVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0112] A25H mutant (as shown in SEQ ID No.99):

[0113] MSAVWSLGAGLLLLLLWVRHRGLEHVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0114] T431D mutant (as shown in SEQ ID No.100):

[0115] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDEDELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0116] T431E mutant (as shown in SEQ ID No.101):

[0117] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDEEELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0118] M60L mutant (as shown in SEQ ID No.102):

[0119] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRLHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0120] M40L mutant (as shown in SEQ ID No.103):

[0121] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLLPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0122] Q51Y mutant (as shown in SEQ ID No.104):

[0123] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYYLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0124] Y145T mutant (as shown in SEQ ID No.105):

[0125] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTATLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0126] S265E mutant (as shown in SEQ ID No.106):

[0127] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENEFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0128] S265D mutant (as shown in SEQ ID No.107):

[0129] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENDFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0130] S265Q mutant (as shown in SEQ ID No.108):

[0131] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENQFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0132] K310G mutant (as shown in SEQ ID No. 109):

[0133] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEGYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0134] S126T mutant (as shown in SEQ ID No.110):

[0135] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDTERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0136] V403E mutant (as shown in SEQ ID No.111):

[0137] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHEDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0138] V403N mutant (as shown in SEQ ID No.112):

[0139] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHNDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0140] V403Q mutant (as shown in SEQ ID No.113):

[0141] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHQDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0142] V403S mutant (as shown in SEQ ID No.114):

[0143] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHSDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0144] M497L mutant (as shown in SEQ ID No. 115):

[0145] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQLNCKDAFPEVYDKICKAARH

[0146] M497Y mutant (as shown in SEQ ID No.116):

[0147] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQYNCKDAFPEVYDKICKAARH

[0148] I238V mutant (as shown in SEQ ID No.117):

[0149] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYVRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0150] M231I mutant (as shown in SEQ ID No.118):

[0151] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKIIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0152] N406E mutant (as shown in SEQ ID No.119):

[0153] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLEVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0154] N406Q mutant (as shown in SEQ ID No.120):

[0155] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLQVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0156] L405I mutant (as shown in SEQ ID No.121):

[0157] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDINVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0158] R295A mutant (as shown in SEQ ID No.122):

[0159] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINAIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0160] R295S mutant (as shown in SEQ ID No.123):

[0161] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINSIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0162] R295C mutant (as shown in SEQ ID No.124):

[0163] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINCIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0164] N298T mutant (as shown in SEQ ID No.125):

[0165] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGTYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0166] M115H mutant (as shown in SEQ ID No.126):

[0167] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIHINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0168] M115Q mutant (as shown in SEQ ID No.127):

[0169] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIQINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0170] Q496K mutant (as shown in SEQ ID No.128):

[0171] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREKMNCKDAFPEVYDKICKAARH

[0172] T445A mutant (as shown in SEQ ID No.129):

[0173] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDMLVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKAKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0174] M188V mutant (as shown in SEQ ID No.130):

[0175] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRPGWLTVSLRVGKYKKTHKNIMINLMDVLEVDSERQ VVRVEPLVTMGQLTAYLNPMGWTIPVVPELDDLTVGGLIMGTGIESSSHIYGLFQHTCVAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEE SKNKENSFVEGLVYSLEEAVIMTGVLTDEAEPSKINRIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYYHRHTRSIFWELQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHVVQDM LVPMKSLEKSIQTFHVDLNVYPLWLCPFLLPNNPGMVHPKGDETELYVDIGAYGEPKTKQFEARASMRQMEKFVRSVHGFQMLYADCYMTREEFWDMFDGSLYHSLREQMNCKDAFPEVYDKICKAARH

[0176] This invention enhances the ability of the chicken-derived DHCR24 gene to transform the substrate cholesterol-5,7,24-trienol through site-directed mutagenesis. By introducing the mutant gene into a high-yield cholesterol-5,7,24-trienol chassis strain, the content of 7-DHC can be ultimately increased. At the same time, this strain itself has good growth characteristics. Optimizing the fermentation process of the 7-DHC genetically engineered strain can shorten the fermentation time, increase the fermentation yield, reduce costs and increase efficiency, thereby realizing industrial production.

[0177] The 24-dehydrocholesterol reductase mutant and engineered strain provided by this invention, as well as the raw materials and reagents used in the application, can all be purchased from the market.

[0178] The present invention will be further illustrated below with reference to the embodiments:

[0179] Example 1: Design of mutation site primers and construction of mutant plasmids

[0180] Identify key amino acid residues:

[0181] Protein simulation analysis was performed on the chicken-derived DHCR24 gene, and molecular docking was performed with the substrate cholesterol-5,7,24-trienol. Forty amino acid mutations were selected for further research based on scoring software. The numbers are listed in order of their scores: S324L, R58F, A25Y, A25F, T431D, M60L, S324V, M40L, Q51Y, T431E, S324I, Y145T, S265E, K310G, S126T, A25H, S265D, V403E, M497L, I238V, S265Q, M231I, M497Y, N406E, L405I, S324T, R295A, N298T, M115H, Q496K, T445A, R295S, N406Q, M115Q, R295C, R58L, V403N, V403Q, M188V, V403S.

[0182] Constructing the DHCR24 E. coli mutant plasmid:

[0183] The chicken-derived DHCR24 gene was synthesized by Anshengda Biotechnology Co., Ltd. (Suzhou) and optimized according to the codon preferences of Saccharomyces cerevisiae (as shown in SEQ ID No. 82), and then ligated into... Figure 2 The plasmid shown contains the expression box consisting of the GAP promoter and the Tdh1 terminator.

[0184] Based on the selected loci S324L, R58F, A25Y, A25F, T431D, M60L, S324V, M40L, Q51Y, T431E, S324I, Y145T, S265E, K310G, S126T, A25H, S265D, V403E, M497L, I238V, S265Q, M231I, M497Y, N406E, L405I, S324T, R295A, N298T, M115H, Q496K, T445A, R295S, N406Q, M115Q, R295C, R58L, V403N, V403Q, M188V, and V403S, the following primers were designed, as shown in Table 1:

[0185] Table 1. Primers required for site-directed saturation mutagenesis

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] The pSCm-N20-G418-GAPp-DHCR24-adh1t plasmid was used as a PCR template, and the mutant primers were used as upstream and downstream primers to prepare the PCR system, as shown in Table 2:

[0193] Table 2. PCR amplification system

[0194]

[0195] The PCR instrument was programmed as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s; 60 °C annealing for 30 s; 72 °C extension (30 s / Kb); repeat the denaturation, annealing, and extension cycles for 30-33 cycles; finally, extend at 72 °C for 5 min and incubate at 16 °C. After PCR, agarose gel electrophoresis was performed to obtain the target fragment. The fragment was then recovered using a gel recovery kit or a liquid PCR recovery kit, and its concentration was determined. The fragment was then digested and recovered using DpnI enzyme, and subsequently transformed into E. coli DH5α competent cells for transformation. Successful transformants were obtained, and the plasmid vectors corresponding to various mutants were thus constructed.

[0196] After recovering and digesting the PCR-obtained fragments and transforming them into E. coli, bacteria were selected for sequencing verification. Figure 3 As shown.

[0197] Example 2 Construction of mutant strains

[0198] The mutant plasmid vector was transformed into strain F-3. After the colonies grew on the culture dish, colony PCR was performed.

[0199] Specifically, the plasmid vectors of the various mutants obtained above were integrated into the Saccharomyces cerevisiae strain (F-6), and the DHCR24 mutant was integrated using yeast electrotransformation. The specific yeast electrotransformation process is as follows:

[0200] ① Take 1 mL test tube and culture the primary seed overnight, then transfer to a 50 mL shake flask the next day for secondary seed culture;

[0201] ② After about 4-5 hours, measure the OD600 to 0.8-1.0, and then place the secondary seeds in an ice bath for 10-15 minutes;

[0202] ③ Centrifuge at 6000 g for 6 min at 4 ℃, discard the supernatant, and collect the bacterial cells;

[0203] ④ Wash the bacterial cells twice with 35-50 mL of sterile water, centrifuge at 6000 g for 4-6 min at 4 ℃, discard the supernatant, and collect the bacterial cells;

[0204] ⑤ Repeat step ④, wash twice with sterile water, resuspend in 35-50 mL of 10% glycerol solution, centrifuge at 6000 g for 4-6 min at 4 ℃, discard the supernatant and collect the bacterial cells;

[0205] ⑥ Add a certain amount of 10% glycerol and mix slowly with a pipette tip, then dispense into 50 μL tubes of competent cells for later use;

[0206] ⑦ Introduce the competent cells into the above mutant plasmid vector, about 3~7 μL, mix well with a pipette tip, add to the pre-dried and pre-cooled electroporation vessel, and place on ice;

[0207] ⑧ Set a voltage of 2.5 kV (breakdown time 4.9~5.5 ms, blank control 6.0 ms); place the electroporation cuvette in the electroporator for electroporation, and immediately after electroporation, add 700-900 μL of YPD culture medium to the electroporation cuvette and transfer it to a 2 mL centrifuge tube. After recovery and incubation at 30 ℃ for 1 h, take an appropriate amount of bacterial culture and spread it on a plate. Then, invert the culture dish and place it in a 30 ℃ constant temperature incubator for incubation.

[0208] After the bacteria have grown on the petri dish, as shown Figure 4 As shown, colony PCR was performed.

[0209] Select a single yeast colony and place it in 20-30 μL of cell lysis buffer (Takara, product number AO91944A). Mix well and place in a PCR instrument. Incubate at 98 °C for 1 h to lyse. The resulting colony can then be used as a template. PCR was then performed using Fly Mix reagent. The corresponding PCR system is shown in Table 3.

[0210] Table 3. PCR reaction system of Saccharomyces cerevisiae colonies

[0211]

[0212] The PCR instrument was programmed as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 20 s; 58℃ annealing for 20 s; 72℃ extension (10 s / Kb); repeat the denaturation, annealing, and extension cycles for a total of 30-35 cycles; finally, extend at 72℃ for 5 min and incubate at 16℃. After PCR, agarose gel electrophoresis was performed, and transformants of the same length as the target were obtained, indicating successful integration. Correct transformants were then selected for culture and subsequent fermentation experiments.

[0213] Example 3: Shake flask treatment and content determination of the above mutant strain.

[0214] YPD shake-flask fermentation was performed on the following mutant strains: S324L, R58F, A25Y, A25F, T431D, M60L, S324V, M40L, Q51Y, T431E, S324I, Y145T, S265E, K310G, S126T, A25H, S265D, V403E, M497L, I238V, S265Q, M231I, M497Y, N406E, L405I, S324T, R295A, N298T, M115H, Q496K, T445A, R295S, N406Q, M115Q, R295C, R58L, V403N, V403Q, M188V, and V403S.

[0215] The obtained site-directed mutagenesis fermentation strain was subjected to shake-flask site-directed fermentation. Single colonies of the desired strain were selected and transferred to test tubes, where they were incubated overnight at 30 °C. Next, the bacterial culture from the test tubes was transferred to YPD shake flasks (250 mL, 50 mL total volume) and incubated at 30 °C for 1-2 days to obtain the seed culture. The seed culture was then transferred to shake-flask YPD fermentation medium, with 2% glucose supplementation every 24 hours, and incubated at 30 °C for 4-5 days. The fermentation broth was then collected. Subsequent processing of the harvested bacteria followed the steps below:

[0216] Weigh 0.2g of dried bacterial powder (after drying treatment, specifically as follows: centrifuge the fermentation broth at 8000 rpm for 5 min, collect the bacterial cells, wash twice with pure water, and freeze-dry the collected bacterial cells in a freeze dryer for 24 h to obtain dried bacterial powder) into a grinding tube, add 1mL of sterile water to resuspend the bacterial cells, add an appropriate amount of grinding beads, and grind in a cell disruptor (grinding frequency 60Hz, grinding interruption time 15s, grinding run time 30s, total grinding times 30 times). After grinding, add the grinding liquid to a 25mL glass tube, add 5mL of saponification solution (composition 20% sodium hydroxide: methanol = 5:3); during this process, add a small portion of the saponification solution to the grinding tube, rinse thoroughly, combine, and then vortex to mix, and saponify at 80 degrees for 2 hours. Remove the test tube, cool it with ice water, and then cool it to room temperature in the dark. Add anhydrous ethanol to approximately 80% of the test tube volume, mix thoroughly, and sonicate at room temperature for 10 minutes. Make up the volume with ethanol to 25 mL, and sonicate again at room temperature for 10 minutes. Pipette the supernatant (centrifugation is optional; record the actual volume, typically around 24.85 mL), and filter it through a 0.22 μm organic filter into a liquid chromatography bottle for subsequent analysis.

[0217] This experiment used the external standard method for liquid chromatography detection. The detection conditions for high performance liquid chromatography were as follows:

[0218] Flow rate: 1 ml / min;

[0219] Mobile phase: pure methanol (isocratic analysis);

[0220] Chromatographic column: C18 reversed-phase column (4.6×250nm, 5μm);

[0221] Time: 22 min;

[0222] Column temperature 30℃;

[0223] The detection wavelength is 282nm.

[0224] Based on the results, the mutant sequence with the highest content was selected to modify subsequent strains.

[0225] Experimental results are as follows Figures 5-6 As shown.

[0226] Table 4. Figure 5 data

[0227]

[0228] Table 5. Figure 6 data

[0229]

[0230] The results show that there are 10 beneficial mutations. The T445A mutation increased 7-DHC production by 4.4%, the S324V mutation by 7.5%, the N406E mutation by 8.5%, the S265Q mutation by 14%, the S126T mutation by 14%, the V403E mutation by 14%, the L405I mutation by 20%, the R295A mutation by 23%, the M231I mutation by 30%, and the M40L mutation by 35.5%. The most beneficial mutation is M40L, and this mutated gene will be subsequently transferred into the chassis strain.

[0231] Example 4: Construction of a high-yield 7-DHC industrial production strain

[0232] Using strain F-3, a high-yielding strain of cholesterol-5,7,24-trien-3β-ol as the starting strain in the applicant's patent CN 119799532 A, "A high-yielding strain of cholesterol-5,7,24-trien-3β-ol and its application," the strain underwent several integrations: integration of two ERG2 and ERG3 genes, knockout of the oxygen-binding protein vhb / transcription factor ROX1, and integration of glucose transport-related genes HXT1 / HXT7 / GCR1. Finally, two copies of the DHCR24 M40L mutant sequence were integrated to obtain the final high-yielding industrial strain F-6B. The relevant integration PCR gel images are shown below. Figures 7-13 As shown.

[0233] The resulting strain was fermented in a 50L fermenter, and the final 7-DHC yield was determined.

[0234] Example 5: Fermentation of the obtained F-6B strain in a 50L fermenter.

[0235] The obtained strain was fermented in a 50L fermenter, and the results are as follows: Figure 14 As shown.

[0236] Fermentation process:

[0237] Test tube (pick a single colony) → 50 / 250ml shake flask (24h, 200rpm, 31℃) → 50 / 250ml primary shake flask (inject 5ml of activated bacterial solution, 24h, 200rpm, 31℃) → 2 / 5L secondary shake flask (24h, 200rpm, 31℃) → 50L container.

[0238] Process control methods:

[0239] 1. During fermentation, the alcohol content should be <0.2%. If the alcohol content is too high, reduce the sugar content and restore it when the alcohol content drops to around 0.1%.

[0240] 2. After fermentation begins, feed the sugar according to the control table design in the early stage. Once the wet weight is >40g / L, the amount of sugar added can be adjusted according to the actual situation (compound vitamins are added to the sugar). Control the RQ to about 1.1-1.15 by adding sugar and maintain a steady increase in CER / OUR. The amount of P fed should be about 1 / 17.6-1 / 10 of the amount of C source fed (rounded up).

[0241] 3. Control dissolved oxygen to above 20%, maximum air volume 50, and stirring 500 (linked).

[0242] Table 6. Figure 14 data

[0243]

[0244] The results after detection by liquid chromatography are as follows:

[0245] The wet weight reached 120 g / L after 24 hours, with a yield of 41.2 mg / g; after 29 hours, the wet weight reached 180 g / L, with a yield of 47.8 mg / g; and after 35 hours, the wet weight reached 230 g / L, with a final yield of 56.2 mg / g. These results demonstrate that this patented strain possesses a high-yield capacity for 7-DHC and can significantly shorten fermentation time, reducing costs and increasing efficiency, thus enabling industrial-scale production.

[0246] Example of effect

[0247] Using strain F-3, a high-yield cholesterol-5,7,24-trien-3β-ol strain from the applicant's patent CN 119799532 A, entitled "A high-yield cholesterol-5,7,24-trien-3β-ol strain and its application," as the starting strain, the strain underwent several modifications: integration of two ERG2 and ERG3 genes, knockout of the oxygen-binding protein vhb / transcription factor ROX1, and integration of glucose transport-related genes HXT1 / HXT7 / GCR1. Finally, two copies of the unmutated DHCR24 sequence were integrated to construct industrial strain F-6A. Strain F-6A and strain F-6B were simultaneously fermented in a 50L fermenter. The specific process is as follows:

[0248] Test tube (pick a single colony) → 50 / 250ml shake flask (24h, 200rpm, 31℃) → 50 / 250ml primary shake flask (inject 5ml of activated bacterial solution, 24h, 200rpm, 31℃) → 2 / 5L secondary shake flask (24h, 200rpm, 31℃) → 50L container.

[0249] Process control methods:

[0250] 1. During fermentation, the alcohol content should be <0.2%. If the alcohol content is too high, reduce the sugar content and restore it when the alcohol content drops to around 0.1%.

[0251] 2. After fermentation begins, feed the sugar according to the control table design in the early stage. Once the wet weight is >40g / L, the amount of sugar added can be adjusted according to the actual situation (compound vitamins are added to the sugar). Control the RQ to about 1.1-1.15 by adding sugar and maintain a steady increase in CER / OUR. The amount of P fed is about 1 / 17.6-1 / 10 of the amount of C source fed (rounded up).

[0252] 3. Control dissolved oxygen to above 20%, maximum air volume 50, and stirring 500 (linked).

[0253] Samples of the two bacterial strains were taken at 24, 29, and 35 hours for 7-DHC content detection. Samples were tested three times for each time period, using the same method as in Example 3. The final experimental results are shown in Table 7. Figure 15 As shown:

[0254] Table 7. Figure 15 data

[0255]

[0256] Compared with the F-6A strain which integrates the unmutated DHCR24 sequence, the 7-DHC content of the constructed high-yield industrial strain F-6B was approximately 35% higher at each time point, as determined by sampling at 24, 29, and 35 hours. This suggests that the obtained high-yield strain F-6B has better potential for industrial application.

[0257] The high-yielding strain F-6B of this patent is compared with the strain from Jiangnan University (the highest-yielding strain in the literature on 7-dehydrocholesterol by rewiring the post-squalene module in lipid droplets of Saccharomyces cerevisiae), as shown in Table 8:

[0258] Table 8

[0259]

[0260] Compared with the Jiangnan University strain, which currently has the highest yield, the F-6B strain has a significantly shorter fermentation time and a slightly higher 7-DHC content.

[0261] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

A mutant of 1,24-dehydrocholesterol reductase, characterized in that, The mutant has an amino acid sequence as shown in SEQ ID No.

103.

2. A nucleic acid molecule encoding the mutant as described in claim 1.

3. An expression box, characterized in that, Includes the nucleic acid molecules as described in claim 2.

4. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 2 or the expression cassette as described in claim 3.

5. A bacterial strain, characterized in that, The strain: (A) Expressing the mutant as described in claim 1; (B) Transformation or transfection of the expression frame as described in claim 3; (C) Transformation or transfection with the expression vector as described in claim 4.

6. The strain according to claim 5, characterized in that, The strains include Saccharomyces cerevisiae.

7. The strain according to claim 6, characterized in that, The chassis strain of the strain is based on strain F-3, and any of the following steps are performed: (I) Integration of two ERG2 and / or ERG3 genes; (II) Knock out oxygen-binding protein vhb and / or transcription factor ROX1; (III) Integrating glucose transport-related genes HXT1, HXT7 and / or GCR1; The F-3 strain is a knockout Saccharomyces cerevisiae FX-2 (… Saccharomyces cerevisiae Saccharomyces cerevisiae obtained from the ERG4, ERG5, and ERG6 genes in FX-2; The preservation number of the brewing yeast FX-2 is CCTCC No: M 2016418.

8. The method for constructing the strain according to any one of claims 5 to 7, characterized in that, In chassis strains: (a) Transformation or transfection of the expression box as described in claim 3; (b) Transformation or transfection of the expression vector as described in claim 4.

9. The construction method as described in claim 8, characterized in that, The strains include Saccharomyces cerevisiae.

10. The construction method as described in claim 8, characterized in that, The chassis strain, starting with strain F-3, was subjected to any of the following: (I) Integration of two ERG2 and / or ERG3 genes; (II) Knock out oxygen-binding protein vhb and / or transcription factor ROX1; (III) Integrating glucose transport-related genes HXT1, HXT7 and / or GCR1; The F-3 strain is a knockout Saccharomyces cerevisiae FX-2 (… Saccharomyces cerevisiae Saccharomyces cerevisiae obtained from the ERG4, ERG5, and ERG6 genes in FX-2; The preservation number of the brewing yeast FX-2 is CCTCC No: M 2016418.

11. Any of the following applications in increasing 7-DHC yield, shortening 7-DHC fermentation time, and / or reducing 7-DHC production costs: (I) The expression vector as described in claim 4; (II) The strain as described in any one of claims 5 to 7; (III) The strain obtained by the construction method according to any one of claims 8 to 10.

12. Any of the following applications in the preparation of 7-DHC: (i) The mutant as described in claim 1; (ii) The nucleic acid molecule as described in claim 2; (iii) The expression box as described in claim 3; (iv) The expression vector as described in claim 4; (v) The strain as described in any one of claims 5 to 7; (vi) The strain obtained by the construction method according to any one of claims 8 to 10. The method for preparing 13.7-DHC is characterized by, include: (a) Cultivate the strain as described in any one of claims 5 to 7, collect the fermentation broth, purify it, and obtain the 7-DHC; (b) Cultivate the strain obtained by the construction method as described in any one of claims 8 to 10, collect the fermentation broth, purify it, and obtain the 7-DHC.

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