Preparation and application of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase mutant

Abstract

The invention discloses preparation and application of a Saccharomyces cerevisiae lanosterol 14 alpha-demethylase mutant, and belongs to the technical field of gene engineering and enzyme engineering. Starting from the Saccharomyces cerevisiae WQ6, the lanosterol 14 alpha-demethylase of the Saccharomyces cerevisiae is mutated by means of genetic engineering and enzyme engineering, the yield of ergosterol of the Saccharomyces cerevisiae can be increased by the obtained single mutation and double mutants, and conditions are created for expanding industrial application of the lanosterol 14 alpha-demethylase of the Saccharomyces cerevisiae.

Description

Technical Field

[0001] The invention relates to the preparation and application of a lanosterol 14α-demethylase mutant of saccharomyces cerevisiae, and belongs to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] Ergosterol is also known as ergosterol, with a molecular formula of C 28 H 44 O. It is produced by lanosterol (Lanosterol) catalyzed by lanosterol 14α-demethylase (Sterol 14α-demethylase, 14DM, CYP51). As a sterol pool, ergosterol can ensure the balance of sterol structures in bacteria and plays a vital role in organisms, such as participating in the composition of membranes to ensure the integrity of cell membranes, promoting the activity of transmembrane proteins, making membranes fluid, and transporting cellular substances. In addition, ergosterol can also affect the absorption and utilization of nutrients by regulating the activity of membrane-bound ATPases, and regulate the transport efficiency of phospholipases by affecting the migration of cell membranes. Ergosterol is a precursor for the synthesis of vitamin D2 and is widely used in the pharmaceutical, food, and feed industries. Since my country started studying ergosterol relatively late, its production method is relatively complicated, but the demand is large and often in short supply. Therefore, improving the supply of ergosterol is of great significance for its subsequent further development in the fields of food and medicine.

[0003] At present, the main method for producing ergosterol in my country is to use microbial fermentation. The main strains used are usually yeast, large fungi and molds. The advantages are fast reproduction, easy cultivation, high yield, safety and speed, but the large difference in ergosterol content between strains is a common problem. Therefore, increasing the ergosterol content of microbial sources is an urgent problem to be solved. The methylation of lanosterol at the 14α position in the ergosterol synthesis pathway is a key step, but the natural lanosterol 14α-demethylase that catalyzes this step has poor enzyme activity and stability. The use of enzyme engineering to obtain lanosterol 14α-demethylase mutants with improved enzyme activity and stability is of great significance for the production of ergosterol by microbial fermentation. Summary of the invention

[0004] Based on the above situation, the present invention utilizes the means of genetic engineering and enzyme engineering to improve the activity of lanosterol 14α-demethylase of Saccharomyces cerevisiae, increase the yield of ergosterol, and create conditions for expanding its industrial application.

[0005] The present invention provides lanosterol 14α-demethylase mutants with improved catalytic efficiency, which are mutants obtained by mutating one or more amino acids at positions 127, 260, 352, 314 or 384 of lanosterol 14α-demethylase derived from Saccharomyces cerevisiae. Compared with their parent lanosterol 14α-demethylase, these mutants have improved catalytic efficiency.

[0006] In one embodiment, the lanosterol 14α-demethylase derived from Saccharomyces cerevisiae contains the amino acids shown in SEQ ID NO.1.

[0007] In one embodiment, the mutant is based on lanosterol 14α-demethylase from Saccharomyces cerevisiae and has one or more of the following mutations:

[0008] (1) Mutate alanine at position 127 to serine;

[0009] (2) mutating alanine at position 260 to lysine;

[0010] (3) mutating lysine at position 352 to leucine;

[0011] (4) mutating glycine at position 314 to valine;

[0012] (5) mutating glycine at position 314 to tryptophan;

[0013] (6) mutating phenylalanine at position 384 to aspartic acid;

[0014] (7) Mutate phenylalanine at position 384 to glutamic acid.

[0015] In one embodiment, the mutant is a mutation of alanine (Ala) at position 127 to serine (Ser), and the mutant is named A127S.

[0016] In one embodiment, the mutant is a mutation of alanine (Ala) at position 260 to lysine (Lys), and the mutant is named A260K.

[0017] In one embodiment, the mutant is a mutation of lysine (Lys) at position 352 to leucine (Leu), and the mutant is named K352L.

[0018] In one embodiment, the mutant is a mutation of glycine (Gly) at position 314 to valine (Val), and the mutant is named G314V.

[0019] In one embodiment, the mutant is a mutation of glycine (Gly) at position 314 to tryptophan (Trp), and the mutant is named G314W.

[0020] In one embodiment, the mutant is a mutation of phenylalanine (Phe) at position 384 to aspartic acid (Asp), and the mutant is named F384D.

[0021] In one embodiment, the mutant is a mutation of phenylalanine (Phe) at position 384 to glutamic acid (Glu), and the mutant is named F384E.

[0022] In one embodiment, the mutant is a mutation of alanine (Ala) at position 127 to serine (Ser), and a mutation of lysine (Lys) at position 352 to leucine (Leu), and the mutant is named A127S / K352L.

[0023] In one embodiment, the mutant is a mutation of alanine (Ala) at position 260 to lysine (Lys), and a mutation of lysine (Lys) at position 352 to leucine (Leu), and the mutant is named A260K / K352L.

[0024] In one embodiment, the mutant is a mutation of alanine (Ala) at position 127 to serine (Ser), and alanine (Ala) at position 260 to lysine (Lys), and the mutant is named A127S / A260K.

[0025] In one embodiment, the mutant is a mutant in which phenylalanine (Phe) at position 384 is mutated to aspartic acid (Asp), and glycine (Gly) at position 314 is mutated to valine (Val), and the mutant is named F384D / G314V.

[0026] In one embodiment, the mutant is a mutation in which phenylalanine (Phe) at position 384 is mutated to aspartic acid (Asp), and glycine (Gly) at position 314 is mutated to tryptophan (Trp), and the mutant is named F384D / G314W.

[0027] In one embodiment, the mutant is a mutation in which phenylalanine (Phe) at position 384 is mutated to valine (Val), and glycine (Gly) at position 314 is mutated to tryptophan (Trp), and the mutant is named F384E / G314V.

[0028] In one embodiment, the mutant is a mutation in which phenylalanine (Phe) at position 384 is mutated to glutamic acid (Glu), and glycine (Gly) at position 314 is mutated to tryptophan (Trp), and the mutant is named F384E / G314W.

[0029] The present invention also provides a gene encoding the lanosterol 14α-demethylase mutant.

[0030] In one embodiment, the nucleotide sequence encoding mutant A127S is shown as SEQ ID NO.3; the nucleotide sequence encoding mutant A260K is shown as SEQ ID NO.4; the nucleotide sequence encoding mutant K352L is shown as SEQ ID NO.5; the nucleotide sequence encoding mutant G314V is shown as SEQ ID NO.6; the nucleotide sequence encoding mutant G314W is shown as SEQ ID NO.7; the nucleotide sequence encoding mutant F384D is shown as SEQ ID NO.8; the nucleotide sequence encoding mutant F384E is shown as SEQ ID NO.9.

[0031] The invention also provides a plasmid carrying the gene.

[0032] In one embodiment, the recombinant plasmid uses pY26 as the starting plasmid.

[0033] The present invention also provides a recombinant microbial cell expressing the Saccharomyces cerevisiae lanosterol 14α-demethylase mutant.

[0034] In one embodiment, the microorganism is Saccharomyces cerevisiae.

[0035] In one embodiment, the Saccharomyces cerevisiae is based on Saccharomyces cerevisiae CENPK2-1D as a starting strain.

[0036] In one embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae having the ability to synthesize ergosterol.

[0037] In one embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae WQ6; the Saccharomyces cerevisiae WQ6 and its construction method have been disclosed in the patent application document with publication number CN114606149A.

[0038] In one embodiment, the Saccharomyces cerevisiae WQ6 is based on Saccharomyces cerevisiae CENPK2-1D, and has the following improvements: NADH-HMG1 is integrated at the GAL2 site on the genome, tHMGR and IDI1-LEU2-Degradation tag are integrated in multiple copies at the Ty2 site, YPL062W, ROX1 and NEM1 are knocked out, ERG4, ERG11, and ERG1 are integrated at the YJL064W site, ERG2, ERG3, CTT1, and ERG4 are integrated at the DOS2 site, tHMG1 and IDI1-spHIS5deg are integrated in multiple copies at the Ty12 site; POS5, ERG4, ERG2, and ERG3 are integrated in multiple copies at the Ty3 site.

[0039] In one embodiment, the recombinant microbial cell uses Saccharomyces cerevisiae WQ6 as a host to express the lanosterol 14α-demethylase mutant to construct an engineered Saccharomyces cerevisiae strain.

[0040] The present invention also provides a method for increasing the ergosterol production of Saccharomyces cerevisiae, wherein the method comprises mutating one or more sites at position 127, position 260, position 352, position 314 or position 384 of the amino acid sequence of lanosterol 14α-demethylase of Saccharomyces cerevisiae.

[0041] In one embodiment, the method is to perform the following steps on lanosterol 14α-demethylase to improve the catalytic ability of Saccharomyces cerevisiae on lanosterol 14α-demethylase:

[0042] (1) mutating alanine at position 127 to serine; or

[0043] (2) mutating alanine at position 260 to lysine; or

[0044] (3) mutating lysine at position 352 to leucine; or

[0045] (4) mutating glycine at position 314 to valine; or

[0046] (5) mutating glycine at position 314 to tryptophan; or

[0047] (6) mutating the phenylalanine at position 384 to aspartic acid; or

[0048] (7) mutating phenylalanine at position 384 to glutamic acid; or

[0049] (8) mutating alanine at position 127 to serine and lysine at position 352 to leucine; or

[0050] (9) mutating alanine at position 260 to lysine and lysine at position 352 to leucine; or

[0051] (10) mutating the alanine at position 127 to serine and mutating the alanine at position 260 to lysine; or

[0052] (11) mutating the phenylalanine at position 384 to aspartic acid and the glycine at position 314 to valine; or

[0053] (12) mutating the phenylalanine at position 384 to aspartic acid and the glycine at position 314 to tryptophan; or

[0054] (13) mutating the phenylalanine at position 384 to glutamic acid and the glycine at position 314 to valine; or

[0055] (14) The phenylalanine at position 384 was mutated to glutamic acid, and the glycine at position 314 was mutated to tryptophan.

[0056] In one embodiment, the lanosterol 14α-demethylase has an amino acid sequence as shown in SEQ ID NO.1.

[0057] The invention also provides application of the engineered yeast Saccharomyces cerevisiae in producing ergosterol.

[0058] In one embodiment, the application is to ferment the engineered strain of Saccharomyces cerevisiae in a culture medium for a period of time and collect ergosterol.

[0059] In one embodiment, the culture medium contains a carbon source, a nitrogen source, and inorganic salts required for the growth of Saccharomyces cerevisiae.

[0060] In one embodiment, the culture medium is YPD medium.

[0061] In one embodiment, the culture medium contains glucose, tryptone, yeast extract, and inorganic salts; the inorganic salts include magnesium salts and iron salts; optionally, the magnesium salt is magnesium sulfate, and the iron salt is ferric sulfate.

[0062] In one embodiment, the pH is controlled at 5.5±0.1 during the fermentation process.

[0063] In one embodiment, feeding is also performed during the fermentation process.

[0064] In one embodiment, the fermentation process is also fed with glucose and trace elements to control the residual sugar in the fermentation system to 0.5 g / L to 1.5 g / L and the ethanol content to 5 g / L to 10 g / L; the trace elements include but are not limited to metal ions and / or vitamins.

[0065] In one embodiment, a feed medium containing an inorganic nitrogen source is also fed during the fermentation process; the inorganic nitrogen source includes but is not limited to ammonium sulfate.

[0066] The present invention also claims to protect the application of the Saccharomyces cerevisiae lanosterol 14α-demethylase mutant in the food, biomedicine, chemical industry, cosmetics, feed and other industries.

[0067] In one embodiment, the use includes but is not limited to the preparation of ergosterol or a product containing ergosterol.

[0068] Beneficial effects:

[0069] (1) The present invention targets lanosterol 14α-demethylase (ERG11) in Saccharomyces cerevisiae CENPK2-1D, transforms the amino acid sequence of ERG11 by site-directed mutagenesis, and obtains lanosterol 14α-demethylase mutants A127S, A260K, K352L, G314V, G314W, F384D, F384E, F384D / G314W, and F384E / G314V with improved catalytic ability;

[0070] (2) The present invention expresses the ERG11 mutants constructed above in Saccharomyces cerevisiae WQ6, and the constructed strains are subjected to shake flask fermentation, so that the ergosterol production of the recombinant strains expressing the mutants A127S, A260K, K352L, G314V, G314W, F384D, F384E, F384D / G314W, and F384E / G314V reaches 645.0 mg / L, 625.8 mg / L, 677.9 mg / L, 622.7 mg / L, 620.1 mg / L, 682.8 mg / L, 733.8 mg / L, 623.9 mg / L, and 536.1 mg / L, which were respectively 31.6%, 27.7%, 38.3%, 27.1%, 26.6%, 39.3%, 49.8%, 27.3%, and 9.4% higher than the 490.0 mg / L produced by the strain expressing the wild-type lanosterol 14α-demethylase.

[0071] The invention utilizes enzyme engineering technology to transform brewer's yeast, so that the yield of ergosterol transformed by metabolic engineering means is further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Schematic diagram of the metabolism of ergosterol in Saccharomyces cerevisiae.

[0073] Figure 2 Figure 2 is a plasmid map carrying the wild-type lanosterol 14α-demethylase gene fragment.

[0074] Figure 3 FIG. 1 is a plasmid diagram for expressing mutants in one embodiment of the present invention.

[0075] Figure 4 Comparison of ergosterol production by the wild type and recombinant strains containing different mutants.

[0076] Figure 5 The fermentation process curve of ergosterol production by wild-type and recombinant bacteria expressing mutant F384E for 80 h. DETAILED DESCRIPTION

[0077] 1. Technical terms

[0078] As used herein, the terms "comprising", "including", "having", and "containing" mean "including but not limited to", "including but not limited to", "having but not limited to", and "containing but not limited to", and can be used interchangeably with the corresponding phrases. Unless the context clearly indicates otherwise, the term "or" used herein is used herein to mean the term "and / or" and can be used interchangeably with it. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, the examples of suitable methods and materials described below are illustrative only and do not limit the present invention in any way.

[0079] The term "fragment" refers to a section of a polynucleotide sequence. Generally, a gene fragment is a polynucleotide containing genetic information, which can encode a protein or RNA molecule, or participate in regulating gene expression. In some embodiments of the present invention, a gene fragment includes but is not limited to an exon encoding a protein, or a non-coding region, such as a promoter, enhancer, and other regulatory elements.

[0080] The term "expression" includes any step involved in the production of the enzyme or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0081] The term "mutant" means a polypeptide having lanosterol 14α-demethylase activity, comprising changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. The mutant of the present invention is based on the amino acid sequence of the polypeptide having SEQ ID NO: 1, and substitutions occur at positions 127, 260, 352, 314, and 384, respectively. The mutant of the present invention has at least 20% of the activity of lanosterol 14α-demethylase, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%.

[0082] In describing the mutants of the invention, the nomenclature described below has been adapted for ease of reference to employ the accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0083] For example:

[0084] A127S: Based on the wild type, the 127th alanine (A) is replaced by glycine (G).

[0085] A260K: Based on the wild type, the 260th alanine (A) is replaced by lysine (K).

[0086] K352L: Based on the wild type, the lysine (K) at position 352 was replaced by leucine (L).

[0087] G314V: Based on the wild type, glycine (G) at position 314 was replaced by valine (V).

[0088] G314W: Based on the wild type, glycine (G) at position 314 was replaced by tryptophan (W).

[0089] F384D: Based on the wild type, the proline (F) at position 384 is replaced by aspartic acid (D).

[0090] F384E: Based on the wild type, the proline (F) at position 384 is substituted with glutamic acid (E).

[0091] A127S / K352L: Based on the wild type, the 127th alanine (A) is replaced by glycine (G), and the 352nd lysine (K) is replaced by leucine (L).

[0092] A260K / K352L: Based on the wild type, the 260th alanine (A) is replaced by lysine (K), and the 352nd lysine (K) is replaced by leucine (L).

[0093] A127S / A260K: Based on the wild type, the 127th alanine (A) is replaced by glycine (G), and the 260th alanine (A) is replaced by lysine (K).

[0094] F384D / G314V: Based on the wild type, the proline (F) at position 384 is replaced by aspartic acid (D), and the glycine (G) at position 314 is replaced by valine (V).

[0095] F384D / G314W: Based on the wild type, the proline (F) at position 384 is replaced by aspartic acid (D), and the glycine (G) at position 314 is replaced by tryptophan (W).

[0096] F384E / G314V: Based on the wild type, the proline (F) at position 384 is replaced by glutamic acid (E), and the glycine (G) at position 314 is replaced by valine (V).

[0097] F384E / G314W: Based on the wild type, the proline (F) at position 384 is replaced by glutamic acid (E), and the glycine (G) at position 314 is replaced by tryptophan (W).

[0098] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid.

[0099] (ii) Culture medium:

[0100] LB medium: 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, add deionized water for sterilization, 121℃, 20min. Add 20g / L agar powder to prepare solid medium.

[0101] YNB medium: Yeast Nutrition Base 67.4 g / L, D(+)-anhydrous glucose 20 g / L, amino acids (5 g / L uracil, 10 g / L tryptophan, 10 g / L leucine, 10 g / L histidine, subtract the corresponding amino acids according to experimental needs).

[0102] YPD medium: tryptone 20 g / L, yeast extract 10 g / L, D(+)-anhydrous glucose 20 g / L.

[0103] Fermentation medium: D(+)-anhydrous glucose 40g / L, tryptone 20g / L, yeast extract 10g / L, MgSO41.5g / L, FeSO4·7H2O (1g added, sterilized by membrane, added to the fermenter at the time of inoculation), defoamer 0.1‰. 5moL / L NaOH was used to control the pH at 5.5±0.1 during the whole fermentation process.

[0104] Feed medium 1 (1.5L): trace element A 20mL / L, trace element B 24mL / L, (filter sterilized); D(+)-anhydrous glucose 800g / L, KH2PO4 18g / L, MgSO4·7H2O 10.24g / L, K2SO4 7g / L, Na2SO4 0.56g / L (sterilized at 115℃ for 15-20min); wherein,

[0105] Trace elements A (metal ions): ZnSO4·7H2O 5.75g / L, MnCl2·4H2O 0.32g / L, CoCl2·6H2O0.47g / L, NaMoO4·2H2O 0.48g / L, CaCl2·2H2O 2.9g / L, FeSO4·7H2O 2.8g / L, 0.5moL / LEDTA80mL / L (filter sterilize, store at 4℃).

[0106] Trace elements B (vitamins): biotin 0.05g / L, calcium pantothenate 1g / L, niacin 1g / L, Myo-inositol 25g / L, pyridoxal hydrochloride 1g / L, p-aminobenzoic acid 0.02g / L (filtered and sterilized, stored at 4°C).

[0107] Feed medium 2 (1 L): (NH4)2SO4 210 g / L, KH2PO4 65 g / L.

[0108] Tryptone and yeast extract were purchased from Shanghai Biotechnology Co., Ltd., and D(+)-anhydrous glucose was purchased from Sinopharm Group.

[0109] (III) Detection methods

[0110] Ergosterol extraction and determination:

[0111] 250 μL of fermentation broth was pipetted into a 2 mL broken tube (MP) containing 1 mL of saponification solution (11.5 g of potassium hydroxide dissolved in 30 mL of 90% ethanol), saponified at 88 °C for 4 h, transferred to a 5 mL EP tube, 600 μL of deionized water and 1980 μL of anhydrous ether were added respectively, and the mixture was shaken and extracted for 10 s. Ergosterol was extracted into the upper organic phase for detection.

[0112] Detection was performed using high performance liquid chromatography. Conditions: Chromatographic column: InertSustain C18 250mm×4.6mmcolumn (particle size 5μm); mobile phase B, methanol containing 1‰ trifluoroacetic acid; flow rate: 1mL / min; column temperature: 30℃; injection volume: 10μL; detector wavelength: ergosterol 280nm; running time 15min.

[0113] (IV) Strain information

[0114] The strain information is shown in Table 1. Saccharomyces cerevisiae WQ6 and its construction method refer to the patent application document with publication number CN114606149A.

[0115] Table 1 Strains involved in the present invention

[0116]

[0117] Table 2 Primer sequences for constructing strains

[0118]

[0119] Table 3 Primer sequences for introducing mutations

[0120]

[0121] Example 1: Construction of expression vector and strain of wild-type Saccharomyces cerevisiae lanosterol 14α-demethylase gene

[0122] The Saccharomyces cerevisiae strain WQ6 (the strain and construction method are disclosed in the patent application document with publication number CN114606149A) was used as the starting strain, and the ERG11 site of the strain was used as the integration site of the ERG11 gene with a uracil amino acid screening tag. The Saccharomyces cerevisiae CENPK2-1D genome was used as a template, and the upstream 500bp homology arm ERG11UP of the target gene ERG11 was amplified using primers UP-F / UP-R, and the promoter P was amplified using primers ADH1p-F / ADH1p-R. ADH1 (nucleotide sequence as shown in SEQ ID NO.10), uracil amino acid screening tag URA was amplified using primers URA-F / URA-R (nucleotide sequence as shown in SEQ ID NO.11), terminator TEFt-F / TEFt-R was amplified using primers TEF (The nucleotide sequence is shown in SEQ ID NO.12), and the promoter P was amplified using primers TEFp-F / TEFp-R. TEF(nucleotide sequence as shown in SEQ ID NO.13), primers ERG11-F / ERG11-R were used to amplify the target gene ERG11 (nucleotide sequence as shown in SEQ ID NO.2), primers DOWN-F / DOWN-R were used to amplify the downstream 500bp homology arm ERG11DOWN of the target gene ERG11; using pY26 plasmid as template, primers KJ-F / KJ-R were used to amplify the pY26 vector integration fragment (nucleotide sequence as shown in SEQ ID NO.14). ADH1 (nucleotide sequence as shown in SEQ ID NO.10), URA (nucleotide sequence as shown in SEQ ID NO.11), T TEF (nucleotide sequence as shown in SEQ ID NO.12) was fused into fragment 1; TEF (nucleotide sequence as shown in SEQ ID NO.13), ERG11 (nucleotide sequence as shown in SEQ ID NO.2), and ERG11DOWN were fused into fragment 2. Then, the fragment 1, fragment 2 and pY26 vector integration fragment were assembled by Gibson assembly to construct plasmid pY26-ERG11 (as shown in Figure 2 ). The constructed plasmid pY26-ERG11 was used as a template to amplify the plasmid containing ERG11UP, P ADH1 、URA、T TEF , P TEF , ERG11, and ERG11DOWN recombinant plasmid fragments, and transforming the recombinant fragments into the competent cells of Saccharomyces cerevisiae WQ-6 strain. The specific steps are as follows:

[0123] Pick a full single colony of Saccharomyces cerevisiae and inoculate it into 5mL of YPD liquid medium, and culture it in a 50mL shake flask at 30℃ and 220rpm for 22h; aspirate 500μL of the above seed solution into 50mL of YPD medium, and culture it at 30℃ and 220rpm for 5h. Transfer it to a 50mL centrifuge tube in the clean bench and place it on ice for 5min. Centrifuge it at 3500rpm and 4℃ for 5min, and discard the supernatant. Wash it with 25mL of sterile water, centrifuge it at 3500rpm and 4℃ for 5min, and discard the supernatant; add 1mL of 0.1 M lithium acetate and resuspend the bacteria in a 1.5mL sterile EP tube. Centrifuge it at 4℃ and 3500rpm for 2.5min, and discard the supernatant. Resuspend the cells with 400μL of pre-cooled 0.1M lithium acetate; centrifuge at 4℃, 3500rpm, 2.5min, discard the supernatant, add 240μL, 50% PEG3350 (m / V), 36μL 1M lithium acetate, 2ng-3ng homologous recombination fragment. Oscillate for 10s, incubate at 30℃ for 50min. Water bath at 42℃ for 25min; centrifuge at 3500rpm, 2.5min, discard the supernatant; resuspend the cells with 200μL of sterile water; spread 70μL of the bacterial solution on YNB medium, culture at 30℃ for 3-4d, and the obtained Saccharomyces cerevisiae strain was named WT.

[0124] Example 2: Preparation of Lanosterol 14α-demethylase Single Mutant

[0125] Using the plasmid pY26-ERG11 constructed in Example 1 as a template, primers for introducing A127S, A260K, K352L, G314V, G314W, F384D, and F384E mutations were designed and synthesized, and site-directed mutagenesis was performed on the template by PCR linearization. After the PCR product was verified to be correct, it was digested with Dpn I at 37°C for 1h and transformed into Escherichia coli JM109 competent cells. The clones were selected for sequencing to confirm whether the coding gene of the lanosterol 14α-demethylase single mutant was correct, and the verified correct gene was the ERG11 single mutant gene; wherein the nucleotide sequence encoding mutant A127S is shown in SEQ ID NO.3; the nucleotide sequence encoding mutant A260K is shown in SEQ ID NO.4; the nucleotide sequence encoding mutant K352L is shown in SEQ ID NO.5; the nucleotide sequence encoding mutant G314V is shown in SEQ ID NO.6; the nucleotide sequence encoding mutant G314W is shown in SEQ ID NO.7. The nucleotide sequence encoding mutant F384D is shown in SEQ ID NO.7; the nucleotide sequence encoding mutant F384D is shown in SEQ ID NO.8; the nucleotide sequence encoding mutant F384E is shown in SEQ ID NO.9.

[0126] Example 3: Construction of a strain expressing a single mutant of lanosterol 14α-demethylase

[0127] According to the method of Example 1, the plasmid pY26-ERG11 constructed in Example 1 was used as a template, and primers ZHUP-F / ZHDW-R were used to amplify the plasmid containing ERG11UP, P ADH1 (nucleotide sequence as shown in SEQ ID NO.10), URA (nucleotide sequence as shown in SEQ ID NO.11), T TEF (nucleotide sequence is shown in SEQ ID NO.12), P TEF (nucleotide sequence as shown in SEQ ID NO.13), the ERG11 single mutant gene prepared in Example 2, and the recombinant plasmid fragment of ERG11DOWN were transformed into strain WQ6 according to the method of Example 1. Saccharomyces cerevisiae strains expressing single mutants A127S, A260K, K352L, G314V, G314W, F384D, and F384E were constructed respectively.

[0128] Example 4: Preparation of lanosterol 14α-demethylase double mutant

[0129] Using the plasmid containing mutants V249A, P342A, and L343A constructed in Example 3 as a template, primers for introducing A127S / K352L, A260K / K352L, A127S / A260K, F384D / G314V, F384D / G314W, F384E / G314V, and F384E / G314W mutants were designed and synthesized. The template was linearized by PCR for site-directed mutagenesis, and the PCR product was transformed into Escherichia coli JM109 competent state after being verified to be correct after being digested with Dpn I at 37°C for 1h, and clones were selected for sequencing to confirm whether the coding gene of the squalene epoxidase double mutant was correct, and the correct gene was verified to be the ERG11 double mutant gene (shown in Table 4).

[0130] Table 4 Double mutant gene sequences

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] Example 5: Preparation of lanosterol 14α-demethylase double mutant

[0138] The constructed plasmid pY26-ERG11 was used as a template to amplify the plasmid containing ERG11UP, P ADH1 (nucleotide sequence as shown in SEQ ID NO.10), URA (nucleotide sequence as shown in SEQ ID NO.11), T TEF (nucleotide sequence is shown in SEQ ID NO.12), P TEF (nucleotide sequence as shown in SEQ ID NO.13), the ERG11 double mutant gene prepared in Example 4 (nucleotide sequences are shown in Table 4), and the recombinant plasmid fragment of ERG11DOWN were transformed into strain WQ6 according to the method of Example 1. The constructed double mutants were named A127S / K352L, A260K / K352L, A127S / A260K, F384D / G314V, F384D / G314W, F384E / G314V, and F384E / G314W.

[0139] Example 6: Fermentation of lanosterol 14α-demethylase and its mutants for the production of ergosterol

[0140] The strains constructed in Example 1, Example 3, and Example 5 were inoculated into a shake flask containing 20 g / L tryptone, 10 g / L yeast extract, and 20 g / L D (+)-anhydrous glucose in 25 mL YPD medium, and 25 μL 25 g / L chloramphenicol was added at the same time. The mixture was shaken at 30°C and 220 rpm for 72 h. 250 μL of the fermentation broth was collected for saponification and ether extraction to detect the ergosterol yield.

[0141] The results showed that the ergosterol production of the WT strain reached 490.0 mg / L, and the ergosterol production of the strains expressing the mutations A127S, A260K, K352L, G314V, G314W, F384D, F384E, F384D / G314W, and F384E / G314V in shake flask fermentation reached 645.0 mg / L, 625.8 mg / L, 677.9 mg / L, and 62 2.7mg / L, 620.1mg / L, 682.8mg / L, 733.8mg / L, 623.9mg / L, and 536.1mg / L, which were respectively 31.6%, 27.7%, 38.3%, 27.1%, 26.6%, 39.3%, 49.8%, 27.3%, and 9.4% higher than the 490.0mg / L produced by the strain expressing the wild-type lanosterol 14α-demethylase.

[0142] Example 7: Lanosterol 14α-demethylase and its mutants were used for the production of ergosterol in a 5L fermenter

[0143] The strains constructed in Example 1, Example 3, and Example 5 and the starting strain WQ6 were inoculated into a shake flask containing 20 g / L tryptone, 10 g / L yeast extract, and 20 g / LD (+)-anhydrous glucose in 1.5 mL YPD medium, and cultured at 30°C and 220 rpm for 19 h as the primary seed liquid. 1% of the primary seed liquid was inoculated into a shake flask containing 200 mL of the same YPD medium, and cultured at 30°C and 220 rpm for 15 h as the secondary seed liquid. The secondary seed liquid was inoculated into a 5L fermentation tank containing 2.5L fermentation medium, and fermented at 30°C for 96 h. When the residual sugar detection value was 0, feed medium 1 was started to control the residual sugar in the range of 0.5 g / L to 1.5 g / L, and the ethanol content was between 5 g / L and 10 g / L. After fermentation for 35 h to 37 h, feed medium 2 was added, and the feed rate was 8 mL / h to 16 mL / h until the fermentation was completed. 5 mL of sample was taken at intervals of 8 to 12 h for saponification and ether extraction to detect ergosterol production.

[0144] The results show that Figure 5 ), the ergosterol production of the recombinant bacteria expressing mutant F384E after 71 h of fermentation could reach 3067.5 mg / L, which was 31.08% higher than the highest production (2340.1 mg / L) of the strain expressing wild-type ERG11.

[0145] Example 8 Method for producing ergosterol by fermentation of recombinant bacteria in different fermentation systems

[0146] This example is used to describe the method for producing ergosterol by fermentation with a recombinant strain:

[0147] (1) Shake flask fermentation of ergosterol:

[0148] Pick a single full colony and culture it in 25 mL YPD medium in a 250 mL shake flask at 30°C and 220 rpm for 72 h.

[0149] (2) Ergosterol fermentation in well plate:

[0150] A single full colony was picked and cultured in 1.5 mL YPD medium in a 48-well round-bottom plate at 30°C and 220 rpm for 72 h.

[0151] (3) Fermentation of ergosterol in a 5L fermentation tank:

[0152] Use a sterile inoculation loop to pick up a full single yeast colony and inoculate it into a shake flask containing 5mL YPD, and culture it at 220rpm, 30℃ for 17-20h as the primary seed liquid. Inoculate the primary seed liquid at an inoculation rate of 1% into a 500mL shake flask containing 200mL YPD, and culture it at 220rpm, 30℃ for 17-20h as the secondary seed liquid, which will be inoculated into a 5L fermentation tank. When the residual sugar detection is 0, start adding feed medium 1, control the residual sugar in the range of 0.5g / L to 1.5g / L, and the ethanol content is between 5g / L and 10g / L. Ferment for 35h to 37h, add feed medium 2, and the feeding rate is 8mL / h to 16mL / h until the end of fermentation. Comparative example:

[0153] Specific implementation method The same as Examples 2 to 6, except that the recombinant bacteria expressing L359D, H392P, I428P, V441D, L483I, H494R, L380A / G314A, and F384A / Y126A were constructed using the primers shown in Table 5. The results showed that the ergosterol production of the recombinant bacteria expressing the above mutations after fermentation for 72 hours according to the method of Example 4 was 404.6 mg / L, 336.7 mg / L, 306.6 mg / L, 340.7 mg / L, 313.1 mg / L, 276.0 mg / L, 439.7 mg / L, and 457.2 mg / L, respectively, which were 17.4%, 31.3%, 37.4%, 30.5%, 36.1%, 43.7%, 10.3%, and 6.7% lower than those of the strain expressing the wild-type lanosterol 14α-demethylase, respectively.

[0154] Table 5 Primer sequences for introducing mutations

[0155]

[0156]

[0157] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Lanosterol 14α-demethylase mutant, It is characterized in that The invention relates to a mutant obtained by mutating one or more amino acids at positions 127, 260, 352, 314 or 384 of lanosterol 14α-demethylase derived from Saccharomyces cerevisiae. The mutant has improved catalytic efficiency compared with its parent lanosterol 14α-demethylase.

2. The mutant according to claim 1, It is characterized in that The lanosterol 14α-demethylase derived from Saccharomyces cerevisiae contains the amino acids shown in SEQ ID NO.

1.

3. The mutant according to claim 1 or 2, It is characterized in that Based on the lanosterol 14α-demethylase from Saccharomyces cerevisiae, the invention has one or more of the following mutations: (1) Mutate alanine at position 127 to serine; (2) mutating alanine at position 260 to lysine; (3) mutating lysine at position 352 to leucine; (4) mutating glycine at position 314 to valine; (5) mutating glycine at position 314 to tryptophan; (6) mutating phenylalanine at position 384 to aspartic acid; (7) Mutate phenylalanine at position 384 to glutamic acid.

4. A gene encoding the lanosterol 14α-demethylase mutant according to any one of claims 1 to 3.

5. A plasmid carrying the gene according to claim 4.

6. A recombinant microbial cell expressing the Saccharomyces cerevisiae lanosterol 14α-demethylase mutant according to any one of claims 1 to 3.

7. An engineered strain of Saccharomyces cerevisiae, It is characterized in that Expressing the lanosterol 14α-demethylase mutant according to any one of claims 1 to 3.

8. The engineered yeast of claim 7, It is characterized in that The mutant according to any one of claims 1 to 3 is expressed in Saccharomyces cerevisiae having the ability to synthesize ergosterol.

9. The engineered yeast of claim 7 or 8, It is characterized in that The Saccharomyces cerevisiae includes but is not limited to Saccharomyces cerevisiae WQ6.

10. A method for increasing the ergosterol production of Saccharomyces cerevisiae. It is characterized in that One or more positions of the amino acid sequence of lanosterol 14α-demethylase derived from Saccharomyces cerevisiae are mutated at position 127, position 260, position 352, position 314 or position 384.

11. The method according to claim 10, It is characterized in that The mutation is any of the following: (1) mutating alanine at position 127 to serine; or (2) mutating alanine at position 260 to lysine; or (3) mutating lysine at position 352 to leucine; or (4) mutating glycine at position 314 to valine; or (5) mutating glycine at position 314 to tryptophan; or (6) mutating the phenylalanine at position 384 to aspartic acid; or (7) mutating phenylalanine at position 384 to glutamic acid; or (8) mutating alanine at position 127 to serine and lysine at position 352 to leucine; or (9) mutating alanine at position 260 to lysine and lysine at position 352 to leucine; or (10) mutating the alanine at position 127 to serine and mutating the alanine at position 260 to lysine; or (11) mutating the phenylalanine at position 384 to aspartic acid and the glycine at position 314 to valine; or (12) mutating the phenylalanine at position 384 to aspartic acid and the glycine at position 314 to tryptophan; or (13) mutating the phenylalanine at position 384 to glutamic acid and the glycine at position 314 to valine; or (14) The phenylalanine at position 384 was mutated to glutamic acid, and the glycine at position 314 was mutated to tryptophan.

12. A method for preparing ergosterol, It is characterized in that The recombinant microbial cell according to claim 6 or the engineered yeast of Saccharomyces cerevisiae according to any one of claims 7 to 9 is fermented in a culture medium for a period of time.

13. The method according to claim 12, It is characterized in that The culture medium contains carbon sources, nitrogen sources and inorganic salts required for the growth of Saccharomyces cerevisiae; optionally, feed supplement is performed during the fermentation process.

14. The method according to claim 13, It is characterized in that Glucose and trace elements are also fed during the fermentation process to control the residual sugar in the fermentation system to 0.5g / L-1.5g / L and the ethanol content to 5g / L-10g / L; the trace elements include but are not limited to metal ions and / or vitamins.

15. The mutant according to any one of claims 1 to 3, or the microbial cell according to claim 6, or the engineered yeast of Saccharomyces cerevisiae according to any one of claims 7 to 9, or the use of any one of claims 10 to 14 in the food, biomedicine, chemical, cosmetics or feed industries.

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