An ancestral carbonyl reductase and uses thereof

Abstract

The application discloses an ancestor carbonyl reductase and application thereof, wherein the amino acid sequence of the ancestor carbonyl reductase is shown as SEQ ID NO. 2, or has single mutation or multiple mutations on SEQ ID NO. 2. The ancestor carbonyl reductase of the application has high alkalinity and high temperature tolerance as a catalyst in synthesis of chiral aromatic alcohol or chiral alcohol ester, has high substrate loading, and the obtained chiral aromatic alcohol or chiral alcohol ester has high optical purity (ee value is more than 99%, and dr value is greater than 100:1).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, in particular to an ancestral carbonyl reductase and application thereof. BACKGROUND

[0002] Vibegron is a selective β3 adrenergic receptor (ADRB3) agonist that increases bladder capacity by relaxing detrusor smooth muscle during bladder filling. Vibegron was first developed by Merck & Co. Inc., and was approved for marketing by the Pharmaceutical and Medical Devices Agency (PMDA) on September 21, 2018, and was marketed in Japan by Kyorin Pharmaceutical Co., Ltd. and Kissei Pharmaceutical Co., Ltd., and was approved for marketing in the United States by the FDA on December 23, 2020, and is the first new oral drug for treating overactive bladder (OAB) since 2012. Vibegron is mainly used for treating urgency urinary incontinence, urinary urgency and urinary frequency symptoms in patients with overactive bladder (OAB).

[0003] The chemical name of vibegron is (6S)-N-[4-[[(2S,5R)-5-[(R)-hydroxy-(phenyl)-methyl]-pyrrolidin-2-yl]methylphenyl]-4-oxo-7,8-dihydro-6H-pyrrolo[1,2-a]-pyrimidine-6-carboxamide, and the molecular structure formula is as follows. The key chiral intermediate 1 is tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl)carbamate, and the molecular structure formula is as follows.

[0004]

[0005] The synthesis of chiral intermediate 1 is mainly through asymmetric reduction of prochiral ketone. Since the substrate can spontaneously racemize under alkaline and high temperature conditions, the theoretical maximum conversion rate is 100%. After screening a plurality of commercial carbonyl reductases, patent WO2014150639A found that KRED-P301 has good enantioselectivity and diastereoselectivity, and the ee value of the final product is 99.4% and the diastereoselectivity dr value is greater than 100:1 under the reaction at 45°C. However, the reaction time is longer and the substrate concentration needs to be further improved.

[0006] In 2024, a research team of Zhejiang University of Technology carried out molecular modification based on functional ring strategy on carbonyl reductase EaSDR6 derived from Exiguobacterium algae, and obtained mutant M5 (A138L / A190V / S193A / Y201F / N204A), which has 868 times higher catalytic efficiency than wild enzyme, and enantioselectivity and diastereoselectivity are both >99%. Using 20 g / L of cells containing the mutant enzyme, 300 g / L of substrate can be completely converted into chiral intermediate 1 in 12 h under organic-water biphasic conditions, which is the highest level so far. However, the enzyme amount of the reaction system is large, and the difficulty of downstream separation and extraction is large. SUMMARY

[0007] In order to solve the problems existing in the prior art, in view of the problems of poor enzyme stability, low substrate loading, and large amount of enzyme addition existing in the reaction of biocatalytic synthesis of chiral aromatic alcohol or chiral alcohol ester, the present application provides an ancestral carbonyl reductase which can tolerate alkaline conditions and has high stability.

[0008] The specific technical scheme of the present application is as follows:

[0009] The present application discloses an ancestral carbonyl reductase, wherein the amino acid sequence of the ancestral carbonyl reductase is shown in SEQ ID NO. 2, or has single mutation or multiple mutations on SEQ ID NO. 2.

[0010] Further, the amino acid sequence of the ancestral carbonyl reductase has mutation at one or more positions of 85th, 197th and 214th positions of SEQ ID NO. 2.

[0011] Further, the phenylalanine at the 85th position of SEQ ID NO. 2 is mutated to alanine; and / or

[0012] The tyrosine at the 197th position of SEQ ID NO. 2 is mutated to alanine; and / or

[0013] The threonine at the 214th position of SEQ ID NO. 2 is mutated to alanine.

[0014] Further, a nucleic acid molecule encoding the ancestral carbonyl reductase as described above;

[0015] Preferably, the nucleic acid molecule is DNA or RNA;

[0016] More preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 3.

[0017] The present application provides an expression vector comprising the nucleic acid molecule as described above;

[0018] The vector is a viral vector or a plasmid vector.

[0019] The present application provides a host cell expressing the ancestor carbonyl reductase as described above.

[0020] Further, the host cell is a bacterium, a fungus, a plant cell or an animal cell.

[0021] Further, the use of the ancestor carbonyl reductase, the nucleic acid molecule, the vector, the host cell as described above in the synthesis of a chiral aromatic alcohol or a chiral alcohol ester.

[0022] Further, the chiral aromatic alcohol or the chiral alcohol ester is selected from one of tert-butyl ((1R, 2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl) carbamate, (R)-phenyl ethanol, (R)-2-chlorophenyl ethanol, (R)-phenylpropanol, (R)-4-chloro-3-hydroxybutyric acid ethyl ester. S R S R

[0023] The present application provides a method for preparing a chiral aromatic alcohol or a chiral alcohol ester, comprising the following steps:

[0024] contacting the ancestor carbonyl reductase as described above with a substrate aromatic ketone or ketone ester in a buffer solution,

[0025] The substrate aromatic ketone or ketone ester is selected from one of 2-(Boc-amino)-1-phenyl-5-hexyn-1-one, acetophenone, 2-chloroacetophenone, phenylpropanone or 4-chloro-3-carbonyl butyric acid ethyl ester;

[0026] Preferably, the buffer solution is a sodium phosphate buffer, a Tris-HCl buffer or a glycine-NaOH buffer; and / or

[0027] The temperature during the contacting process is 20-60℃; and / or

[0028] The pH during the contacting process is 7-11.

[0029] The ancestor carbonyl reductase of the present application as a catalyst in the synthesis of a chiral aromatic alcohol or a chiral alcohol ester has high alkalinity and high temperature tolerance, high substrate loading, high optical purity of the obtained chiral aromatic alcohol or chiral alcohol ester (ee value of more than 99%, dr value > 100: 1), mild reaction conditions, simple operation, easy industrial amplification, and good industrial application development prospects. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 ​​​​is a schematic diagram of a reaction of an ancestral carbonyl reductase catalyzing dynamic kinetic resolution to synthesize tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl)carbamate. DETAILED DESCRIPTION

[0031] The exemplary embodiments of this application are described herein with reference to the drawings, in which various details are set forth to facilitate an understanding of the embodiments of the application. However, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made and equivalents employed without departing from the scope and spirit of the application. Also, for the purpose of clarity and the brevity, the description below is focused on the differences from the prior art.

[0032] It should be noted that certain terms have been used throughout the specification to refer to particular components. As one skilled in the art will appreciate, different persons can refer to the same component by different names. This document does not intend to distinguish between components that differ in name but not in function. As used throughout this document, the terms "comprise" or "comprising" are to be interpreted as "including but not limited to." The description herein is intended to be illustrative of the best mode and not as an exhaustive list of alternatives, modifications, constructions and permutations. The scope of the application is therefore intended to be defined by the appended claims.

[0033] Ancestral enzymes refer to special enzymes at the early stage of the evolution of the earth, which often have higher catalytic activity, stability, and substrate spectrum than modern enzymes, providing more possibilities for industrial biological catalysis. By collecting homologous sequences, multiple sequence alignment, phylogenetic tree construction, ancestral sequence inference, and other ancestral sequence reconstruction techniques, ancestral enzymes can be easily reconstructed and "revived", and further through heterologous expression and functional screening, more high-quality property catalysts can be provided for industrial biological catalysis. Since the biosynthesis of the weinreb intermediate needs to be carried out under alkaline and high temperature conditions, by constructing an ancestral carbonyl reductase with alkali-resistant conditions and high temperature, efficient preparation of tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl)carbamate can be realized.

[0034] The present application provides an ancestral carbonyl reductase, wherein the amino acid sequence of the ancestral carbonyl reductase is shown in SEQ ID NO. 2, or has a single mutation or multiple mutations on SEQ ID NO. 2.

[0035] In the present application, the amino acid sequence of the ancestral carbonyl reductase has a mutation at one or more of positions 85, 197, and 214 of SEQ ID NO. 2.

[0036] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85 on SEQ ID NO. 2.

[0037] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 197 on SEQ ID NO. 2.

[0038] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 214 on SEQ ID NO. 2.

[0039] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85 and position 197 on SEQ ID NO. 2.

[0040] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85 and position 214 on SEQ ID NO. 2.

[0041] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 197 and position 214 on SEQ ID NO. 2.

[0042] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85, position 197 and position 214 on SEQ ID NO. 2.

[0043] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85 on SEQ ID NO. 2.

[0044] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 197 on SEQ ID NO. 2.

[0045] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 214 on SEQ ID NO. 2.

[0046] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85 on SEQ ID NO. 2.

[0047] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 197 on SEQ ID NO. 2.

[0048] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 214 on SEQ ID NO. 2.

[0049] In some embodiments, the amino acid sequence of the ancestral carbonyl reductase has a mutation at position 85 on SEQ ID NO. 2.

[0050] In some embodiments, the SEQ ID NO. 2 is mutated at position 85 phenylalanine to alanine, and position 214 threonine to alanine.

[0051] In some embodiments, the SEQ ID NO. 2 is mutated at position 197 tyrosine to alanine, and position 214 threonine to alanine.

[0052] In some embodiments, the SEQ ID NO. 2 is mutated at position 85 phenylalanine to alanine, position 197 tyrosine to alanine, and position 214 threonine to alanine. The amino acid sequence thereof is SEQ ID NO. 4.

[0053] The source of the ancestral carbonyl reductase described in the present application preferably includes: constructing an ancestral carbonyl reductase by using ancestral sequence reconstruction method, obtaining a carbonyl reductase by artificially synthesizing a full amino acid sequence, and obtaining a carbonyl reductase by cloning and expressing through genetic engineering method.

[0054] In the present application, the ancestral sequence reconstruction (ASR) is discussed in Randall et al. (Nat. Commun. 7:12847 doi:10.1038 / ncomms 12847 (2016)). The authors define ASR as "a process of analyzing modern sequences in the context of evolution / phylogeny to infer ancestral sequences at specific nodes of a tree". Ancestral sequence reconstruction (ASR) is used in molecular evolution studies. Unlike the traditional evolution method of studying proteins by comparing homologous proteins at the ends of different branches of a phylogenetic tree in a horizontal manner, ASR explores statistically inferred ancestral proteins within the nodes of the tree in a vertical manner. A phylogenetic tree is a branching diagram showing evolutionary relationships among various species of biological organisms or other entities based on similarities and differences in their physical or genetic characteristics. In a rooted phylogenetic tree, each node with descendants represents the inferred most recent common ancestor of those descendants. In ASR, multiple related homologs of a protein of interest are selected and aligned in a multiple sequence alignment (MSA), a phylogenetic tree is constructed, which has statistically inferred sequences at the nodes of the branches. These sequences are so-called "ancestors". The process of synthesizing the corresponding DNA, transforming it into cells and producing proteins is so-called "reconstruction".

[0055] The ancestor carbonyl reductase described in the present application is preferably derived from the genus Kluyveromyces. The carbonyl reductase is obtained by the method of ancestor sequence reconstruction, and the cloned enzyme is repeatedly compared and screened by determining and comparing the activity of the carbonyl reductase, its activity, enantioselectivity, diastereoselectivity, etc. on racemic 2-(Boc-amino)-1-phenyl-5-hexyn-1-one, and finally the ancestor carbonyl reductase with the best catalytic performance is obtained.

[0056] The gene source of the carbonyl reductase described in the present application includes: the carbonyl reductase gene obtained by the method of artificial whole gene synthesis. The carbonyl reductase gene source is preferably: the ancestor sequence No. 61 obtained by the method of ancestor sequence reconstruction. The specific preparation method preferably includes: obtaining the full-length gene sequence by codon optimization according to the corresponding amino acid sequence, and submitting the gene sequence to a gene synthesis company for artificial synthesis. After obtaining the corresponding gene, the gene is amplified by PCR means, and the sequence is connected into pET28a. The primers are as follows:

[0057] Upstream primer: 5'-gtgccgcgcggcagccatatgagcgtgctggtgagcg-3' (SEQ ID NO. 5)

[0058] Downstream primer: 5'-acggagctcgaattcggatccttacagtttgccttcttttttcaga-3' (SEQ ID NO. 6)

[0059] The underlined part of the nucleotide sequence of the upstream primer is Ndel The underlined part of the downstream primer is BamHI The enzyme digestion site. Then, using the artificially synthesized gene as a template, the polymerase chain reaction (PCR) is used for gene amplification to obtain the complete carbonyl reductase full-length gene DNA fragment. The full-length carbonyl reductase gene (nucleotide sequence as shown in SEQ ID NO. 1 in the sequence listing), named ADH61, has a full length of 1038 nucleotide bases. The coding sequence starts from the 1st base to the 1038th base, the start codon is ATG, and the stop codon is TAA. The sequence has no intron, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2 in the sequence listing.

[0060] Due to the degeneracy of the genetic code, the nucleic acid molecule encoding the above-mentioned carbonyl reductase (the amino acid sequence is shown as SEQ ID NO. 2 in the sequence listing) is not limited to the nucleic acid molecule shown as SEQ ID NO. 1. A homolog of the polynucleotide can also be provided by introducing appropriate substitutions, deletions, alterations, insertions or additions of nucleotides. The homolog of the polynucleotide in the present application can be obtained by substituting, deleting or adding one or more bases of the nucleic acid molecule shown as SEQ ID NO. 1 in the sequence listing within the range of maintaining the activity of the carbonyl reductase. The mutation of the carbonyl reductase gene is preferably a mutation of 3 bases, i.e., mutation of phenylalanine (abbreviated as F) at position 85 of the coding sequence of the aforementioned carbonyl reductase gene to alanine (abbreviated as A), mutation of tyrosine (abbreviated as Y) at position 197 to alanine, and mutation of threonine (abbreviated as T) at position 214 to alanine, thereby obtaining a mutant of the carbonyl reductase gene, the nucleotide sequence of which is shown as SEQ ID NO. 3, and the amino acid sequence of which is shown as SEQ ID NO. 4.

[0061] Due to the degeneracy of the genetic code, the nucleic acid molecule encoding the above-mentioned carbonyl reductase (the amino acid sequence is shown as SEQ ID NO. 4 in the sequence listing) is not limited to the nucleic acid molecule shown as SEQ ID NO. 3. A homolog of the polynucleotide can also be provided by introducing appropriate substitutions, deletions, alterations, insertions or additions of nucleotides.

[0062] The present application also provides a nucleic acid molecule encoding the aforementioned ancestral carbonyl reductase, preferably, the nucleic acid molecule is DNA or RNA. More preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1 or SEQ ID NO. 3.

[0063] Table 1 is the sequence listing

[0064]

[0065] The present application also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0066] The expression vector can be constructed by cloning the aforementioned ancestral carbonyl reductase gene into various expression vectors by conventional methods in the art, wherein the expression vector includes various vectors in the art, such as commercially available plasmids, cosmids, phage or viral vectors, etc., and the expression vector is preferably a pET-28a plasmid.

[0067] Further, the expression vector can be prepared by the following method: the product of the ancestral carbonyl reductase gene amplified by PCR is digested with restriction enzymes Ndel and BamHIdouble enzyme digestion, at the same time, the expression vector pET-28a is digested by restriction enzymes Ndel and BamHI double enzyme digestion, forming complementary sticky ends, recovering the above-mentioned ancestral carbonyl reductase gene enzyme digestion product and the enzyme-digested pET-28a plasmid, connecting by using T4 DNA ligase, constructing a recombinant expression vector pET28a containing the ancestral carbonyl reductase gene adh61 .

[0068] The application also provides a host cell expressing the aforementioned ancestral carbonyl reductase.

[0069] The host cell is a conventional host cell in the art, as long as it can meet the requirements that the recombinant expression vector can be stably self-replicated, and the ancestral carbonyl reductase gene carried by it can be effectively expressed, such as bacteria, fungi, plant cells, animal cells, etc.

[0070] Further, the bacteria are preferably Escherichia coli, and more preferably Escherichia coli E. coli BL21 (DE3) or Escherichia coli E. coli DH5α. The aforementioned recombinant expression vector pET28a-adh61 is transformed into Escherichia coli E. coli BL21 (DE3), and a genetically engineered strain, i.e., Escherichia coli E. coli BL21 (DE3) / pET28a- adh61 .

[0071] Further, the aforementioned ancestral carbonyl reductase is prepared by the following steps: inoculating the aforementioned host cell expressing the ancestral carbonyl reductase into a culture medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to collect the bacterial cells; crushing the bacterial cells and centrifuging to obtain a cell crushing supernatant; and extracting the cell crushing supernatant to obtain the aforementioned ancestral carbonyl reductase.

[0072] Further, the culture medium can be any culture medium in the art that can make the transformant grow and produce the ancestral carbonyl reductase. The culture medium is preferably LB medium: peptone 5-15 g / L, yeast extract 1-10 g / L, NaCl 5-15 g / L, pH 6.0-8.0. The culture method and culture conditions are not particularly limited, and can be appropriately selected according to the type of host cell and culture method and other factors according to the ordinary knowledge in the art, as long as the transformant can grow and produce the ancestral carbonyl reductase. Other specific operations for culturing the transformant can be performed according to the conventional operations in the art. The preferred strain culture method includes: inoculating the recombinant Escherichia coli (preferably E. coliThe BL21(DE3) is inoculated into LB medium containing kanamycin for culture, and when the optical density OD600 of the culture solution reaches 0.6-0.8 (preferably 0.6), the ancestor carbonyl reductase of the application can be efficiently expressed under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1-1.0 mmol / L (preferably 0.1-0.3 mmol / L).

[0073] The application also provides the use of the aforementioned ancestor carbonyl reductase, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned host cell in the synthesis of a chiral aromatic alcohol or a chiral alcohol ester.

[0074] Further, the chiral aromatic alcohol or chiral alcohol ester is selected from one of tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl)carbamate, S )-phenyl ethanol, S )-phenylpropanol, R )-2-chlorophenyl ethanol, R )-4-chloro-3-hydroxybutyric acid ethyl ester.

[0075] The application provides a method for preparing a chiral aromatic alcohol or a chiral alcohol ester, comprising the step of contacting the ancestor carbonyl reductase with a substrate aromatic ketone or ketone ester in a buffer solution.

[0076] The ancestor carbonyl reductase used in the above method can exist in a crude enzyme solution, a freeze-dried cell or a cell disruption supernatant, or can be a purified enzyme.

[0077] Further, the substrate aromatic ketone or ketone ester is selected from one of 2-(Boc-amino)-1-phenyl-5-hexyn-1-one, acetophenone, propiophenone, 2-chloroacetophenone, 4-chloro-3-carbonyl butyric acid ethyl ester.

[0078] Further, the contacting condition comprises a temperature of 20-60℃, for example, 20℃, 23℃, 25℃, 28℃, 30℃, 33℃, 35℃, 38℃, 40℃, 43℃, 45℃, 48℃, 50℃, 53℃, 55℃, 58℃, 60℃, and any range and any value in any range consisting of any of the above-mentioned numerical values, preferably 40-58℃, and more preferably 45℃.

[0079] Further, the contacting condition comprises a pH of 7-11, preferably 9-11, and further preferably 10.

[0080] Further, the buffer solution is a sodium phosphate buffer solution, a Tris-HCl buffer solution or a glycine-NaOH buffer solution.

[0081] Specifically, in glycine-NaOH buffer, under the catalysis of the carbonyl reductase, a dynamic kinetic reduction synthesis reaction is carried out using a substrate aromatic ketone or ketone ester to form an optically active chiral aromatic alcohol or chiral alcohol ester.

[0082] Further, the conditions for the dynamic kinetic reduction reaction of the carbonyl reductase catalyzing aromatic ketone or ketone ester are as follows: the ancestral carbonyl reductase is dissolved in a buffer, and the substrate aromatic ketone or ketone ester is added to a final concentration of 100-2000 mM. The reaction is carried out at 20-60°C, mechanically stirred, and the pH is controlled by supplementing NaOH until the ee of the substrate approaches 99%. After the reaction is completed, the chiral aromatic alcohol or chiral alcohol ester is extracted with dichloromethane three times, the extract is combined, a drying agent such as anhydrous sodium sulfate is added, and dried overnight, and finally the solvent is removed by rotary evaporation or the like to obtain the chiral aromatic alcohol or chiral alcohol ester.

[0083] In some embodiments, 2-(Boc-amino)-1-phenyl-5-hexyn-1-one compound is used as a substrate, and a dynamic kinetic reduction synthesis reaction is carried out in glycine-NaOH buffer under the catalysis of the carbonyl reductase using 2-(Boc-amino)-1-phenyl-5-hexyn-1-one compound to form optically active tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl) carbamate.

[0084] Further, the conditions for the dynamic kinetic reduction reaction of the carbonyl reductase catalyzing 2-(Boc-amino)-1-phenyl-5-hexyn-1-one are as follows: the ancestral carbonyl reductase is dissolved in a buffer, and the substrate 2-(Boc-amino)-1-phenyl-5-hexyn-1-one is added to a final concentration of 100-2000 mM. The reaction is carried out at 20-60°C, mechanically stirred, and the pH is controlled by supplementing NaOH until the ee of the substrate approaches 99%. After the reaction is completed, tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl) carbamate is extracted with dichloromethane three times, the extract is combined, a drying agent such as anhydrous sodium sulfate is added, and dried overnight, and finally the solvent is removed by rotary evaporation or the like to obtain the chiral tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl) carbamate.

[0085] In this context, "ee" is an abbreviation for Enantiomeric Excess, which is a key indicator for measuring the purity of a single enantiomer in a chiral compound. When the ee value of the substrate approaches 99%, it means that the content of one enantiomer in the substrate is much higher than that of its mirror image isomer (another enantiomer), and the chiral purity is extremely high.

[0086] In this article, dr value is the abbreviation of Diastereomeric Ratio, which is a key indicator to measure the composition of diastereomeric mixture in chiral compounds, mainly used to describe the ratio relationship between different combinations of diastereomers in compounds containing multiple chiral centers.

[0087] The dr value represents the content ratio of the two main diastereomers in the mixture, usually expressed in the form of "major diastereomer: minor diastereomer". For example: if the contents of two diastereomers A and B in the substrate are 90% and 10% respectively, then dr = A:B = 9:1; if the ratio is 100:1, then dr = 100:1, indicating that the major diastereomer is absolutely dominant.

[0088] Examples

[0089] The materials used in the experiments and the experimental methods are generally and / or specifically described in this application. In the following examples, % means wt%, i.e. weight percentage, unless otherwise specified. The reagents or instruments used, if not specified by the manufacturer, are conventional reagent products that can be obtained commercially.

[0090] Example 1 Cloning of Ancestral Carbonyl Reductase Gene

[0091] Ancestral sequence reconstruction was performed using online software FireProt-ASR (FireProt-ASR (muni.cz)). According to the constructed ancestral carbonyl reductase, its coding gene was obtained by codon optimization, and the full-length sequence was further artificially synthesized by a gene synthesis company. The PCR primers were designed as follows:

[0092] Upstream primer: 5'-gtgccgcgcggcagccatatgagcgtgctggtgagcg-3'

[0093] Downstream primer: 5'-acggagctcgaattc g gatccttacagtttgccttcttttttcaga-3'

[0094] Among them, the underlined part of the upstream primer is Ndel the enzyme digestion site, and the underlined part of the downstream primer is BamHI the enzyme digestion site.

[0095] The ancestral carbonyl reductase DNA synthesized above was used as a template for PCR amplification. The PCR system was as follows: 2x Taq PCR MasterMix 10 μL, 1 μL (0.3 μmol / L) of each of the upstream primer and the downstream primer, 1 μL (0.1 μg) of the DNA template, and 7 μL of ddH2O. The PCR amplification procedure was as follows: (1) 95°C, pre-denaturation for 3 min; (2) 94°C, denaturation for 30 s; (3) 55°C, annealing for 30 s; (4) 72°C, elongation for 2 min; steps (2)-(4) were repeated for 30 cycles; (5) 72°C, continued elongation for 10 min, and cooling to 4°C. The PCR product was purified by agarose gel electrophoresis, and the PCR product was recovered by using an agarose gel DNA recovery kit. An intact ancestral carbonyl reductase full-length gene sequence was obtained, which was 1038 bp in length after DNA sequencing, and was named ADH61. The nucleotide sequence of the gene is shown in SEQ ID NO. 1 in the sequence listing.

[0096] Example 2 Preparation of an ancestral carbonyl reductase recombinant expression vector and a recombinant expression transformant

[0097] The ancestral carbonyl reductase gene DNA fragment obtained in Example 1 and a pET-28a empty plasmid (from Invitrogen) were subjected to double digestion with restriction endonucleases Nde I and Xho I at 37°C for 2 h, and the target fragment was purified by agarose gel electrophoresis and recovered by using an agarose gel DNA recovery kit. Ndel and BamHI The target fragment was ligated under the action of T4 DNA ligase at 4°C overnight to obtain a recombinant expression plasmid pET28a-ADH61. adh61 .

[0098] The above recombinant expression plasmid was transformed into E. coli DH5α competent cells, and positive recombinants were screened on a kanamycin-containing resistance plate. Single colonies were picked, and positive clones were verified by colony PCR. The recombinant bacteria were cultured, and the plasmid was extracted after amplification, and was re-transformed into E. coli BL21 (DE3) competent cells. The transformation liquid was spread on an LB plate containing kanamycin, and was cultured at 37°C overnight to obtain positive recombinant transformant E. coli BL21 (DE3) / pET28a-ADH61. E. coli E. coli BL21 (DE3) / pET28a-ADH61. E. coli adh61 The positive clones were verified by colony PCR and gene sequencing.

[0099] Example 3 Expression and purification of the ancestral carbonyl reductase

[0100] ​​The recombinant E. coli obtained in Example 2 was inoculated into LB medium (10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, pH 7.0) containing kanamycin, and was cultured at 37°C overnight with shaking. The culture was inoculated into a 500 mL flask containing 100 mL of LB medium at a ratio of 1% (v / v), and was cultured at 37°C with shaking at 180 rpm. When the OD600 of the culture reached 0.6, 0.2 mmol / L of IPTG was added as an inducer, and the culture was induced at 25°C for 12 h. The culture was centrifuged to collect the cells, which were washed twice with physiological saline. The resting cells were obtained by freeze-drying for 24 h, and were stored at 4°C after collection. The resting cells were also suspended in a buffer at pH 7.0, and were sonicated in an ice bath. The supernatant was collected by centrifugation, and was used as a crude enzyme solution of the recombinant ancestral carbonyl reductase.

[0101] The crude enzyme solution was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was loaded onto a 1.0 mL Ni-NTA gravity column, and the protein was eluted using 50, 100, 150, 200, 250, 300, 350, 400, and 450 mmol / L of imidazole and Buffer B solution (0.5 mol / L of NaCl, 500 mmol / L of imidazole, 20 mmol / L of PBS, pH 7.4) at a gradient. The eluted sample was collected. The eluate containing the target protein was concentrated using a 10 kDa ultrafiltration tube. After concentration, the protein was replaced and desalted with 20 mmol / L of Tris-HCl, and was obtained as a pure enzyme of the ancestral carbonyl reductase. The enzyme was stored in a refrigerator at -80°C after being rapidly frozen in liquid nitrogen. -1 -1 -1 -1 -1

[0102] Example 4: Activity determination of the ancestral carbonyl reductase on different substrates

[0103] ​​​​​The reductive activity of the ancestral carbonyl reductase was determined by detecting the change in absorbance at 340 nm using an enzyme marker. The activity determination method was as follows: in a 200 μL reaction system (100 mmol / L sodium phosphate buffer, pH 8.0), 1 mmol / L of the substrate (20 mmol / L, dissolved in ethanol) was added, and after incubation at 30°C for 2 min, an appropriate amount of the ancestral carbonyl reductase prepared in Example 3 was added, mixed rapidly, and the change in absorbance at 340 nm was detected. The enzyme activity (U) was defined as the amount of enzyme required to catalyze the reduction of 1 μmol of NADPH per minute under the above conditions. The results of the catalytic activity of the carbonyl reductase on different substrates are shown in Table 1. The specific activity of the ancestral carbonyl reductase ADH61 on 2-(Boc-amino)-1-phenyl-5-hexyn-1-one was 4.8 U / mg, and the activity on 4-chloro-3-carbonyl butyl acetate was the highest, reaching 198.6 U / mg. The above results show that ADH61 has a wide substrate spectrum and can be used to synthesize various chiral aromatic alcohols or chiral alcohol esters of different structures.

[0104] Table 1 Specific activity of wild-type ADH61 on different prochiral ketone substrates

[0105]

[0106] Example 5 Conversion reaction at different pH values

[0107] In 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.0, 7.5, 8.0, 9.0, 10.0, 11.0), 10 g / L of the wild-type ancestral carbonyl reductase prepared in Example 3 and 100 mmol / L of the substrate (2-(Boc-amino)-1-phenyl-5-hexyn-1-one) were added, and the reaction was carried out at 30°C and 120 rpm oscillation. After 12 h of reaction, the reaction was extracted and monitored, and after drying with anhydrous sodium sulfate overnight, the substrate conversion rate, ee value, and dr value were determined and analyzed. The results are shown in Table 2. The wild-type ancestral carbonyl reductase has high alkaline resistance and has higher conversion rate under alkaline conditions. It has the highest conversion rate at pH 10.0, which can reach 99.0%, and the ee value and dr value of the product are 99.9% and >100:1, respectively.

[0108] Table 2 Reaction effect of wild-type ADH61 at different pH values

[0109]

[0110] Example 6 Conversion reaction at different temperatures

[0111] In 10 mL of sodium phosphate buffer (100 mmol / L, pH 10.0), 5 g / L of wild-type ancestor carbonyl reductase prepared in Example 3 and 100 mmol / L of substrate (2-(Boc-amino)-1-phenyl-5-hexyn-1-one) were added, and the reaction was carried out at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C with 120 rpm shaking. After 12 h of reaction, the reaction was extracted and monitored, and the substrate conversion rate, ee value, and dr value were determined after drying with anhydrous sodium sulfate overnight. The results are shown in Table 3. The wild-type ancestor carbonyl reductase has high thermal stability and higher conversion rate at high temperature. It has the highest conversion rate at 45°C, which can reach 99.9%, and the ee value and dr value of the product are 99.9% and >100:1, respectively.

[0112] Table 3 Reaction effect of wild-type ADH61 at different temperatures

[0113]

[0114] Example 7 Preparation of ancestor carbonyl reductase with mutations

[0115] The full-length gene sequence of the carbonyl reductase ADH61 obtained in Example 1 (nucleotide sequence shown as SEQ ID NO. 1) was subjected to 3-base mutations. The mutation positions of the mutant were F mutated to A at position 85, Y mutated to A at position 197, and T mutated to A at position 214 of the coding sequence of the carbonyl reductase gene. The sequence of the mutant gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4. The mutant gene of the carbonyl reductase was used to prepare the ancestor carbonyl reductase mutant by the method described in Examples 2-3. Compared with the wild type, the ADH61 mutant enzyme has higher specific activity, and the specific activity of the pure enzyme for the substrate 2-(Boc-amino)-1-phenyl-5-hexyn-1-one is 27.8 U / mg, which is 5.8 times that of the wild-type ancestor carbonyl reductase prepared in Example 3.

[0116] Table 4 Specific activity of ADH61 mutant enzyme for different prochiral ketone substrates

[0117]

[0118] Example 8 Conversion reaction at different pH values

[0119] In 10 mL of sodium phosphate buffer (100 mmol / L, pH 7.0, 7.5, 8.0, 9.0, 10.0, 11.0), 2 g / L of the ancestor carbonyl reductase mutant prepared in Example 7 and 100 mmol / L of the substrate (2-(Boc-amino)-1-phenyl-5-hexyn-1-one) were added, and the reaction was carried out at 30°C with 120 rpm oscillation. After 6 h of reaction, the reaction was extracted with shaking and monitored. After drying overnight with anhydrous sodium sulfate, the substrate conversion rate, ee value, and dr value were determined by analysis, and the results are shown in Table 5. The carbonyl reductase mutant had the highest conversion rate of 99.9% at pH 10.0, and the ee value and dr value of the product were 99.9% and >100:1, respectively.

[0120] Table 5 Reaction effects of ADH61 mutants at different pH

[0121]

[0122] Example 9 Conversion reaction at different temperatures

[0123] In 10 mL of sodium phosphate buffer (100 mmol / L, pH 10.0), 2 g / L of the ancestor carbonyl reductase mutant prepared in Example 7 and 100 mmol / L of the substrate (2-(Boc-amino)-1-phenyl-5-hexyn-1-one) were added, and the reaction was carried out at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C with 120 rpm oscillation. After 6 h of reaction, the reaction was extracted with shaking and monitored. After drying overnight with anhydrous sodium sulfate, the substrate conversion rate, ee value, and dr value were determined by analysis, and the results are shown in Table 6. The carbonyl reductase mutant had the highest conversion rate of 99.9% at 45°C, and the ee value and dr value of the product were 99.9% and >100:1, respectively.

[0124] Table 6 Reaction effects of ADH61 mutants at different temperatures

[0125]

[0126] Example 10 Dynamic kinetic reduction reaction of 2-(Boc-amino)-1-phenyl-5-hexyn-1-one with different concentrations catalyzed by carbonyl reductase mutants

[0127] The ancestral carbonyl reductase mutant prepared in Example 7 was added in 10 mL of sodium phosphate buffer (100 mmol / L, pH 10.0) at different concentrations, and the reaction was carried out at 45°C with 120 rpm oscillation. The reaction was monitored by sampling at intervals. After the reaction was completed, equal volume of ethyl acetate was used for extraction, and the extraction was repeated twice. The combined extract was dried overnight with anhydrous sodium sulfate, and then the substrate conversion rate, ee value and dr value were determined. The results are shown in Table 7. The ancestral carbonyl reductase mutant can completely convert the highest 2000 mmol / L substrate (574 g / L) in 12 h, with a conversion rate of 99.9%, an ee value of >99.9%, and a dr value of >100:1. When the substrate concentration is further increased to 2500 mol / L, the conversion rate is 90.2% after 24 h.

[0128] Table 7 Reaction effect of ADH61 mutant catalysis of different substrate concentrations

[0129]

[0130] The highest level of the current research is the carbonyl reductase mutant M5 (A138L / A190V / S193A / Y201F / N204A) of EaSDR6 developed by the research team of Zhejiang University of Technology. The mutant enzyme is applied to 20 g / L cells, and under organic-aqueous two-phase conditions, 300 g / L substrate can be completely converted to tert-butyl ((1R, 2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl) carbamate in 12 h, and the S / C is 15. Compared with the above research, the substrate concentration of the present research is 574 g / L, the enzyme addition amount is 12 g / L, and the S / C is 47.8. All the parameters are better than the above research.

[0131] The above is only the preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments without departing from the technical solution content of the present application, and according to the technical essence of the present application, still belongs to the protection scope of the present application.

Claims

1. An ancestral carbonyl reductase, wherein, the amino acid sequence of the ancestor carbonyl reductase is shown as SEQ ID NO. 2, or has multiple mutations on SEQ ID NO. 2, the multiple mutations are: the SEQ ID NO. 2 is mutated from phenylalanine to alanine at position 85; and the SEQ ID NO. 2 is mutated from tyrosine to alanine at position 197; and the SEQ ID NO. 2 is mutated from threonine to alanine at position 214.

2. A nucleic acid molecule encoding the ancestor carbonyl reductase according to claim 1.

3. The nucleic acid molecule of claim 2, wherein, the nucleic acid molecule is DNA.

4. The nucleic acid molecule of claim 2, wherein, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

3.

5. An expression vector comprising the nucleic acid molecule according to any one of claims 2-4; the vector is a viral vector or a plasmid vector.

6. A host cell expressing the ancestor carbonyl reductase according to claim 1.

7. The host cell of claim 6, wherein, the host cell is a bacterium, a fungus, a plant cell or an animal cell.

8. Use of the ancestor carbonyl reductase according to claim 1, the nucleic acid molecule according to any one of claims 2-4, the vector according to claim 5 or the host cell according to claim 6 or 7 in the synthesis of tert-butyl ((1R,2R)-1-hydroxy-1-phenyl-5-hexyn-2-yl)carbamate.

9. A process for the preparation of tert-butyl ((1 R,2R)-1 -hydroxy-1 -phenyl-5- hexyn-2-yl)carbamate, wherein, comprising the following steps: contacting the ancestor carbonyl reductase according to claim 1 with 2-(Boc-amino)-1-phenyl-5-hexyn-1-one in a buffer solution.

10. The method according to claim 9, wherein, the buffer solution is a sodium phosphate buffer, a Tris-HCl buffer or a glycine-NaOH buffer; and / or the temperature during the contacting is 20-60℃; and / or the pH during the contacting is 7-11.

Images