A cholesterol 7-dehydrogenase and a method for synthesizing 7-dehydrocholesterol using it

Abstract

This invention belongs to the field of bioengineering, specifically relating to a cholesterol 7-dehydrogenase and a method for synthesizing 7-dehydrocholesterol using the same. This invention provides a cholesterol dehydrogenase and a method for synthesizing 7-dehydrocholesterol using said cholesterol dehydrogenase. By utilizing a highly efficient cholesterol 7-dehydrogenase evolved through enzyme engineering and optimized reaction conditions, this invention reduces dependence on expensive chemical reagents, lowers energy consumption, shortens the production cycle, and improves overall production efficiency.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a cholesterol 7-dehydrogenase and a method for synthesizing 7-dehydrocholesterol using it. Background Technology

[0002] 7-Dehydrocholesterol (7-DHC) is a precursor to cholesterol in its chemical structure. In the human body, it is converted into vitamin D3 by ultraviolet radiation, which is crucial for maintaining bone health and immune system function. This compound is not only biologically significant but also commercially valuable, particularly as a key raw material for the production of vitamin D3. The widespread use of vitamin D3, including as a food additive, feed additive, and in the medical field, has led to a growing demand for 7-dehydrocholesterol.

[0003] Traditionally, the production of 7-dehydrocholesterol has primarily relied on extracting cholesterol from lanolin and then converting it to 7-dehydrocholesterol using chemical or photochemical methods. Specifically, in the field of chemical synthesis, the production of 7-dehydrocholesterol mainly depends on the precise modification of the cholesterol molecule. This process involves multiple chemical reactions, including the selective introduction of double bonds to form 7-dehydrocholesterol. The challenge of this method lies in controlling reaction conditions such as temperature, pressure, reaction time, and catalyst selection, all of which require precise control to ensure high yields and product purity. Furthermore, byproducts that may arise during chemical synthesis need to be removed through subsequent purification steps, which not only increases production costs but also imposes an environmental burden.

[0004] Photochemical irradiation is another method for producing 7-dehydrocholesterol. It utilizes ultraviolet (UV) light to irradiate cholesterol, inducing an internal chemical change within the molecule to form 7-dehydrocholesterol. The advantages of this method are its simplicity and avoidance of toxic chemical reagents. However, the conversion efficiency of photochemical irradiation is relatively low, and it has strict requirements on the wavelength and intensity of the light source. Furthermore, UV irradiation may cause other undesirable side reactions, affecting the purity and yield of the product.

[0005] While these methods are feasible, they often come with problems such as high cost, low efficiency, numerous byproducts, and environmental pollution. In modern production, despite technological advancements, challenges remain regarding environmental friendliness, cost-effectiveness, and production efficiency.

[0006] In recent years, microbial transformation, as an emerging production method, has attracted widespread attention by utilizing engineered microorganisms to convert cholesterol into 7-dehydrocholesterol. The advantages of this method lie in its relative environmental friendliness and the ability to optimize microbial strains through genetic engineering, thereby improving conversion rates and product specificity. The main challenges of microbial transformation lie in the construction and optimization of microbial strains, requiring a deeper understanding of the biological pathway of cholesterol to 7-dehydrocholesterol conversion and how to effectively enhance the activity of this pathway. Currently, there are no relevant research reports. Summary of the Invention

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

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

[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 from the amino acid sequence shown in SEQ ID NO:1.

[0010] This invention screened and identified a novel cholesterol dehydrogenase that exhibits catalytic activity towards cholesterol, converting it into 7-dehydrocholesterol. Based on this cholesterol dehydrogenase, the inventors conducted enzyme engineering evolution, obtaining 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 with the amino acid sequence shown in SEQ ID NO:1.

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

[0013] In some preferred embodiments, the cholesterol dehydrogenase exhibits an enzyme activity that is 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%, and at least about 95% higher than the cholesterol dehydrogenase shown in the amino acid sequence of SEQ ID NO:1. % or more, 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 that is increased by about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% compared to the cholesterol dehydrogenase with the amino acid sequence shown in SEQ ID NO:1.

[0015] In some embodiments, the amino acid difference is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 1-5, more preferably 3.

[0016] In some implementations, the amino acid difference is an amino acid substitution.

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

[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 at the fourth amino acid position is selected from Y4A, Y4R, Y4Q, Y4T, Y4V, or Y4W.

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

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

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

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

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

[0026] In some embodiments, the cholesterol dehydrogenase has an amino acid sequence as 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 also linked to a second polypeptide.

[0032] In some embodiments, the second polypeptide includes a tagged polypeptide.

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

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

[0035] In some preferred embodiments, the recruitment tag peptide includes 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 chaperone contains a RIDD tag sequence.

[0040] In some preferred embodiments, the N-terminus of the reductase chaperone contains 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 implementations, the reductase chaperone includes TsaB, VanB, or KshB.

[0045] In some preferred embodiments, the reductase chaperone 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 implementations, the cholesterol dehydrogenase and the second polypeptide are directly linked or indirectly linked through a connector.

[0057] In some embodiments, the cholesterol dehydrogenase contains amino acid modifications.

[0058] In some embodiments, the amino acid modification can improve the lipid solubility, water solubility, biocompatibility, hydrophilicity, stability, biological activity, 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 implementations, the polynucleotide comprises a codon-optimized nucleic acid.

[0061] Thirdly, the present invention provides a carrier comprising any of the aforementioned polynucleotides.

[0062] In some implementations, the vector includes an expression regulatory element operatively linked to the nucleic acid.

[0063] In some implementations, the expression regulation element includes a promoter, an enhancer, and / or a terminator.

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

[0065] Fourthly, the present invention provides a host cell comprising any of the aforementioned polynucleotides or vectors.

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

[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, Bacillus subtilis, and / or yeast.

[0069] Fifthly, the present invention provides a composition comprising one or more of the aforementioned cholesterol dehydrogenase, polynucleotide, carrier, and host cell.

[0070] In some embodiments, the composition further includes a cholesterol-solventizing agent, such as Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof.

[0071] In some preferred embodiments, the co-solvent is Tween 80.

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

[0073] In some embodiments, the reductase chaperone includes TsaB, VanB, or KshB, preferably KshB.

[0074] In some preferred embodiments, the reductase chaperone contains a RIDD tag.

[0075] In some preferred embodiments, the N-terminus of the reductase chaperone contains 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 includes a reaction additive that promotes cholesterol catalysis, 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 includes NADH, glucose, and glucose dehydrogenase.

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

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

[0081] In a seventh aspect, the present invention provides the use of any of the foregoing cholesterol dehydrogenase polynucleotides, carriers, host cells and / or compositions in the preparation of products for the synthesis of 7-dehydrocholesterol.

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

[0083] In some implementations, the product includes reagents or kits.

[0084] Eighthly, the present invention provides a method for synthesizing 7-dehydrocholesterol, the method comprising:

[0085] Under suitable reaction conditions, any of the aforementioned cholesterol dehydrogenases is brought into contact with cholesterol to catalyze a reaction, yielding 7-dehydrocholesterol.

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

[0087] (I) Culture 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 brought into contact with cholesterol to undergo a catalytic reaction, resulting in 7-dehydrocholesterol.

[0089] In some implementations, (II) further includes 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 implementations, (II) further includes the following conditions:

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

[0094] In some implementations, (II) further includes the following conditions:

[0095] A co-solvent is added to the catalytic reaction. The co-solvent includes Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and combinations thereof, preferably Tween 80, more preferably 10% Tween 80.

[0096] In some implementations, (II) further includes the following conditions:

[0097] Polymyxin B (PMBS) is 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-dry any of the aforementioned cholesterol dehydrogenases to obtain a freeze-dried powder;

[0100] (ii) Under suitable reaction conditions, the lyophilized powder is brought into contact with the reductase chaperone lyophilized powder and cholesterol to undergo a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

[0101] In some implementations, (ii) further includes the following conditions:

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

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

[0104] In some implementations, (ii) further includes the following conditions:

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

[0106] In some implementations, (ii) further includes the following conditions:

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

[0108] In some implementations, the reductase chaperone includes TsaB, VanB, or KshB.

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

[0110] This invention proposes a one-step in vitro method for the efficient synthesis of 7-dehydrocholesterol from cholesterol using cholesterol 7-dehydrogenase. The key to this method lies in utilizing a newly identified cholesterol 7-dehydrogenase and, through enzyme engineering evolution and carefully designed enzymatic reactions, obtaining a cholesterol 7-dehydrogenase mutant. This mutant achieves highly efficient conversion of cholesterol to 7-dehydrocholesterol, with enzyme activity increased by approximately 9 times compared to wild-type cholesterol 7-dehydrogenase, ultimately yielding over 2 g / L of 7-dehydrocholesterol. This invention, by using a highly efficient cholesterol 7-dehydrogenase evolved through enzyme engineering and optimized reaction conditions, reduces dependence on expensive chemical reagents, lowers energy consumption, shortens the production cycle, and improves overall production efficiency. Attached Figure Description

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

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

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

[0114] Figure 4 The figure shows the yield of 7-dehydrocholesterol when different amounts of KshB enzyme powder were added under in vitro reaction conditions. The horizontal axis represents the amount of KshB enzyme powder added, in g / L.

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

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

[0117] Figure 7 The effects of different reductase chaperones on 7-dehydrocholesterol production were shown.

[0118] Figure 8 The effects of different reaction additives on the production of 7-dehydrocholesterol are shown.

[0119] Figure 9 The effects of different auxiliary solvents on the yield of 7-dehydrocholesterol are shown.

[0120] Figure 10 The effects of different electron transport systems on 7-dehydrocholesterol production are shown. Detailed Implementation

[0121] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the scope of protection of this invention.

[0122] This disclosure may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this disclosure may be combined with technical features of any or more other embodiments to obtain further embodiments. This disclosure includes such further embodiments obtained through combination.

[0123] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If any use or terminology in any publication or patent incorporated by reference conflicts with that used in this disclosure, the use and terminology of this disclosure shall prevail.

[0124] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[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 pertains. For purposes 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 indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0127] As illustrated in this invention, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0128] The range of numbers used in this disclosure should be understood as including all numbers within that range. For example, the range 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 this disclosure indicates, the terms “comprising,” “having,” “including,” or “containing” can mean inclusive or open-ended and do not exclude additional, uncited elements or method steps. At the same time, “comprising,” “having,” “including,” or “containing” can also mean closed-ended and exclude additional, uncited elements or method steps.

[0130] As indicated in this disclosure, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used in this disclosure, “a” and “an” refer to one or more grammatical objects.

[0131] As this disclosure illustrates, “and / or” should be understood to mean any one of the options or a combination of any two or more of the options.

[0132] As used in this disclosure, the term "nucleic acid" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from naturally occurring genes, modified in a way not originally present in nature to contain nucleic acid segments, or is synthetic, and may contain one or more control sequences. Nucleic acids can be codon-optimized nucleic acids, such as those codon-optimized 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 disclosed herein, “sequence identity” or “identity” refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same bases or amino acid monomer subunits, for example, if every position in 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 two sequences divided by the number of homologous positions at that position × 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences. According to some specific embodiments of this disclosure, “identity” refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or nucleotide sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the procedure, 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 performing a search for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0135] The twenty common amino acids involved in this disclosure are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (Second Edition, ESGolub and DRGren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this 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 disclosed herein, the terms "recruitment tag" and "recruitment tag polypeptide" are used interchangeably to refer to a protein tag that can be used to guide a target protein to bind, assemble, or approach in vivo or in vitro based on protein-protein interactions. In some instances, the recruitment tag is fused to the N-terminus or C-terminus of a target protein (e.g., the cholesterol dehydrogenase or cholesterol dehydrogenase mutant of this disclosure) to achieve recruitment through specific interactions with other proteins or molecules. In some instances, the recruitment tags described in this disclosure include RIAD and RIDD tags, with RIAD tags specifically referring to in certain technical solutions.

[0137] As used in this disclosure, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0138] As used in this disclosure, the terms "recombinant cell" and "host cell" are used interchangeably and refer to cells that can be used to introduce a vector, 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 a protein with the molecular formula C6H2O. 27 H 44 Compounds of O (CAS#:434-16-2).

[0140] I. Overview of the Invention

[0141] This invention selects a novel, newly identified cholesterol 7-dehydrogenase protein sequence as the starting sequence. Through screening and testing of approximately 1000 cholesterol 7-dehydrogenase sequences in a gene library, a sequence with initiating activity and lacking transmembrane structure was identified as our starting sequence. After obtaining the starting sequence (wild-type) of cholesterol 7-dehydrogenase, this invention performs multiple rounds of enzyme engineering evolution (including point saturation mutations and combinatorial mutations) to continuously improve its unit enzyme activity.

[0142] The present invention also designs and optimizes reaction conditions, including:

[0143] a. Optimization of substrate solvent:

[0144] Since cholesterol is a hydrophobic lipid, it is insoluble in water but readily soluble in organic solvents. However, excessive organic solvents can affect enzyme activity. The inventors screened various organic solvents and surfactants to improve cholesterol solubility while minimizing damage to enzyme activity.

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

[0146] Since reductase chaperones for cholesterol 7-dehydrogenase are not readily available in nature, the inventors screened and obtained three reductase chaperones (TsaB, VanB, and KshB) and then optimized them.

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

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

[0149] d. Optimize other reaction additives

[0150] Even with the use of organic solvents or surfactants, the substrate cholesterol remains difficult for enzymes to catalyze. Therefore, the inventors optimized the reaction additives by adding hydroxypropyl-β-cyclodextrin to significantly improve the reaction conversion rate.

[0151] Furthermore, this invention also co-optimizes cholesterol 7-dehydrogenase and its corresponding reductase chaperone:

[0152] Since this reaction requires the binding or proximity of cholesterol 7-dehydrogenase and its corresponding reductase chaperone to transfer electrons, and the two enzymes in wild-type do not spontaneously combine 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 chaperone 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 approximately 1,000 sequences of cholesterol 7-dehydrogenase from a gene bank and ultimately obtained a cholesterol 7-dehydrogenase that catalyzes the synthesis of 7-dehydrocholesterol from cholesterol, and characterized its amino acid sequence.

[0157] This invention provides a cholesterol dehydrogenase based on screened cholesterol dehydrogenases through enzyme engineering evolution. 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 enhanced enzyme activity compared to the cholesterol dehydrogenase with the amino acid sequence shown in SEQ ID NO:1.

[0159] In some preferred embodiments, the cholesterol dehydrogenase exhibits an enzyme activity that is 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%, and at least about 95% higher than the cholesterol dehydrogenase shown in the amino acid sequence of SEQ ID NO:1. % or more, 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 that is increased by about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% compared to the cholesterol dehydrogenase with the amino acid sequence shown in SEQ ID NO:1.

[0161] In some instances, the cholesterol dehydrogenase differs from the cholesterol dehydrogenase shown in SEQ ID NO:1 by at least one amino acid.

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

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

[0164] In some preferred embodiments, the amino acid differences are 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0165] In some preferred embodiments, the amino acid differences are 1-5.

[0166] In some preferred embodiments, the amino acid differences are three.

[0167] In some instances, the amino acid substitutions include amino acids at the following sites:

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

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

[0170] In some preferred embodiments, the amino acid substitution sites are selected from the 4th, 6th, and 313th amino acids.

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

[0172] In some preferred embodiments, the amino acid at position 4 is replaced with Y4A.

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

[0174] In some preferred embodiments, the amino acid at position 6 is replaced by L6R.

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

[0176] In some preferred embodiments, the amino acid at position 313 is substituted with Y313I.

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

[0178] In addition to the preferred combinations of mutation sites provided above, the amino acid substitutions 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 based on general technical knowledge:

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

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

[0181] In some instances, the second polypeptide comprises a tag polypeptide, which includes, but is not limited to: recruitment tag polypeptides, fluorescent protein polypeptides, luminescent polypeptides, influenza hemagglutinin tag polypeptides, c-myc tag polypeptides, herpes simplex virus glycoprotein D (gD) tag polypeptides, multihistidine tag polypeptides, FLAG tag polypeptides, KT3 epitope tag polypeptides, tubulin epitope tag polypeptides, T7 gene 10 protein tag polypeptides, streptavidin tag polypeptides, vesicular stomata virus glycoprotein (VSV-G) epitope tag polypeptides, small epitopes (Pk) found on the P and V proteins of the simian paramyxovirus 5 (V5) tag polypeptide, alkaline phosphatase (AP) tag polypeptides, bluetongue virus tag (B-tag) polypeptides, calmodulin-binding peptide (CalBP) tag polypeptides, and chloramphenicol acetyltransferase. (CAT) tagged peptides, choline-binding domain (CholBD) tagged peptides, chitin-binding domain (ChitBD) tagged peptides, cellulose-binding domain (CellBP) tagged peptides, dihydrofolate reductase (DHFR) tagged peptides, galactose-binding protein (GBP) tagged peptides, maltose-binding protein (MBP) tagged peptides, glutathione S-transferase (GST) tagged peptides, Glu-Glu (EE) tagged peptides, human influenza hemagglutinin (HA) tagged peptides, horseradish peroxidase (HRP) tagged peptides, NE-tagged peptides, HSV-tagged peptides, ketosterone isomerase (KSI) tagged peptides, LacZ tagged peptides, NusA tagged peptides, PDZ domain tagged peptides, AviTag peptides, SBP-tagged peptides, Softag 1-peptide, Softag 3-peptide, TC-tagged peptide, VSV-tagged peptide, Xpress-tagged peptide, Isopeptag peptide, SpyTag peptide, SnoopTag peptide, Profinity eXact-tagged peptide, Protein C-tagged peptide, 51-tagged peptide, S-tagged peptide, Biotin-Carboxyl Carrier Protein (BCCP)-tagged peptide, Small Ubiquitin-like Modifier (SUMO)-tagged peptide, Tandem Affinity Purification (TAP)-tagged peptide, HaloTag peptide, Nus-tagged peptide, Thioredoxin-tagged peptide, CYD-tagged peptide, HPC-tagged peptide, TrpE-tagged peptide, or ubiquitin-tagged peptide, etc.

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

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

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

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

[0186] In some instances, the cholesterol dehydrogenase contains amino acid modifications. In some preferred instances, the amino acid modifications can improve the lipophilicity, water solubility, biocompatibility, hydrophilicity, stability, biological activity, and / or protect the cholesterol dehydrogenase from hydrolysis and / or enzymatic degradation.

[0187] Polynucleotides

[0188] This invention provides a polynucleotide encoding any of the aforementioned cholesterol dehydrogenases.

[0189] The polynucleotides may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. When purifying nucleic acids from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, using 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 acids are "isolated" or "substantially pure." The nucleic acid molecules of this disclosure 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 disclosed herein may be prepared or obtained by known means (e.g., by self-DNA synthesis and / or recombinant DNA technology) based on information regarding the amino acid sequence of the polypeptides of this disclosure, and / or may 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 of the aforementioned polynucleotides.

[0194] The vectors of this invention are polynucleotide vectors for introducing genetic material into cells. Vectors used in the methods described herein can be linear or circular. Vectors can be integrated into the target genome of a host cell or replicate independently within the host cell. For many applications, integrative vectors that produce stable transformants are preferred. Vectors may include, for example, origins of replication, multiple cloning sites (MCS), and / or selection markers. Expression vectors typically include an expression cassette containing regulatory elements that promote the expression of a polynucleotide sequence (typically a coding sequence) in a specific host cell. Vectors include, but are not limited to, integrative vectors, prokaryotic plasmids, episomes, viral vectors, entrapments, and artificial chromosomes.

[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, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology, 185, Academic Press, San Diego, Calif. (1990).

[0196] The vector or other polynucleotide can be introduced into microbial cells by a variety of standard methods, such as transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipid transfection-mediated or DEAE-dextrin-mediated transfection or transfection using recombinant phage viruses), incubation with calcium phosphate DNA precipitate, high-speed bombardment with DNA-coated microbolites, 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 of the aforementioned polynucleotides or vectors.

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

[0200] In some preferred embodiments, the host cell is an *Escherichia coli* strain. In some preferred embodiments, the host cell is a genetically modified *E. coli* strain. In some preferred embodiments, the modified *E. coli* strain has a background of JM109(DE3), W3110(DE3), BL21(DE3), C41(DE3), and / or C43(DE3). For more information on modified *E. coli* strains, please 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," *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 the aforementioned cholesterol dehydrogenase polynucleotides, a carrier, and a host cell.

[0203] The inventors discovered in practice that cholesterol is insoluble in water but readily soluble in organic solvents. However, excessive organic solvents can affect enzyme activity. The inventors screened various organic solvents and surfactants to improve cholesterol solubility while minimizing damage to enzyme activity, ultimately obtaining a preferred co-solvent.

[0204] In some instances, the composition further includes a cholesterol-solventizing agent, such as Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof.

[0205] In some preferred embodiments, the co-solvent is Tween 80. Those skilled in the art can select the content of Tween 80 according to the specific circumstances, for example, 5%, 10%, or 15%. In some instances, 10% Tween 80 has proven to be preferred, as it facilitates cholesterol dissolution.

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

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

[0208] In some examples, the composition further includes a reaction additive that promotes cholesterol catalysis, preferably cyclodextrin or a derivative thereof. 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), hepta(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 instances, the composition also includes NADH, glucose, and glucose dehydrogenase.

[0210] Methods for synthesizing 7-dehydrocholesterol

[0211] This invention provides the use of any of the foregoing cholesterol dehydrogenase polynucleotides, carriers, host cells, and / or compositions in the synthesis of 7-dehydrocholesterol, or in the preparation of products for the synthesis of 7-dehydrocholesterol. In some examples, the products include reagents or kits.

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

[0213] Under suitable reaction conditions, any of the aforementioned cholesterol dehydrogenases is brought into contact with cholesterol to catalyze a reaction, yielding 7-dehydrocholesterol.

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

[0215] (I) Culture 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 brought into contact with cholesterol to undergo a catalytic reaction, resulting in 7-dehydrocholesterol.

[0217] In some instances, step (II) further includes the addition of a reaction additive to the catalytic reaction. In some preferred instances, the reaction additive comprises cyclodextrin or a derivative thereof, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin.

[0218] In some instances, (II) further includes the following condition: the addition of NADH, glucose, and glucose dehydrogenase (GDH) to the catalytic reaction.

[0219] In some instances, step (II) further includes the addition of an auxiliary solvent to the catalytic reaction, the auxiliary solvent comprising Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof. In some preferred instances, the auxiliary solvent is Tween 80.

[0220] In some instances, (II) further includes the following condition: the addition of polymyxin B (PMBS) to the catalytic reaction.

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

[0222] (i) Freeze-dry any of the aforementioned cholesterol dehydrogenases to obtain a freeze-dried powder;

[0223] (ii) Under suitable reaction conditions, the lyophilized powder is brought into contact with the reductase chaperone lyophilized powder and cholesterol to undergo a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

[0224] In some instances, (ii) further includes the condition that a reaction additive is added to the catalytic reaction.

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

[0226] In some instances, (ii) further includes the following condition: the addition of NADH, glucose, and glucose dehydrogenase (GDH) to the catalytic reaction.

[0227] In some instances, step (ii) further includes the addition of a co-solvent to the catalytic reaction, the co-solvent comprising Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof. In some preferred instances, the co-solvent is Tween 80.

[0228] In some instances, the reductase chaperone includes TsaB, VanB, or KshB, with KshB being preferred.

[0229] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in numerous publications.

[0230] Example 1: Mutant Screening

[0231] Enzyme activity assay: The C7DAC25 gene sequence (encoding the amino acid sequence shown in SEQ ID NO:1) was added to the C-terminus with a RIAD sequence and integrated into the pET28a plasmid. This plasmid was transformed into C43 *E. coli* via chemical transformation, and the revived cell slurry was spread onto agar plates containing kanamycin for selection. Single colonies were picked and cultured overnight in LB-Kan medium, then transferred to TB-Kan medium and cultured until OD 0.8. 1 mM IPTG was added, and the culture continued overnight for 18 hours. After centrifugation, the supernatant was discarded, and the prepared reaction solution and substrate cholesterol were added to the bottom cell pellet. After reacting for 4 hours, extraction was performed, and the supernatant was collected for HPLC analysis. The substrate cholesterol was premixed with Tween-80 and subjected to high-pressure and high-temperature treatment to obtain a mixed solution; this solution was added to the reaction system to begin the reaction.

[0232] Enzyme engineering evolution: Point saturation mutations and combinatorial mutations were performed on the C7DAC25 gene to construct corresponding plasmid libraries. These plasmid libraries were transformed into C43 *E. coli* via chemical transformation. The enzyme activity assay procedure was repeated, and the enzyme activities of different mutants were detected. The mutant with the highest enzyme activity was screened out, such as... Figure 1 As shown. Finally, a cholesterol 7-dehydrogenase mutant with three beneficial mutations compared to the wild type (the amino acid sequence of which is shown in SEQ ID NO:2) was selected, and the enzyme activity was increased by 9-fold (compared to the wild-type cholesterol 7-dehydrogenase with the amino acid sequence shown in SEQ ID NO:1).

[0233] In addition, the inventors measured the enzyme activity of wild-type cholesterol 7-dehydrogenase (without the RIAD sequence added to the C-terminus), and found that the enzyme activity was similar to that of wild-type cholesterol 7-dehydrogenase with the RIAD sequence added to the C-terminus, with no significant difference.

[0234] Example 2: In vivo catalytic synthesis of 7-dehydrocholesterol

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

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

[0237] 3.1: Enzyme powder preparation

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

[0239] 3.2: In vitro catalysis of enzyme powder

[0240] After expressing the evolved C7DAC25 shown in 3.1 into a cholesterol 7-dehydrogenase mutant using E. coli C43, a similar method can be used to prepare the cholesterol 7-dehydrogenase reductase chaperone kshB (with a RIDD tag fused to its N-terminus) enzyme powder. The two enzyme powders were mixed separately to form enzyme solutions (10 g / L cholesterol 7-dehydrogenase solution and 10 g / L reductase chaperone kshB solution), which were then added to a 2% HPbCD, 0.1 g / L GDH / NAD, and 10 g / L glucose and 10 g / L cholesterol-Tween 80 mixed solution, and reacted for 4 hours. Figure 4 As shown, under the conditions of in vitro reaction of this enzyme powder, when the amount of KshB enzyme powder is increased to 50 g / L, the yield of 7-dehydrocholesterol can 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 point saturation mutagenesis and combinatorial mutagenesis of the C7DAC25 gene, and corresponding plasmid libraries were constructed. The plasmid libraries were transformed into C43 *E. coli* via chemical transformation, and the enzyme activity assay procedure described in Example 1 was repeated to detect the enzyme activity of different mutants. The results are as follows: Figure 5 As shown.

[0243] Comparative Example 2: The effect of recruitment tags on 7-dehydrocholesterol production

[0244] To determine the effects of recruitment tags RIAD and RIDD on 7-dehydrocholesterol production, the following experiment was designed, differing from Example 3 only in the experimental groups:

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

[0246] (2) Blank control group;

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

[0248] (4) The C7D+ reductase group, which also includes cholesterol 7-dehydrogenase mutant and reductase chaperone kshB, does not contain recruitment tags.

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

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

[0251] Comparative Example 3: Effects of different reductase chaperones on 7-dehydrocholesterol production

[0252] The inventors screened suitable reductase chaperones, which differed from Example 3 only in the type of reductase chaperone, including 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, as a reductase chaperone, participates in the reaction together with the cholesterol 7-dehydrogenase mutant, resulting in the highest production of 7-dehydrocholesterol, which is far superior to other reductase chaperones.

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

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

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

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

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

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

[0260] The experimental design differs from Example 3 only in the auxiliary solvents: 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, and isopropanol.

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

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

[0263] To further optimize reaction conditions and improve reaction efficiency, the inventors screened a series of electron transport systems to study the effects of 7-dehydrocholesterol production.

[0264] The experimental design differs from Example 3 only in the electron transport system: blank control (containing 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 Figure 10 As shown, the reaction efficiency of NADH+GDH+Glucose as an electron transport system is significantly better than that of 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, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A cholesterol dehydrogenase, characterized in that, The amino acid sequence of the cholesterol dehydrogenase is SEQ ID NO:

2.

2. The cholesterol dehydrogenase according to claim 1, characterized in that, The cholesterol dehydrogenase has three amino acid substitutions of Y4A, L6R, and Y313I compared to the amino acid sequence shown in SEQ ID NO: 1, and the cholesterol dehydrogenase has improved enzyme activity compared to the cholesterol dehydrogenase shown in SEQ ID NO:

1.

3. The cholesterol dehydrogenase according to claim 1 or 2, characterized in that, The cholesterol dehydrogenase is also linked to a second polypeptide, which is a tag polypeptide.

4. The cholesterol dehydrogenase according to claim 3, wherein the tag peptide is a recruitment tag peptide, and the recruitment tag peptide recruits a reductase chaperone to approach or bind to the cholesterol dehydrogenase.

5. The cholesterol dehydrogenase according to claim 4, wherein the recruitment tag polypeptide is a RIAD tag.

6. The cholesterol dehydrogenase according to claim 5, characterized in that, The amino acid sequence of the RIAD tag is shown in SEQ ID NO:

3.

7. The cholesterol dehydrogenase according to claim 3, characterized in that, The cholesterol dehydrogenase is directly linked to the second polypeptide or indirectly linked through a connector.

8. The cholesterol dehydrogenase according to claim 7, characterized in that, The C-terminus of the cholesterol dehydrogenase is linked to the N-terminus of the second polypeptide.

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

10. A carrier, characterized in that, It contains the polynucleotide as described in claim 9.

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

12. A composition, characterized in that, It comprises any one of the cholesterol dehydrogenases of claims 1-8, the polynucleotide of claim 9, the vector of claim 10, and the host cell of claim 11.

13. The composition according to claim 12, characterized in that, The composition also includes a cholesterol-solventizing agent.

14. The composition according to claim 13, characterized in that, The co-solvents include Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol, and combinations thereof.

15. The composition according to claim 13, characterized in that, The co-solvent is Tween 80.

16. The composition according to claim 12, characterized in that, The composition also includes a reductase chaperone that is close to or bound to the cholesterol dehydrogenase.

17. The composition according to claim 16, characterized in that, The reductase chaperones include TsaB, VanB, or KshB.

18. The composition according to claim 17, characterized in that, The reductase chaperone is KshB.

19. The composition according to any one of claims 16-18, characterized in that, The N-terminus of the reductase chaperone contains a RIDD tag sequence.

20. The composition according to claim 12, characterized in that, The composition also includes reaction additives that promote cholesterol catalysis.

21. The composition according to claim 20, characterized in that, The reaction additives include cyclodextrin or its derivatives.

22. The composition according to claim 21, characterized in that, The reaction additives include β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

23. The composition according to claim 22, characterized in that, The reaction additive is hydroxypropyl-β-cyclodextrin.

24. The composition according to claim 12, characterized in that, The composition also includes NADH, glucose, and glucose dehydrogenase.

25. Use of the cholesterol dehydrogenase according to any one of claims 1-8, the polynucleotide according to claim 9, the carrier according to claim 10, the host cell according to claim 11, and / or the composition according to any one of claims 12-24 in the synthesis of 7-dehydrocholesterol and / or in the preparation of reagents or kits for the synthesis of 7-dehydrocholesterol.

26. A method for synthesizing 7-dehydrocholesterol, characterized in that, The method includes: Under suitable reaction conditions, the cholesterol dehydrogenase of any one of claims 1-8 or the composition of any one of claims 22-24 is brought into contact with cholesterol to undergo a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

27. A method for synthesizing 7-dehydrocholesterol, characterized in that, The method includes: (I) Culture the host cells of claim 11 under suitable culture conditions to express the cholesterol dehydrogenase of any one of claims 1-8; (II) The host cells are resuspended in a buffer solution and brought into contact with cholesterol to undergo a catalytic reaction, resulting in 7-dehydrocholesterol.

28. The method according to claim 27, characterized in that, The (II) further includes the following condition: adding a reaction additive to the catalytic reaction.

29. The method according to claim 28, characterized in that, The reaction additives include cyclodextrin or its derivatives.

30. The method according to claim 29, characterized in that, The reaction additives include β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

31. The method according to claim 30, characterized in that, The reaction additive is hydroxypropyl-β-cyclodextrin.

32. The method according to claim 27, characterized in that, The (II) further includes the following conditions: the addition of NADH, glucose and glucose dehydrogenase to the catalytic reaction.

33. The method according to claim 32, characterized in that, The (II) further includes the following condition: a co-solvent is added to the catalytic reaction, the co-solvent including Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and combinations thereof.

34. The method according to claim 33, characterized in that, The co-solvent is Tween 80.

35. The method according to claim 32, characterized in that, The (II) further includes the following condition: the addition of polymyxin B in the catalytic reaction.

36. A method for synthesizing 7-dehydrocholesterol, characterized in that, The method includes: (i) Freeze-dry the cholesterol dehydrogenase according to any one of claims 1-8 to obtain a freeze-dried powder; (ii) Under suitable reaction conditions, the lyophilized powder is brought into contact with the reductase chaperone lyophilized powder and cholesterol to undergo a catalytic reaction, and 7-dehydrocholesterol is obtained after the reaction.

37. The method according to claim 36, characterized in that, The (ii) also includes the following condition: adding a reaction additive to the catalytic reaction.

38. The method according to claim 37, characterized in that, The reaction additives include cyclodextrin or its derivatives.

39. The method according to claim 38, characterized in that, The reaction additives include β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

40. The method according to claim 39, characterized in that, The reaction additive is hydroxypropyl-β-cyclodextrin.

41. The method according to claim 36, characterized in that, The (ii) further includes the following condition: the addition of NADH, glucose and glucose dehydrogenase to the catalytic reaction.

42. The method according to claim 41, characterized in that, The (ii) further includes the following condition: a co-solvent is added to the catalytic reaction, the co-solvent including Tween 80, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropanol and combinations thereof.

43. The method according to claim 42, characterized in that, The co-solvent is Tween 80.

44. The method according to claim 36, characterized in that, The reductase chaperones include TsaB, VanB, or KshB.

45. The method according to claim 44, characterized in that, The reductase chaperone is KshB.

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