Cholesterol 7-dehydrogenase and method for synthesizing 7-dehydrocholesterol by using cholesterol 7-dehydrogenase

Abstract

The invention belongs to the field of bioengineering, and particularly relates to cholesterol 7-dehydrogenase and a method for synthesizing 7-dehydrocholesterol by using the cholesterol 7-dehydrogenase. The invention provides a cholesterol dehydrogenase, and also provides a method for synthesizing 7-dehydrocholesterol by using the cholesterol dehydrogenase. By means of high-efficiency cholesterol 7-dehydrogenase evolved by using enzyme engineering and optimized reaction conditions, dependence on expensive chemical reagents is reduced, meanwhile, energy consumption is reduced, the production cycle is shortened, and the overall production efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to cholesterol 7-dehydrogenase and a method for synthesizing 7-dehydrocholesterol by using the same. Background Art

[0002] 7-Dehydrocholesterol (7-DHC) is a precursor of cholesterol in chemical structure. It is converted into vitamin D3 in the human body through ultraviolet radiation and is essential for maintaining bone health and immune system function. This compound is not only of great biological significance, but also of great commercial value, especially as a key raw material for the production of vitamin D3. The wide application of vitamin D3, including food additives, feed additives and applications in the medical field, has led to an increasing demand for 7-dehydrocholesterol.

[0003] Traditionally, the production of 7-dehydrocholesterol mainly relies on the extraction of cholesterol from lanolin and the subsequent conversion of cholesterol to 7-dehydrocholesterol by chemical or photochemical methods. Specifically, in the field of chemical synthesis, the production of 7-dehydrocholesterol mainly relies on the precise modification of cholesterol molecules. This process involves multi-step chemical reactions, including the selective introduction of double bonds to form 7-dehydrocholesterol. The challenge of this method lies in the control of reaction conditions, such as temperature, pressure, reaction time, and the choice of catalyst, which all need to be precisely controlled to ensure high yield and product purity. In addition, the by-products that may be produced during the chemical synthesis process need to be removed through subsequent purification steps, which not only increases production costs but also imposes a burden on the environment.

[0004] Photochemical method is another method for producing 7-dehydrocholesterol, which uses ultraviolet light to irradiate cholesterol to induce chemical changes inside the molecule to form 7-dehydrocholesterol. The advantages of this method are simple operation and avoidance of the use of toxic chemical reagents. However, the conversion efficiency of the photochemical method is relatively low, and there are strict requirements on the wavelength and intensity of the light source. In addition, ultraviolet irradiation may cause other undesirable side reactions, affecting the purity and yield of the product.

[0005] Although these methods are feasible, they are often accompanied by problems such as high cost, low efficiency, many by-products and environmental pollution. In modern production, despite technological advances, there are still challenges in terms of environmental friendliness, cost-effectiveness and production efficiency.

[0006] In recent years, microbial transformation has attracted widespread attention as an emerging production method that uses engineered microorganisms to convert cholesterol into 7-dehydrocholesterol. The advantage of this method is that it is relatively environmentally friendly and can optimize microbial strains through genetic engineering technology to improve conversion rate and product specificity. The challenges of microbial transformation mainly lie in the construction and optimization of strains, which requires a deep understanding of the biological pathway for the conversion of cholesterol to 7-dehydrocholesterol and how to effectively enhance the activity of this pathway. There are currently no related research reports. Summary of the invention

[0007] The present invention discovers and identifies a new cholesterol 7-dehydrogenase, and provides a process route for obtaining 7-dehydrocholesterol from cholesterol by a one-step enzymatic method in vitro. The present invention reduces the dependence on expensive chemical reagents, reduces energy consumption, shortens the production cycle, and improves overall production efficiency by using a highly efficient cholesterol 7-dehydrogenase evolved by enzyme engineering and optimized reaction conditions.

[0008] In order to achieve the above technical objectives, the present invention hereby proposes the following technical solutions:

[0009] In a first aspect, the present invention provides a cholesterol dehydrogenase (cholesterol 7-dehydrogenase) having an amino acid sequence as shown in SEQ ID NO: 1, or having at least one amino acid difference with the amino acid sequence as shown in SEQ ID NO: 1.

[0010] The present invention screened and identified a new cholesterol dehydrogenase, which has catalytic activity on cholesterol and can convert cholesterol into 7-dehydrocholesterol. The inventor carried out enzyme engineering evolution based on this cholesterol dehydrogenase and obtained a series of cholesterol dehydrogenases with enhanced or improved catalytic performance.

[0011] In some embodiments, the cholesterol dehydrogenase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0012] In some embodiments, the cholesterol dehydrogenase has improved enzyme activity compared to the cholesterol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:1.

[0013] In some preferred embodiments, the cholesterol dehydrogenase has an enzyme activity that is increased by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 10 ... %, at least about 100% or more, at least about 150% or more, at least about 200% or more, at least about 250% or more, at least about 300% or more, at least about 350% or more, at least about 400% or more, at least about 450% or more, at least about 500% or more, at least about 550% or more, at least about 600% or more, at least about 650% or more, at least about 700% or more, at least about 750% or more, at least about 800% or more, at least about 850% or more, at least about 900% or more, at least about 950% or more, or at least about 1000% or more.

[0014] In some preferred embodiments, the cholesterol dehydrogenase has an enzyme activity increased by about 500%, about 600%, about 700%, about 800%, about 900% or about 1000% compared to the cholesterol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:1.

[0015] In some embodiments, the amino acid differences are 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 1-5, and more preferably 3.

[0016] In some embodiments, the amino acid differences are amino acid substitutions.

[0017] In some preferred embodiments, the amino acid substitution includes the amino acids at the following positions:

[0018] 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 20th, 256th, 313th.

[0019] In some preferred embodiments, the amino acid substitution site is selected from the 4th, 6th and 313th amino acids.

[0020] In some preferred embodiments, the amino acid substitution of the amino acid at position 4 is selected from Y4A, Y4R, Y4Q, Y4T, Y4V or Y4W.

[0021] In some preferred embodiments, the amino acid substitution of the amino acid at position 4 is Y4A.

[0022] In some preferred embodiments, the amino acid substitution of the amino acid at position 6 is selected from L6R, L6D, L6C or L6T.

[0023] In some preferred embodiments, the amino acid substitution of the amino acid at position 6 is L6R.

[0024] In some preferred embodiments, the amino acid substitution at amino acid position 313 is selected from Y313L, Y313N, Y313I, Y313A, Y313K, Y313R, Y313T, Y313H or Y313C.

[0025] In some preferred embodiments, the amino acid substitution at amino acid position 313 is Y313I.

[0026] In some embodiments, the cholesterol dehydrogenase has the amino acid sequence shown in SEQ ID NO:2.

[0027] SEQ ID NO: 1:

[0028] MTLYKLTMLTQQATDRTDRCWRPPRTLKSMPPVFPNGWIPVLESHQLCTGQVKSLDVLGLHLVAFRSEDGEAHVMDAHCPHLGAHMGHMGRVVGDCIECPFHGWRFRGKDGACTHVPYSAKVPEFIRAKTWLSCELLGLLFIWYHAEDEPPSWHLVDCPEISSGQWKVERRFEHTVHCHIQDIAENGA DVGHFNKLHKASCLMTSDQFAKTGGLCWWGQLATHSWDAKWTSNGHAASVKVDTSVSLLGFSPDFLKQHVDVRQVGPALVILHMKGRHGDTLIVQALIPEGPLRIRVLHSFFPEPGQPWLMRWIYVAGFRSMVERDIAIWNQKTYLKQPCLVKEERSIAAFRKWYSQFYSTNSPTWQDVRDQSLEW*.

[0029] SEQ ID NO:2:

[0030] MTLAKRTMLTQQATDRTDRCWRPPRTLKSMPPVFPNGWIPVLESHQLCTGQVKSLDVLGLHLVAFRSEDGEAHVMDAHCPHLGAHMGHMGRVVGDCIECPFHGWRFRGKDGACTHVPYSAKVPEFIRAKTWLSCELLGLLFIWYHAEDEPPSWHLVDCPEISSGQWKVERRFEHTVHCHIQDIAENGA DVGHFNKLHKASCLMTSDQFAKTGGLCWWGQLATHSWDAKWTSNGHAASVKVDTSVSLLGFSPDFLKQHVDVRQVGPALVILHMKGRHGDTLIVQALIPEGPLRIRVLHSFFPEPGQPWLMRWIIVAGFRSMVERDIAIWNQKTYLKQPCLVKEERSIAAFRKWYSQFYSTNSPTWQDVRDQSLEW*.

[0031] In some embodiments, the cholesterol dehydrogenase is further linked to a second polypeptide.

[0032] In some embodiments, the second polypeptide comprises a tag polypeptide.

[0033] In some embodiments, the tag polypeptide includes, but is not limited to: a recruitment tag polypeptide, a fluorescent protein polypeptide, a luminescent polypeptide, an influenza hemagglutinin tag polypeptide, a c-myc tag polypeptide, a herpes simplex virus glycoprotein D (gD) tag polypeptide, a polyhistidine tag polypeptide, a FLAG tag polypeptide, a KT3 epitope tag polypeptide, a tubulin epitope tag polypeptide, a T7 gene 10 protein tag polypeptide, a streptavidin tag polypeptide, a vesicular ostomy virus glycoprotein (VSV-G) epitope tag polypeptide, a small epitope (Pk) found on the P and V proteins of the simian virus paramyxovirus 5 (V5) tag polypeptide, an alkaline phosphatase (AP) tag polypeptide, a bluetongue virus tag (B-tag) polypeptide, a calmodulin binding peptide (CalBP) tag polypeptide, a chloramphenicol acetyltransferase (CACT) T) tag polypeptide, choline binding domain (CholBD) tag polypeptide, chitin binding domain (ChitBD) tag polypeptide, cellulose binding domain (CellBP) tag polypeptide, dihydrofolate reductase (DHFR) tag polypeptide, galactose binding protein (GBP) tag polypeptide, maltose binding protein (MBP) polypeptide, glutathione-S-transferase (GST) polypeptide, Glu-Glu (EE) tag polypeptide, human influenza hemagglutinin (HA) tag polypeptide, horseradish peroxidase (HRP) tag polypeptide, NE-tag polypeptide, HSV tag polypeptide, ketosteroid isomerase (KSI) tag, LacZ tag polypeptide, NusA tag polypeptide, PDZ domain tag polypeptide, AviTag polypeptide, SBP-tag polypeptide, Softag 1 peptide, Softag 3 peptide, TC tag peptide, VSV-tag peptide, Xpress tag peptide, Isopeptag peptide, SpyTag peptide, SnoopTag peptide, Profinity eXact tag peptide, Protein C tag peptide, 51-tag peptide, S-tag peptide, biotin-carboxyl carrier protein (BCCP) tag peptide, small ubiquitin-like modifier (SUMO) tag peptide, tandem affinity purification (TAP) tag peptide, HaloTag peptide, Nus-tag peptide, thioredoxin-tag peptide, CYD tag peptide, HPC tag peptide, TrpE tag peptide or ubiquitin tag peptide, etc.

[0034] In some preferred embodiments, the second polypeptide is a recruitment tag polypeptide, which recruits a reductase partner to approach or bind to the cholesterol dehydrogenase.

[0035] In some preferred embodiments, the recruitment tag polypeptide comprises a RIAD tag.

[0036] In some preferred embodiments, the RIAD tag has an amino acid sequence as shown in SEQ ID NO:3.

[0037] SEQ ID NO:3:

[0038] GGGGSGGGGSGGGGCGLEQYANQLADQIIKEATEGC.

[0039] In some preferred embodiments, the reductase partner comprises a RIDD tag sequence.

[0040] In some preferred embodiments, the N-terminus of the reductase partner comprises a RIDD tag sequence.

[0041] In some preferred embodiments, the RIDD tag has an amino acid sequence as shown in SEQ ID NO:4.

[0042] SEQ ID NO:4:

[0043] GGGGSGGGGSGGGGCGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYF ERLEKEEAK.

[0044] In some embodiments, the reductase chaperone comprises TsaB, VanB, or KshB.

[0045] In some preferred embodiments, the reductase partner is KshB.

[0046] The amino acid sequence of KshB is shown in SEQ ID NO:5.

[0047] The amino acid sequence of VanB is shown in SEQ ID NO:6.

[0048] The amino acid sequence of TsaB is shown in SEQ ID NO:7.

[0049] SEQ ID NO:5:

[0050] MTEAIGDEPLGDHVLELQIAEVVDETDEARSLVFAVPDGSDDPEIPPRRLRYAPGQFLTLRVPSERTGSVARCYSLCSSPYTDDALAVTVKRTADGYASNWLCDHAQVGMRIHVLAPSGNFVPTTLDADFLLLAAGSGITPIMSICKSALAEGGGQVTLLYANRDDRSVIFGDALRELAAKYPDRLTVLHWLESLQGLPSASALAKLVAPYTDRPVFICGPGPFMQAARDALAALKVPAQQVHIEVFKSLESDPFAAVKVDDSGDEAPATAVVELDGQTHTVSWPRTAKLLDVLLAAGLDAPFSCREGHCGACACTLRAGKVNMGVNDVLEQQDLDEGLILACQSRPESDSVEVTYDE。

[0051] SEQ ID NO:6:

[0052] MIEVIVGAIRLEAQDIHSFELFRADGAALPSFEPGAHIDLHLPNGLVRQYSLCGPAERPRHYRIAVLRCRDSRGGSATLHAELRVGQRLHIGEPRNLFPLSPEPGPHLLFAGGIGITPLLAMAERLARDGADFQLHYCAHSGERAAFVDYLGRCAFADRVHCHFDHGESSRRADLRALLATSPRDAQLYLCGPAGFMQWIEESARELGWEASRLHREHFAAAPRDASADGTFEVQLASNGALIRVAAGQTVLAALREAGVDLPASCEQGICGTCLTRVLDGEPEHRDLYLSEEEQAANDCFTPCCSRSRSPRLVLDL。

[0053] SEQ ID NO:7:

[0054] MSADVPVTVAAVRAVARDVLALELRHANGQPLPGASAGAHIDLALPNGLVRQYSLVNATGQATMDCYQVAVGWDANSRGGSVWIHEKLKVGQALRVTHRATCSEMAPEHRRVLLLAGGIGVTPIYAMAQACAQQGVDVELWASARSAPRLAYLEELKAL LGQRLHLHADDEQGGPMNLTERLATQRWDAVYACGPAPMLDALTAATAHWAPGSVRMERFKGAEQPASERQPFELVLQRAGLSTTVDAHESVLDAMERVGVDFPWSCREGICGTCEAPVLEGEVQHLDYVLSPEERAEQRRMMVCVSRCGGGRLVLDI.

[0055] In some embodiments, the C-terminus of the cholesterol dehydrogenase is linked to the N-terminus of the second polypeptide.

[0056] In some embodiments, the cholesterol dehydrogenase and the second polypeptide are linked directly or indirectly via a linker.

[0057] In some embodiments, the cholesterol dehydrogenase comprises an amino acid modification.

[0058] In some embodiments, the amino acid modification can improve the lipid solubility, water solubility, biocompatibility, hydrophilicity, stability, biological activity of cholesterol dehydrogenase, and / or protect cholesterol dehydrogenase from hydrolysis and / or enzymatic degradation.

[0059] In a second aspect, the present invention provides a polynucleotide comprising a nucleic acid encoding any of the aforementioned cholesterol dehydrogenases.

[0060] In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid.

[0061] In a third aspect, the present invention provides a vector comprising any of the aforementioned polynucleotides.

[0062] In some embodiments, the vector comprises an expression control element operably linked to the nucleic acid.

[0063] In some embodiments, the expression control element comprises a promoter, an enhancer and / or a terminator.

[0064] In some embodiments, the vector comprises an expression vector, a cloning vector, a shuttle vector, or a viral vector.

[0065] In a fourth aspect, the present invention provides a host cell comprising any one of the aforementioned polynucleotides or vectors.

[0066] In some embodiments, the host cell includes a eukaryotic cell and / or a prokaryotic cell.

[0067] In some preferred embodiments, the eukaryotic cells include yeast cells, animal cells and / or insect cells.

[0068] In some preferred embodiments, the prokaryotic cells include Escherichia coli, Rhodococcus erythrorhizium, Bacillus subtilis and / or yeast.

[0069] In a fifth aspect, the present invention provides a composition comprising one or more of any of the aforementioned cholesterol dehydrogenases, polynucleotides, vectors and host cells.

[0070] In some embodiments, the composition further comprises a co-solvent for solubilizing cholesterol, such as Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof.

[0071] In some preferred embodiments, the cosolvent is Tween 80.

[0072] In some embodiments, the composition further includes a reductase chaperone that is proximal to or associated with the cholesterol 7-dehydrogenase.

[0073] In some embodiments, the reductase partner comprises TsaB, VanB or KshB, preferably KshB.

[0074] In some preferred embodiments, the reductase partner comprises a RIDD tag.

[0075] In some preferred embodiments, the N-terminus of the reductase partner comprises a RIDD tag.

[0076] In some preferred embodiments, the RIDD tag has an amino acid sequence as shown in SEQ ID NO:4.

[0077] In some preferred embodiments, the composition further comprises an additive that promotes the cholesterol-catalyzed reaction, such as cyclodextrin or a derivative thereof, preferably β-cyclodextrin or hydroxypropyl-β-cyclodextrin, more preferably hydroxypropyl-β-cyclodextrin.

[0078] In some preferred embodiments, the composition further comprises NADH, glucose and glucose dehydrogenase.

[0079] In a sixth aspect, the present invention provides use of any of the aforementioned cholesterol dehydrogenase polynucleotides, vectors, host cells and / or compositions in the synthesis of 7-dehydrocholesterol.

[0080] In some embodiments, the use is to synthesize 7-dehydrocholesterol in one step using cholesterol as a substrate.

[0081] In a seventh aspect, the present invention provides use of any of the aforementioned cholesterol dehydrogenase polynucleotides, vectors, host cells and / or compositions in the preparation of a product for synthesizing 7-dehydrocholesterol.

[0082] In some embodiments, the use is to synthesize 7-dehydrocholesterol in one step using cholesterol as a substrate.

[0083] In some embodiments, the product comprises a reagent or a kit.

[0084] In an eighth aspect, the present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0085] Under suitable reaction conditions, any of the above cholesterol dehydrogenases is brought into contact with cholesterol to undergo a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

[0086] In a ninth aspect, the present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0087] (I) culturing any of the aforementioned host cells under suitable culture conditions to express any of the aforementioned cholesterol dehydrogenases;

[0088] (II) The host cells are resuspended in a buffer solution and contacted with cholesterol to undergo a catalytic reaction, thereby obtaining 7-dehydrocholesterol.

[0089] In some embodiments, said (II) further comprises the following conditions:

[0090] A reaction additive is added to the catalytic reaction.

[0091] In some preferred embodiments, the reaction additive includes cyclodextrin or its derivatives, such as β-cyclodextrin (bCD) and hydroxypropyl-β-cyclodextrin (HPbCD).

[0092] In some embodiments, said (II) further comprises the following conditions:

[0093] NADH, glucose and glucose dehydrogenase (GDH) are added to the catalytic reaction.

[0094] In some embodiments, said (II) further comprises the following conditions:

[0095] A cosolvent is added in the catalytic reaction, and the cosolvent includes Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof, preferably Tween 80, and more preferably 10% Tween 80.

[0096] In some embodiments, said (II) further comprises the following conditions:

[0097] Polymyxin B (PMBS) was added to the catalytic reaction.

[0098] In a tenth aspect, the present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0099] (i) freeze-drying any of the above cholesterol dehydrogenases to obtain a lyophilized powder;

[0100] (ii) under conditions suitable for the reaction, the lyophilized powder is contacted with the lyophilized powder of the reductase partner and cholesterol to carry out a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

[0101] In some embodiments, the (ii) further comprises the following conditions:

[0102] A reaction additive is added to the catalytic reaction.

[0103] In some preferred embodiments, the reaction additive includes cyclodextrin or a derivative thereof, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin.

[0104] In some embodiments, the (ii) further comprises the following conditions:

[0105] NADH, glucose and glucose dehydrogenase (GDH) are added to the catalytic reaction.

[0106] In some embodiments, the (ii) further comprises the following conditions:

[0107] An auxiliary solvent is added in the catalytic reaction, and the auxiliary solvent includes Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof, preferably Tween 80, and more preferably 10% Tween 80.

[0108] In some embodiments, the reductase chaperone comprises TsaB, VanB, or KshB.

[0109] In some preferred embodiments, the reductase partner is KshB.

[0110] The present invention proposes a method for efficiently synthesizing 7-dehydrocholesterol from cholesterol by an in vitro one-step method using cholesterol 7-dehydrogenase. The key to this method is to use a newly identified cholesterol 7-dehydrogenase, and through enzyme engineering evolution and carefully designed enzymatic reactions, a cholesterol 7-dehydrogenase mutant is obtained to achieve efficient conversion of cholesterol to 7-dehydrocholesterol. The enzyme activity is increased by about 9 times compared with the wild-type cholesterol 7-dehydrogenase, and the final yield of 7-dehydrocholesterol can reach more than 2g / L. The present invention reduces the dependence on expensive chemical reagents by using the highly efficient cholesterol 7-dehydrogenase evolved by enzyme engineering and optimized reaction conditions, while reducing energy consumption, shortening the production cycle, and improving overall production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 The comparison of enzyme activities of cholesterol 7-dehydrogenase mutant and wild-type cholesterol 7-dehydrogenase is shown.

[0112] Figure 2 The yield of 7-dehydrocholesterol under in vivo reaction conditions with different cell loads and different electron sources is shown.

[0113] Figure 3 The enzyme powder of the cholesterol 7-dehydrogenase mutant prepared by the present invention is shown.

[0114] Figure 4 The graph shows the production of 7-dehydrocholesterol when different amounts of KshB enzyme powder are added under in vitro reaction conditions, wherein the abscissa is the amount of KshB enzyme powder added, in g / L.

[0115] Figure 5 The enzyme activities of different cholesterol 7-dehydrogenase mutants are shown, wherein the relative enzyme activity refers to the percentage of the enzyme activity of each mutant relative to the wild-type enzyme activity.

[0116] Figure 6 The effect of recruitment tag on 7-dehydrocholesterol production is shown.

[0117] Figure 7 The effect of different reductase partners on the yield of 7-dehydrocholesterol is shown.

[0118] Figure 8 The effect of different reaction additives on the yield of 7-dehydrocholesterol is shown.

[0119] Fig. 9 The effect of different auxiliary solvents on the yield of 7-dehydrocholesterol is shown.

[0120] Fig.10 The effect of different electron transport systems on the yield of 7-dehydrocholesterol is shown. DETAILED DESCRIPTION

[0121] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0122] The present disclosure may be implemented in other specific forms without departing from the basic attributes of the present disclosure. It should be understood that, without conflict, any and all embodiments of the present disclosure may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present disclosure includes other embodiments obtained by such combinations.

[0123] All publications and patents mentioned in this disclosure are hereby incorporated by reference into the present invention in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0124] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0125] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0126] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0127] As shown in the present invention, the term "about" means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0128] The numerical ranges used in the present disclosure should be understood to have enumerated all the numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0129] As shown in the present disclosure, the terms "comprising", "having", "including" or "containing" can mean inclusive or open-ended, not excluding additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" can also mean closed, excluding additional, uncited elements or method steps.

[0130] As used in this disclosure, the terms "optional", "either", "any", or "any" mean that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. As used in this disclosure, "a" and "an" are used in this disclosure to refer to one or more than one of the grammatical objects.

[0131] As shown in the present disclosure, “and / or” should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0132] As used in the present disclosure, the term "nucleic acid" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to a segment containing a nucleic acid in a manner that does not originally exist in nature, or is synthetic, and the nucleic acid molecule may contain one or more control sequences. The nucleic acid may be a codon-optimized nucleic acid, such as a codon-optimized nucleic acid for expression in E. coli cells.

[0133] As used in this disclosure, the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0134] As shown in the present disclosure, "sequence identity" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the percentage of identity between the two sequences is a function of the number of matching or homologous positions shared by the compared position where the molecules are homologous at that position × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of identity. According to some specific embodiments of the present disclosure, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0135] The compilation of the twenty conventional amino acids involved in the present disclosure follows conventional usage. See, for example, Immunology-A Synthesis (Second Edition, E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0136] As shown in the present disclosure, the terms "recruitment tag" and "recruitment tag polypeptide" are used interchangeably and refer to a protein tag that can be used to guide the target protein to bind, assemble or approach in vivo or in vitro based on protein interactions. In some examples, the recruitment tag is fused to the N-terminus or C-terminus of the target protein (such as the cholesterol dehydrogenase or cholesterol dehydrogenase mutant of the present disclosure) to achieve recruitment through specific interactions with other proteins or molecules. In some examples, the recruitment tags described in the present disclosure include RIAD and RIDD tags, which specifically refer to RIAD tags in certain technical solutions.

[0137] As used in the present disclosure, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0138] As used in the present disclosure, the terms "recombinant cell" and "host cell" are used interchangeably and refer to cells that can be used to introduce vectors, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0139] As used in this disclosure, the term "7-dehydrocholesterol" refers to 27 H 44 O (CAS#: 434-16-2).

[0140] I. Summary of the Invention

[0141] The present invention selects a specific newly identified protein sequence of cholesterol 7-dehydrogenase as the starting sequence, and screens and tests about 1,000 sequences of cholesterol 7-dehydrogenase in the gene library, and finally finds a cholesterol 7-dehydrogenase with initial activity and without a transmembrane structure as our starting sequence. After obtaining the starting sequence (wild type) of cholesterol 7-dehydrogenase, the present invention performs multiple rounds of enzyme engineering evolution (including point saturation mutation and combination mutation) to continuously improve its unit enzyme activity.

[0142] The present invention also designs and optimizes the reaction conditions, which include:

[0143] a. Optimization of substrate solvent:

[0144] Since the substrate cholesterol is a hydrophobic lipid substance, it is insoluble in water but soluble in organic solvents. However, excessive organic solvents will affect the enzyme activity. The inventors screened a variety of organic solvents and surfactants to increase the solubility of cholesterol while minimizing the damage to the enzyme activity.

[0145] b. Optimization of the reductase partner of cholesterol 7-dehydrogenase

[0146] Since the reductase partners of cholesterol 7-dehydrogenase in nature are not easy to obtain, the inventors screened and obtained three reductase partners (TsaB, VanB, KshB) and performed screening and optimization.

[0147] c. Optimize the electron transfer system (NADH cycle)

[0148] Since the reaction requires NADH as a coenzyme, and NADH is expensive and not suitable for large-scale addition, the inventors cleverly combined glucose dehydrogenase and glucose cycle to ensure that NADH can be repeatedly recycled in the reaction system.

[0149] d. Optimize other reaction additives

[0150] Even with organic solvents or surfactants, the substrate cholesterol is still difficult to be catalyzed by the enzyme. Therefore, the inventors optimized the reaction additives and significantly increased the conversion rate of the reaction by adding hydroxypropyl-β-cyclodextrin.

[0151] In addition, the present invention also co-optimizes cholesterol 7-dehydrogenase and its corresponding reductase partner:

[0152] Since the reaction requires cholesterol 7-dehydrogenase and its corresponding reductase partner to bind or approach and transfer electrons, and the two wild-type enzymes do not spontaneously bind together in the reaction system, the inventors designed a specific RIAD sequence at the C-terminus of cholesterol 7-dehydrogenase and a specific RIDD sequence at the N-terminus of the reductase partner to ensure that the two enzymes can approach or bind to each other in the reaction system to catalyze the reaction.

[0153] II. Detailed Description of the Invention

[0154] Cholesterol dehydrogenase

[0155] The present invention provides a cholesterol dehydrogenase having an amino acid sequence as shown in SEQ ID NO:1.

[0156] The inventors screened about 1,000 cholesterol 7-dehydrogenase sequences from a gene library and ultimately obtained a cholesterol 7-dehydrogenase that catalyzes the synthesis of 7-dehydrocholesterol from cholesterol, and characterized its amino acid sequence.

[0157] The present invention conducts enzyme engineering evolution based on the screened cholesterol dehydrogenase, and provides a cholesterol dehydrogenase, wherein the cholesterol dehydrogenase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0158] In some embodiments, the cholesterol dehydrogenase has improved enzyme activity compared to the cholesterol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:1.

[0159] In some preferred embodiments, the cholesterol dehydrogenase has an enzyme activity that is increased by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 10 ... %, at least about 100% or more, at least about 150% or more, at least about 200% or more, at least about 250% or more, at least about 300% or more, at least about 350% or more, at least about 400% or more, at least about 450% or more, at least about 500% or more, at least about 550% or more, at least about 600% or more, at least about 650% or more, at least about 700% or more, at least about 750% or more, at least about 800% or more, at least about 850% or more, at least about 900% or more, at least about 950% or more, or at least about 1000% or more.

[0160] In some preferred embodiments, the cholesterol dehydrogenase has an enzyme activity increased by about 500%, about 600%, about 700%, about 800%, about 900% or about 1000% compared to the cholesterol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:1.

[0161] In some examples, the cholesterol dehydrogenase has at least one amino acid difference from the cholesterol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:1.

[0162] In some preferred embodiments, the amino acid differences include sequence variants obtained by amino acid deletion, addition, substitution and / or replacement, which fully or partially retain the activity of the original amino acid sequence, preferably sequence variants obtained by amino acid substitution, and more preferably sequence variants obtained by conservative amino acid substitution.

[0163] In some preferred embodiments, the amino acid difference is an amino acid substitution.

[0164] In some preferred examples, the amino acid differences are 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0165] In some preferred examples, the amino acid difference is 1-5.

[0166] In some preferred examples, the amino acid difference is 3.

[0167] In some examples, the amino acid substitutions include amino acids at the following positions:

[0168] 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 20th, 256th, 313th.

[0169] The amino acid sites described in the present invention are all counted sequentially from the N-terminus to the C-terminus of the protein.

[0170] In some preferred examples, the amino acid substitution site is selected from the 4th, 6th and 313th amino acids.

[0171] In some preferred examples, the amino acid substitution of the amino acid at position 4 is selected from Y4A, Y4R, Y4Q, Y4T, Y4V or Y4W.

[0172] In some preferred examples, the amino acid substitution of the amino acid at position 4 is Y4A.

[0173] In some preferred examples, the amino acid substitution of the amino acid at position 6 is selected from L6R, L6D, L6C or L6T.

[0174] In some preferred examples, the amino acid substitution at amino acid position 6 is L6R.

[0175] In some preferred examples, the amino acid substitution of the amino acid at position 313 is selected from Y313L, Y313N, Y313I, Y313A, Y313K, Y313R, Y313T, Y313H or Y313C.

[0176] In some preferred examples, the amino acid substitution of amino acid at position 313 is Y313I.

[0177] In some preferred examples, the cholesterol dehydrogenase has an amino acid sequence as shown in SEQ ID NO:2.

[0178] In addition to the preferred mutation site combinations provided above, the amino acid substitution may also be selected from one or more of the following sites, which can be selected or freely combined by those skilled in the art according to common technical knowledge:

[0179] L3G, L9P, L9K, L9T, K5Q, Q256C, Q256A, Q256W, T10D, T10R, T10H, M8D, C20L, C20I, T14S, Q11E, A13R, Q12R.

[0180] In some examples, the cholesterol dehydrogenase is further linked to a second polypeptide.

[0181] In some examples, the second polypeptide comprises a tag polypeptide, including, but not limited to: a recruitment tag polypeptide, a fluorescent protein polypeptide, a luminescent polypeptide, an influenza hemagglutinin tag polypeptide, a c-myc tag polypeptide, a herpes simplex virus glycoprotein D (gD) tag polypeptide, a polyhistidine tag polypeptide, a FLAG tag polypeptide, a KT3 epitope tag polypeptide, a tubulin epitope tag polypeptide, a T7 gene 10 protein tag polypeptide, a streptavidin tag polypeptide, a vesicular ostomy virus glycoprotein (VSV-G) epitope tag polypeptide, a small epitope (Pk) found on the P and V proteins of the simian virus paramyxovirus 5 (V5) tag polypeptide, an alkaline phosphatase (AP) tag polypeptide, a bluetongue virus tag (B-tag) polypeptide, a calmodulin binding peptide (CalBP) tag polypeptide, a chloramphenicol acetyltransferase (CAT) tag polypeptide, choline binding domain (CholBD) tag polypeptide, chitin binding domain (ChitBD) tag polypeptide, cellulose binding domain (CellBP) tag polypeptide, dihydrofolate reductase (DHFR) tag polypeptide, galactose binding protein (GBP) tag polypeptide, maltose binding protein (MBP) peptide, glutathione-S-transferase (GST) peptide, Glu-Glu (EE) tag polypeptide, human influenza hemagglutinin (HA) tag polypeptide, horseradish peroxidase (HRP) tag polypeptide, NE-tag polypeptide, HSV tag polypeptide, ketosteroid isomerase (KSI) tag, LacZ tag polypeptide, NusA tag polypeptide, PDZ domain tag polypeptide, AviTag polypeptide, SBP-tag polypeptide, Softag 1 peptide, Softag 3 peptide, TC tag peptide, VSV-tag peptide, Xpress tag peptide, Isopeptag peptide, SpyTag peptide, SnoopTag peptide, Profinity eXact tag peptide, Protein C tag peptide, 51-tag peptide, S-tag peptide, biotin-carboxyl carrier protein (BCCP) tag peptide, small ubiquitin-like modifier (SUMO) tag peptide, tandem affinity purification (TAP) tag peptide, HaloTag peptide, Nus-tag peptide, thioredoxin-tag peptide, CYD tag peptide, HPC tag peptide, TrpE tag peptide or ubiquitin tag peptide, etc.

[0182] In some preferred examples, the second polypeptide is a recruitment tag polypeptide, and the recruitment tag polypeptide recruits the reductase partner to approach or bind to the cholesterol dehydrogenase. In some preferred examples, the recruitment tag polypeptide approaches or binds to another recruitment tag polypeptide in the reductase partner, so that the C-terminus of the cholesterol dehydrogenase approaches or binds to the N-terminus of the reductase partner.

[0183] In some preferred examples, the recruitment tag polypeptide includes a RIAD tag. In some preferred examples, the RIAD tag has an amino acid sequence as shown in SEQ ID NO: 3. In some preferred examples, the RIAD tag has an amino acid sequence with at least one or more amino acid differences (preferably amino acid substitutions) from the amino acid sequence as shown in SEQ ID NO: 3.

[0184] In some examples, the C-terminus of the cholesterol dehydrogenase is linked to the N-terminus of the second polypeptide.

[0185] In some examples, the cholesterol dehydrogenase and the second polypeptide are directly connected or indirectly connected through a linker. In some preferred examples, the linker is a flexible linker (including but not limited to a hydrocarbon linker and a peptide linker). In some preferred examples, the linker is a polypeptide rich in glycine (G) and serine (S), such as GGGGS, (GGGGS) n 、(GGGGGS) n etc., wherein n may be 1-100, preferably 1-50, more preferably 1-10.

[0186] In some examples, the cholesterol dehydrogenase comprises an amino acid modification. In some preferred examples, the amino acid modification can improve the lipid solubility, water solubility, biocompatibility, hydrophilicity, stability, biological activity of the fusion protein, and / or protect the cholesterol dehydrogenase from hydrolysis and / or enzymolysis.

[0187] Polynucleotide

[0188] The present invention provides a polynucleotide encoding any one of the aforementioned cholesterol dehydrogenases.

[0189] The polynucleotides may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard well-known techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. The nucleic acid molecules disclosed herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0190] The nucleic acids of the present disclosure can be prepared or obtained by known means (e.g., by self-DNA synthesis and / or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the present disclosure given herein, and / or can be isolated from suitable natural sources.

[0191] In some instances, the polynucleotide comprises a codon-optimized nucleotide sequence. In some preferred instances, the polynucleotide comprises a nucleotide sequence optimized for eukaryotic or prokaryotic codons. In some preferred instances, the polynucleotide comprises a nucleotide sequence optimized for prokaryotic (preferably Escherichia coli) codons.

[0192] Carrier

[0193] The present invention provides a vector comprising any one of the aforementioned polynucleotides.

[0194] The vector of the present invention is a polynucleotide vector for introducing genetic material into a cell. The vector used for the method described herein can be linear or circular. The vector can be integrated into the target genome of the host cell or replicate independently in the host cell. For many applications, it is preferred to produce an integration vector of a stable transformant. The vector may include, for example, an origin of replication, a multiple cloning site (MCS) and / or a screening marker, etc. The expression vector generally includes an expression cassette containing a regulatory element that can promote the expression of a polynucleotide sequence (usually a coding sequence) in a specific host cell. The vector includes, but is not limited to, an integration vector, a prokaryotic plasmid, an episome, a viral vector, a cosmid, and an artificial chromosome.

[0195] Exemplary regulatory elements that can be used in expression cassettes include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described in, e.g., Goeddel, Gene Expression Technology: Methods In Enzymology, 185, Academic Press, San Diego, Calif. (1990).

[0196] The vector or other polynucleotides can be introduced into microbial cells by various standard methods, such as transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated or DEAE-dextrin-mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitates, high-speed bombardment with DNA-coated microprojectiles, and protoplast fusion. Transformants can be selected by any method known in the art.

[0197] Host cells

[0198] The present invention provides a host cell comprising any one of the aforementioned polynucleotides or vectors.

[0199] Any host cell that can be used to express the introduced gene can be modified for fermentation production of 7-dehydrocholesterol as described above. In some examples, the host cell includes eukaryotic cells and / or prokaryotic cells. In some examples, the host cell is a prokaryotic cell. In some preferred examples, the prokaryotic cell is a microorganism that is naturally unable to ferment and produce 7-dehydrocholesterol, including Gram-positive or Gram-negative bacteria, which can be modified as described above. Examples include Corynebacterium glutamicum, Bacillus subtilis, subtilus), B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp. (e.g., Pseudomonas putida), putida), Pseudomonas alcaligenes, P. citrea, Lactobacillus spp. (e.g., Lactococcus lactis, Lactobacillus plantarum), L. grayi, E. coli, E. faecium, E. gallinarum, E. casseliflavus, and / or E. faecalis cells.

[0200] In some preferred instances, the host cell is an Escherichia coli (E.coli) strain. In some preferred instances, the host cell is a genetically modified Escherichia coli strain. In some preferred instances, the modified Escherichia coli strain has a background of JM109 (DE3), W3110 (DE3), BL21 (DE3), C41 (DE3) and / or C43 (DE3). For detailed information on the modified Escherichia coli strain, refer to the article ("Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels" by Miroux B. and Walker JE, Journal of Molecular Biology, the entire contents of which are incorporated herein by reference.

[0201] Composition

[0202] The present invention provides a composition comprising one or more of any one of the aforementioned cholesterol dehydrogenase polynucleotides, vectors and host cells.

[0203] The inventors found in practice that cholesterol is insoluble in water but soluble in organic solvents. However, excessive organic solvents will affect enzyme activity. The inventors screened a variety of organic solvents and surfactants to improve the solubility of cholesterol while minimizing the damage to enzyme activity, and finally obtained a preferred cosolvent.

[0204] In some examples, the composition further includes a co-solvent for solubilizing cholesterol, such as Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof.

[0205] In some preferred examples, the cosolvent is Tween 80. Those skilled in the art can select the content of Tween 80 according to actual conditions, such as 5%, 10%, or 15%. In some examples, 10% Tween 80 is proven to be better and is conducive to the dissolution of cholesterol.

[0206] In some examples, the composition further comprises a reductase partner that is close to or bound to the cholesterol dehydrogenase. In some preferred examples, the reductase partner comprises another recruitment tag polypeptide that is close to or bound to the recruitment tag polypeptide of the cholesterol dehydrogenase, preferably a RIDD tag sequence. In some preferred examples, the RIDD tag sequence is located at the N-terminus of the reductase partner.

[0207] In some examples, the reductase chaperone includes TsaB, VanB or KshB. In some preferred examples, the reductase chaperone is KshB.

[0208] In some examples, the composition further comprises a reaction additive that promotes cholesterol catalysis, preferably cyclodextrin or its derivatives. In some preferred examples, the reaction additive includes but is not limited to: α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl β-cyclodextrin (HPBCD), methyl-β-cyclodextrin (RAMEB), heptath (2,6-dimethyl)-β-cyclodextrin (DIMEB), sulfobutyl-β-cyclodextrin (SBECD), hydroxypropyl-γ-cyclodextrin (HPGCD), etc. In some preferred examples, the reaction additive is hydroxypropyl β-cyclodextrin.

[0209] In some examples, the composition further includes NADH, glucose, and glucose dehydrogenase.

[0210] Method for synthesizing 7-dehydrocholesterol

[0211] The present invention provides the use of any of the aforementioned cholesterol dehydrogenase polynucleotides, vectors, host cells and / or compositions in synthesizing 7-dehydrocholesterol or preparing products for synthesizing 7-dehydrocholesterol. In some examples, the product includes a reagent or a kit.

[0212] The present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0213] Under suitable reaction conditions, any of the above cholesterol dehydrogenases is brought into contact with cholesterol to undergo a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

[0214] The method provided by the present invention can synthesize 7-dehydrocholesterol in one step, and only needs to catalyze the substrate cholesterol to generate 7-dehydrocholesterol under suitable reaction conditions (including in vivo and in vitro). The present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0215] (I) culturing any of the aforementioned host cells under suitable culture conditions to express any of the aforementioned cholesterol dehydrogenases;

[0216] (II) The host cells are resuspended in a buffer solution and contacted with cholesterol to undergo a catalytic reaction, thereby obtaining 7-dehydrocholesterol.

[0217] In some examples, the (II) further comprises the following condition: adding a reaction additive to the catalytic reaction. In some preferred examples, the reaction additive comprises cyclodextrin or its derivatives, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin.

[0218] In some examples, the (II) further comprises the following condition: adding NADH, glucose and glucose dehydrogenase (GDH) into the catalytic reaction.

[0219] In some examples, the (II) further comprises the following condition: an auxiliary solvent is added to the catalytic reaction, and the auxiliary solvent includes Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof. In some preferred examples, the auxiliary solvent is Tween 80.

[0220] In some examples, the (II) further comprises the following condition: adding polymyxin B (PMBS) into the catalytic reaction.

[0221] The present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0222] (i) freeze-drying any of the above cholesterol dehydrogenases to obtain a lyophilized powder;

[0223] (ii) under conditions suitable for the reaction, the lyophilized powder is contacted with the lyophilized powder of the reductase partner and cholesterol to carry out a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

[0224] In some examples, the (ii) further comprises the following condition: adding a reaction additive into the catalytic reaction.

[0225] In some preferred examples, the reaction additive includes cyclodextrin or its derivatives, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin.

[0226] In some examples, the (ii) further comprises the following condition: adding NADH, glucose and glucose dehydrogenase (GDH) into the catalytic reaction.

[0227] In some examples, the (ii) further comprises the following condition: a cosolvent is added to the catalytic reaction, and the cosolvent comprises Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof. In some preferred examples, the cosolvent is Tween 80.

[0228] In some examples, the reductase partner comprises TsaB, VanB or KshB, preferably KshB.

[0229] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. If the specific conditions are not specified in the embodiment, it is carried out according to the conditions of normal conditions or manufacturer's suggestion. If the manufacturer is not specified in all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in the publication of many publications.

[0230] Example 1: Mutant screening

[0231] Determination of enzyme activity: After adding the RIAD sequence to the C-terminus of the C7DAC25 gene sequence (which encodes the amino acid sequence shown in SEQ ID NO: 1), it was integrated into the pET28a plasmid. The plasmid was transformed into C43 Escherichia coli by chemical transformation, and the revived cell fluid was smeared on an agar plate containing kanamycin for selection and screening. The monoclonal colony was picked to LB-Kan medium for overnight culture, and then transferred to TB-Kan medium for culture to OD 0.8, and 1mM IPTG was added to continue overnight culture for 18 hours. Centrifuge, discard the supernatant, add the prepared reaction solution and substrate cholesterol to the bottom cell precipitation, extract after 4 hours of reaction, take the supernatant, and analyze it by HPLC. The substrate cholesterol is pre-mixed with Tween-80, and a mixed solution is obtained after high pressure and high temperature treatment; the solution is added to the reaction system to start the reaction.

[0232] Enzyme engineering evolution: Perform point saturation mutation and combinatorial mutation on C7DAC25 gene to construct corresponding plasmid library. Transform the plasmid library into C43 Escherichia coli by chemical transformation, repeat the above enzyme activity determination process, detect the enzyme activity of different mutants, and screen out the mutant with the highest enzyme activity, such as Figure 1 Finally, a cholesterol 7-dehydrogenase mutant with three beneficial mutations compared to the wild type was screened out (its amino acid sequence is shown in SEQ ID NO: 2), and the enzyme activity was increased by 9 times (relative to the wild type cholesterol 7-dehydrogenase with the amino acid sequence shown in SEQ ID NO: 1).

[0233] In addition, the inventors determined the enzyme activity of wild-type cholesterol 7-dehydrogenase (without RIAD sequence added to the C-terminus) and compared it with the wild-type cholesterol 7-dehydrogenase with RIAD sequence added to the C-terminus, the enzyme activities were similar and there was no significant difference.

[0234] Example 2: Catalytic synthesis of 7-dehydrocholesterol in vivo

[0235] The evolved C7DAC25 (cholesterol 7-dehydrogenase mutant with amino acid sequence as shown in SEQ ID NO: 2, with RIAD tag fused to its C-terminus) was expressed by C43 E. coli, centrifuged, and the cell pellet was collected. 2% HPbCD, 0.5g / LPMBS, 0.1g / L GDH / NAD and 10g / L glucose were added to 0.5 times, 1 times and 2 times the mass of the cell pellet (400μL of fermentation broth is 1 times the cell, i.e. 1×cell), respectively, and then 10g / L cholesterol-Tween 80 mixed solution was added. Figure 2 As shown, under the conditions of the in vivo reaction, the yield of 7-dehydrocholesterol can reach >2 g / L, and the conversion rate (yield of 7-dehydrocholesterol / mass of raw materials) can reach >20%.

[0236] Example 3: In vitro catalytic synthesis of 7-dehydrocholesterol

[0237] 3.1: Enzyme powder preparation

[0238] The evolved C7DAC25 (cholesterol 7-dehydrogenase mutant with amino acid sequence as shown in SEQ ID NO: 2, with RIAD tag fused to its C-terminus) was expressed by C43 Escherichia coli, centrifuged, and the cell pellet was collected. 120 g of the fermented cell pellet was redissolved in phosphate buffer solution, lysed with a homogenizer, centrifuged, and the supernatant was collected and dried with a freeze dryer to obtain cholesterol 7-dehydrogenase enzyme powder (about 7-8 g) ( Figure 3 ).

[0239] 3.2: In vitro catalysis of enzyme powder

[0240] After the evolved C7DAC25 shown in 3.1 is expressed by C43 Escherichia coli and the enzyme powder of the cholesterol 7-dehydrogenase mutant is obtained, the enzyme powder of the reductase partner kshB (with a RIDD tag fused to its N-terminus) corresponding to cholesterol 7-dehydrogenase can be obtained by a similar method. The two enzyme powders are mixed into enzyme powder solutions (10g / L cholesterol 7-dehydrogenase enzyme powder solution, 10g / L reductase partner kshB enzyme powder solution) and then added to 2% HPbCD, 0.1g / L GDH / NAD and 10g / L glucose, 10g / L cholesterol-Tween 80 mixed solution, and reacted for 4 hours. Figure 4 As shown, under the conditions of the in vitro reaction of the enzyme powder, when the dosage of KshB enzyme powder was increased to 50 g / L, the production of 7-dehydrocholesterol could reach >1 g / L.

[0241] Comparative Example 1: Enzyme activities of different cholesterol 7-dehydrogenase mutants

[0242] A series of cholesterol-7-dehydrogenase mutants were obtained by performing point saturation mutation and combination mutation on the C7DAC25 gene, and the corresponding plasmid library was constructed. The plasmid library was transformed into C43 Escherichia coli by chemical transformation, and the enzyme activity determination process described in Example 1 was repeated to detect the enzyme activity of different mutants. The results are shown in FIG. Figure 5 shown.

[0243] Comparative Example 2: Effect of recruitment tag on 7-dehydrocholesterol production

[0244] In order to determine the effect of recruitment tags RIAD and RIDD on the production of 7-dehydrocholesterol, the following experiment was designed, which differed from Example 3 only in that the experimental grouping was different:

[0245] (1) Reductase (chaperone) group, only the reductase chaperone kshB was added;

[0246] (2) blank control group;

[0247] (3) C7D group, only cholesterol 7-dehydrogenase mutant was added;

[0248] (4) C7D+reductase group, with both the cholesterol 7-dehydrogenase mutant and the reductase chaperone kshB added, neither containing a recruitment tag;

[0249] (5) C7D-RIAD+reductase-RIDD group, RIAD tag was fused to the C-terminus of cholesterol 7-dehydrogenase mutant; RIDD tag was fused to the N-terminus of reductase partner kshB.

[0250] The results are as follows Figure 6 As shown, the cholesterol 7-dehydrogenase mutant and the reductase chaperone kshB were fused with the recruitment tags RIAD and RIDD, respectively, and the production of 7-dehydrocholesterol was significantly improved.

[0251] Comparative Example 3: Effect of different reductase partners on 7-dehydrocholesterol production

[0252] The inventors screened for suitable reductase partners, which differed from Example 3 only in the type of reductase partners, and included the following experimental groups: blank control (containing the cholesterol 7-dehydrogenase mutant shown in Example 3), TsaB, VanB, and KshB.

[0253] The results are as follows Figure 7 As shown, KshB participated in the reaction together with the cholesterol 7-dehydrogenase mutant as a reductase chaperone, and the yield of 7-dehydrocholesterol was the highest, far superior to other reductase chaperones.

[0254] Comparative Example 4: Effect of different reaction additives on the yield of 7-dehydrocholesterol

[0255] In order to further optimize the reaction conditions and improve the reaction efficiency, the inventors screened the effects of a series of reaction additives on the yield of 7-dehydrocholesterol.

[0256] The experimental design differed from that of Example 3 only in the reaction additives (blank (containing the cholesterol 7-dehydrogenase mutants shown in Example 3 and kshB), bCD or HPbCD).

[0257] The results are as follows Figure 8 As shown, when HPbCD is used as a reaction additive, the reaction efficiency is significantly better than that of bCD, and the yield of 7-dehydrocholesterol is higher.

[0258] Comparative Example 5: Effect of different auxiliary solvents on the yield of 7-dehydrocholesterol

[0259] In order to further optimize the reaction conditions and improve the reaction efficiency, the inventors screened a series of auxiliary solvents for their effects on the yield of 7-dehydrocholesterol.

[0260] The difference between the experimental design and Example 3 is that the auxiliary solvents are different: water, water + 10% Tween-80, water + 10% Tween-80 + 10% dimethyl sulfoxide, water + 10% Tween-80 + 10% methanol, water + 10% Tween-80 + 10% ethanol, water + 10% Tween-80 + 10% acetonitrile, water + 10% Tween-80 + 10% isopropanol, isopropanol.

[0261] The results are as follows Fig. 9 As shown, the reaction efficiency when Tween 80 solution was used as an auxiliary solvent was significantly better than that of other auxiliary solvents, and the yield of 7-dehydrocholesterol was higher.

[0262] Comparative Example 6: Effects of different electron transport systems on the yield of 7-dehydrocholesterol

[0263] In order to further optimize the reaction conditions and improve the reaction efficiency, the inventors screened the effects of a series of electron transport systems on the yield of 7-dehydrocholesterol.

[0264] The experimental design differed from Example 3 only in the electron transport system: blank control (comprising the cholesterol 7-dehydrogenase mutant and kshB shown in Example 3), NADH, NADH+ADH+IPA, and NADH+GDH+Glucose.

[0265] The results are as follows Fig.10 As shown, the reaction efficiency of NADH+GDH+Glucose as an electron transport system is significantly better than other electron transport systems, and the yield of 7-dehydrocholesterol is higher.

[0266] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A cholesterol dehydrogenase, characterized in that The cholesterol dehydrogenase has an amino acid sequence as shown in SEQ ID NO: 1, or has at least one amino acid difference from the amino acid sequence as shown in SEQ ID NO:

1.

2. The cholesterol dehydrogenase according to claim 1, characterized in that The cholesterol dehydrogenase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1; Preferably, the cholesterol dehydrogenase has improved enzyme activity compared to the cholesterol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:

1.

3. The cholesterol dehydrogenase according to claim 1 or 2, characterized in that The cholesterol dehydrogenase has 1-5 amino acid differences from the amino acid sequence shown in SEQ ID NO: 1, preferably 3 amino acid differences, and the amino acid difference is preferably an amino acid substitution.

4. The cholesterol dehydrogenase according to claim 3, characterized in that The amino acid substitutions include the following amino acids: 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 20th, 256th, 313th; Preferably, the amino acid substitution site is selected from the 4th, 6th and 313th amino acids.

5. The cholesterol dehydrogenase according to claim 4, characterized in that The amino acid substitution of the amino acid at position 4 is selected from Y4A, Y4R, Y4Q, Y4T, Y4V or Y4W, preferably Y4A; and / or The amino acid substitution of the amino acid at position 6 is selected from L6R, L6D, L6C or L6T, preferably L6R; and / or The amino acid substitution of the amino acid at position 313 is selected from Y313L, Y313N, Y313I, Y313A, Y313K, Y313R, Y313T, Y313H or Y313C, preferably Y313I; Preferably, the cholesterol dehydrogenase has an amino acid sequence as shown in SEQ ID NO:

2.

6. The cholesterol dehydrogenase according to any one of claims 1 to 5, characterized in that The cholesterol dehydrogenase is also linked to a second polypeptide; Preferably, the second polypeptide comprises a tag polypeptide; More preferably, the tag polypeptide comprises a recruitment tag polypeptide, which recruits a reductase partner to approach or bind to the cholesterol dehydrogenase.

7. The cholesterol dehydrogenase according to claim 6, characterized in that The recruitment tag polypeptide is a RIAD tag; Preferably, the RIAD tag has the amino acid sequence shown in SEQ ID NO:

3.

8. The cholesterol dehydrogenase according to any one of claims 6-7, characterized in that The cholesterol dehydrogenase is directly linked to the second polypeptide or indirectly linked via a linker; Preferably, the C-terminus of the cholesterol dehydrogenase is linked to the N-terminus of the second polypeptide.

9. A polynucleotide, characterized in that It comprises a nucleic acid encoding the cholesterol dehydrogenase according to any one of claims 1-8.

10. A carrier, characterized in that It comprises the polynucleotide according to claim 9.

11. A host cell, characterized in that It comprises the polynucleotide according to claim 9 or the vector according to claim 10.

12. A composition, characterized in that It comprises one or more of the cholesterol dehydrogenase according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10, and the host cell according to claim 11; Preferably, the composition further comprises a cosolvent for solubilizing cholesterol, such as Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof; more preferably, the cosolvent is Tween 80; Preferably, the composition further comprises a reductase chaperone that is close to or bound to the cholesterol dehydrogenase; more preferably, the reductase chaperone comprises TsaB, VanB or KshB; further, the reductase chaperone is KshB; further, the N-terminus of the reductase chaperone comprises a RIDD tag sequence; Preferably, the composition further comprises an additive that promotes the cholesterol-catalyzed reaction, such as cyclodextrin or a derivative thereof, preferably β-cyclodextrin or hydroxypropyl-β-cyclodextrin, more preferably hydroxypropyl-β-cyclodextrin; Preferably, the composition further comprises NADH, glucose and glucose dehydrogenase.

13. Use of the cholesterol dehydrogenase according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10, the host cell according to claim 11 and / or the composition according to claim 12 in synthesizing 7-dehydrocholesterol and / or preparing a reagent or kit for synthesizing 7-dehydrocholesterol.

14. A method for synthesizing 7-dehydrocholesterol, characterized in that: The method comprises: Under suitable reaction conditions, the cholesterol dehydrogenase described in any one of claims 1 to 8 or the composition described in claim 12 is contacted with cholesterol to carry out a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

15. A method for synthesizing 7-dehydrocholesterol, characterized in that: The method comprises: (I) culturing the host cell according to claim 11 under suitable culture conditions to express the cholesterol dehydrogenase according to any one of claims 1 to 8; (II) resuspending the host cells in a buffer solution and contacting them with cholesterol to cause a catalytic reaction, thereby obtaining 7-dehydrocholesterol; Preferably, the (II) further comprises the following conditions: adding a reaction additive to the catalytic reaction; more preferably, the reaction additive comprises cyclodextrin or its derivatives, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin; Preferably, the (II) further comprises the following conditions: adding NADH, glucose and glucose dehydrogenase to the catalytic reaction; Preferably, the (II) further comprises the following conditions: a cosolvent is added to the catalytic reaction, and the cosolvent comprises Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof; more preferably, the cosolvent is Tween 80; Preferably, the (II) further comprises the following condition: adding polymyxin B into the catalytic reaction.

16. A method for synthesizing 7-dehydrocholesterol, characterized in that: The method comprises: (i) freeze-drying the cholesterol dehydrogenase according to any one of claims 1 to 8 to obtain a freeze-dried powder; (ii) contacting the lyophilized powder with a reductase partner lyophilized powder and cholesterol under conditions suitable for the reaction to cause a catalytic reaction, thereby obtaining 7-dehydrocholesterol; Preferably, the (ii) further comprises the following conditions: adding a reaction additive to the catalytic reaction; more preferably, the reaction additive comprises cyclodextrin or a derivative thereof, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin; Preferably, the (ii) further comprises the following conditions: adding NADH, glucose and glucose dehydrogenase to the catalytic reaction; Preferably, the (ii) further comprises the following conditions: adding a cosolvent to the catalytic reaction, the cosolvent comprising Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and a combination thereof; more preferably, the cosolvent is Tween 80; Preferably, the reductase chaperone comprises TsaB, VanB or KshB; more preferably, the reductase chaperone is KshB.

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