WO2019011237A1 - Use of lactate dehydrogenase in asymmetric synthesis of chiral hydroxyl compound - Google Patents

Abstract

Provided is the use of a lactate dehydrogenase in the asymmetric synthesis of a chiral hydroxyl compound. In particular, provided is the use of a polypeptide in the production of a compound of formula A or a downstream product with the compound of formula A as a precursor. Further provided is a method for producing a compound of formula A, the method comprising culturing a bacterial strain that expresses the polypeptide to obtain the compound of formula A. Further provided are a bacterial strain that produces the compound of formula A, and a method for constructing the bacterial strain that produces the compound of formula A. By means of the method, the compound of formula A can be efficiently and inexpensively produced. The method prepares the obtained compound of formula A at a high concentration, and has the advantages of a high product optical purity, mild reaction conditions, being environmentally friendly, having a simple operation and easy industrial amplification.

Description

Application of a lactate dehydrogenase in asymmetric synthesis of chiral hydroxy compounds Technical field

The invention belongs to the technical field of bioengineering, and particularly relates to a lactate dehydrogenase, a preparation method of the lactate dehydrogenase, and the use of the lactate dehydrogenase as a catalyst in asymmetric synthesis of a chiral hydroxy compound.

Background technique

(R)-2-Hydroxy-4-phenylbutanoic acid ethyl ester (R-HPBE, CAS No.: 90315-82-5) is a chiral secondary alcohol which synthesizes numerous angiotensin converting enzymes (ACE). A key chiral building block for inhibitors (ie, Ply).

Ply drugs are mainly used to treat heart failure and high blood pressure. At present, there are mainly four types of antihypertensive drugs such as ACE inhibitors, calcium antagonists, angiotensin II receptor antagonists and β-blockers in the Chinese antihypertensive drug market. In recent years, Puli drugs have been affected by sartan drugs, and sales have declined. However, due to the new EU standards and the withdrawal of some domestic manufacturers, the prices are relatively stable.

Since Ondetti et al. developed the first generation of captopril in 1977, the Ply-like drugs have been expanded to more than 80 derivatives. Several common Ply drugs are shown above. Because of its advantages of significant curative effect, small adverse reactions and long duration of action, Ply-like drugs still have broad application prospects in clinical practice. Since R-HPBE is a key chiral intermediate of this drug, the research on the new method of R-HPBE synthesis still has great practical significance and industrial application prospects.

Methods of synthesizing R-HPBE include chemical methods and biological methods. Among them, the chemical method is based on cheap and readily available raw materials, and finally synthesizes R-HPBE through a multi-step reaction. For example, benzoic acid and pyruvic acid are subjected to a multi-step reaction to form 2-hydroxy-4-phenylbutyric acid, followed by racemization and esterification to synthesize (R)-HPBE; and as by diethyl oxalate and Ethyl phenylpropionate was used as a starting material to synthesize ethyl 2-carbonyl-4-phenylbutanoate and then subjected to asymmetric hydrogenation to obtain (R)-HPBE. The chemical method has obvious advantages in product scale and can reach a production scale of 500 kg. However, the chemical method uses a catalyst contaminated with heavy metals such as Pt, which cannot meet the requirements of green chemistry and has high cost. Further, chemical methods have disadvantages such as low yield, relatively harsh reaction conditions, high purity of the substrate, and low optical activity of the obtained product, and thus are not suitable for scale production.

At present, there are two main ways to synthesize (R)-HPBE by biological method. One is to use lipase to resolve rac-HPBE to obtain (R)-HPBE, or to split rac-HPBA and then ethylate to obtain (R)- HPBE. Another approach is to asymmetrically reduce OPBE or OPBA with a ketoreductase to form (R)-HPBE or (R)-HPBA, and (R)-HPBA can be further esterified to synthesize (R)-HPBE. With ketone reductase for asymmetric reduction, the theoretical yield can reach 100%, while the theoretical yield of lipase can only reach 50%. Ketoreductase has certain advantages, but ketoreductase catalysis usually requires additional price. Expensive coenzyme NAD ⁺ /NADP ⁺ .

The Applicant has applied for a patent for the asymmetric reduction of ketone reductase (R)-HPBE (CN2014105418998), wherein the ketone ester can achieve an optical purity of 98% or more by the action of the ketoreductase. However, the stability of the raw material OPBE is poor, the separation and purification process of the product is complicated, and the production cost is high.

Summary of the invention

The technical problem to be solved by the present invention is that

One of the objects of the present invention is to provide a use of a polypeptide in the production of a compound of formula A or a downstream product of a compound of formula A as a precursor.

Another object of the invention is to provide a process for the production of a compound of formula A.

A further object of the invention is to provide a compound producing strain of formula A.

A further object of the present invention is to provide a method of constructing a strain of a compound of formula A.

In a first aspect of the invention there is provided a use of a polypeptide for the production of a compound of formula A or a downstream product of a compound of formula A, wherein:

(a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

(b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- a polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) formed by substitution, deletion or addition of three, most preferably one amino acid residues;

In the formula,

R ₁ represents hydrogen, or 1, 2, 3 or 4 substituents selected from the group consisting of halogen, -OH, substituted or unsubstituted C ₁ -C ₈ alkyl, substituted or unsubstituted C ₃ -C ₈ a cycloalkyl, substituted or unsubstituted C ₁ -C ₈ alkoxy group, or a substituted or unsubstituted C ₃ -C ₈ cycloalkoxy group, wherein said substitution has one or more selected from the group consisting of Substituents: halogen, -OH, -NH ₂ , -CN, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, -NH(C ₁ -C ₃ alkyl), -N(C ₁ -C ₃ alkyl) ₂ ;

R ₂ is a substituted or unsubstituted C ₁ -C ₁₀ linear alkylene group, a substituted or unsubstituted C ₃ -C ₈ cycloalkyl group, a substituted or unsubstituted C ₂ -C ₆ straight or branched alkenyl group And -(CH ₂ ) _n -, -(CH=CH) _p - and a substituted or unsubstituted C ₂ -C ₁₀ linear or branched alkynyl group, wherein n is selected from any integer from 0 to 10, p is A positive integer of 1-5.

In another preferred embodiment, the compound of formula A is formed by asymmetric reduction of a prochiral carbonyl acid compound.

In another preferred embodiment, the prochiral carbonyl acid compound is a compound of formula B:

Wherein R ₁ and R ₂ are as defined above.

In another preferred embodiment, the prochiral carbonyl acid compound comprises the following compound or a pharmaceutically acceptable salt thereof:

In another preferred embodiment, R ₁ is H or Cl.

In another preferred embodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another preferred embodiment, R ₁ is H and n is 0, ₁ , 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another preferred embodiment, R ₁ is Cl and n is 0, ₁ , 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another preferred embodiment, R ₁ is H and p is 1.

In another preferred embodiment, R ₁ is Cl and p is 1.

In another preferred embodiment, the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.

In another preferred embodiment, the compound of formula A comprises the following compound or a pharmaceutically acceptable salt thereof:

In another preferred embodiment, the downstream products of the compound of formula A include: enalapril, benazepril, ramipril, cilazapril, cepril, and spironolide.

In a second aspect of the invention, there is provided a compound of formula A producing a strain which expresses the following polypeptide:

(a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

(b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), which is formed by substitution, deletion or addition of three, most preferably one amino acid residues.

In another preferred embodiment, the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.

In another preferred embodiment, the production strain is a bacterium.

In another preferred embodiment, the production strain is Escherichia coli.

In another preferred embodiment, the E. coli is selected from the group consisting of E. coli BL21 (DE3), E. coli C2566.

In a third aspect of the invention, there is provided a method of producing a compound of formula A, the method comprising:

(a) in the liquid reaction system, using the compound of the formula B as a substrate, in the presence of a coenzyme, under the catalysis of a stereoselective lactate dehydrogenase (D-lactate dehydrogenase), the reaction shown in the reaction formula I is carried out. Thereby forming a compound of formula A;

In Reaction Scheme I, R ₁ and R ₂ are as defined above;

(b) optionally isolating the compound of formula A from the reaction system after the reaction of the previous step.

In another preferred embodiment, the concentration of the compound of the formula B in the reaction system is from 1 g/L to 1000 g/L, preferably from 5 g/L to 500 g/L, more preferably from 10 g/L to 100 g. /L, optimally 20g/L-80g/L.

In another preferred embodiment, the concentration of the stereoselective lactate dehydrogenase in the reaction system is 1 × 10 ² U / L - 1 × 10 ⁵ U / L, preferably 1 × 10 ³ U /L-1 × 10 ⁵ U / L, more preferably 5 × 10 ³ U / L - 1 × 10 ⁵ U / L, and even more preferably 1 × 10 ⁴ U / L - 1 × 10 ⁵ U / L, preferably 5 × 10 ⁴ U - 1 × 10 ⁵ U / L.

In another preferred embodiment, the stereoselective lactate dehydrogenase is:

(a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

(b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), which is formed by substitution, deletion or addition of three, most preferably one amino acid residues.

In another preferred embodiment, the stereoselective lactate dehydrogenase is:

At one end of the amino acid sequence shown in SEQ ID NO: 1, one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) formed by the addition of three, most preferably one amino acid residues.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.

In another preferred embodiment, the compound of formula A is R-HPBA.

In another preferred embodiment, the compound of formula A is (R)-(E)-2-hydroxy-4-phenyl-3-butenoic acid.

In another preferred embodiment, a coenzyme is also present in the reaction system.

In another preferred embodiment, the coenzyme is selected from the group consisting of NADH, NADPH, NAD, NADP, or a combination thereof.

In another preferred embodiment, the concentration of the coenzyme in the reaction system is from 5 mg/L to 1000 mg/L, preferably from 10 mg/L to 800 mg/L, more preferably from 20 mg/L to 600 mg/L. More preferably, it is 50 mg/L to 500 mg/L, and most preferably 100 mg/L to 300 mg/L.

In another preferred embodiment, an enzyme for coenzyme regeneration is also present in the reaction system.

In another preferred embodiment, the concentration of the enzyme for coenzyme regeneration is 1 × 10 ² U / L - 1 × 10 ⁶ U / L, preferably 1 × 10 ³ U / L - 1 × 10 ⁵ U/L, more preferably 5 × 10 ³ U / L - 2 × 10 ⁵ U / L, most preferably 1.5 × 10 ⁴ U / L - 1.5 × 10 ⁵ U / L.

In another preferred embodiment, the enzyme for coenzyme regeneration is selected from the group consisting of formate dehydrogenase, glucose dehydrogenase, or a combination thereof.

In another preferred embodiment, the concentration of the glucose dehydrogenase is 1 × 10 ² U / L - 1 × 10 ⁶ U / L, preferably 1 × 10 ³ U / L - 5 × 10 ⁵ U / L, more preferably 1 × 10 ⁴ U / L - 1 × 10 ⁵ U / L, most preferably 4 × 10 ⁴ U / L - 5 × 10 ⁴ U / L.

In another preferred embodiment, the formate dehydrogenase concentration is 1 × 10 ² U / L - 1 × 10 ⁶ U / L, preferably 1 × 10 ³ U / L - 5 × 10 ⁵ U / L, more preferably 1 × 10 ⁴ U / L - 1 × 10 ⁵ U / L, most preferably 4 × 10 ⁴ U / L - 5 × 10 ⁴ U / L.

In another preferred embodiment, in the step (a), the temperature is from 10 ° C to 50 ° C, preferably from 15 ° C to 40 ° C, more preferably from 20 ° C to 30 ° C.

In another preferred embodiment, in step (a), the pH is from 6 to 10, preferably from 6.5 to 9.0, more preferably from 7.5 to 8.0.

In a fourth aspect of the invention, there is provided a method of producing a compound of formula A, the method comprising:

1) cultivating the production strain described in the second aspect of the invention under production conditions, thereby obtaining a compound of formula A;

2) Optionally, the compound of formula A is isolated from the culture system of 1).

In a fifth aspect of the invention, there is provided a method of constructing a strain of a compound of formula A, the method comprising:

The strain is made to comprise an expression vector expressing a polypeptide or such that a gene expressing the following polypeptide is integrated into the genome of the strain, the polypeptide being:

(a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

(b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), which is formed by substitution, deletion or addition of three, most preferably one amino acid residues.

In another preferred embodiment, the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.

In another preferred embodiment, the sequence of the gene is selected from the group consisting of:

(i) the sequence set forth in SEQ ID NO. 5 or SEQ ID NO: 7;

(ii) a polynucleotide complementary to any of the sequences defined in (i); or

(iii) having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more of any of the sequences defined in (i) Any polynucleotide or complementary sequence of sequence identity.

In another preferred embodiment, the sequence of the gene is set forth in SEQ ID NO: 7.

In another preferred embodiment, the gene is constructed on an expression vector.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Detailed ways

Through extensive and intensive research, the inventors have unexpectedly discovered a polypeptide (D-lactate dehydrogenase) which is capable of stereoselectively catalyzing the reaction of formula I, such as OPBA, E. -2--Oxo-4-phenyl-3-butenoic acid) is efficiently converted to a compound of formula A (eg R-HPBA, (R)-(E)-2-hydroxy-4-phenyl-3-butyl Acetate), conversion rate ≥98%, chiral ee value ≥99%, thereby greatly improving production efficiency and reducing production costs. The raw material for the reduction reaction by using the lactate dehydrogenase of the present invention is easy to obtain, has high yield, low cost, and is easy to be enlarged, and is suitable for large-scale production. The present invention has been completed on this basis.

Definition of Terms

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined.

As used herein, when used in reference to a particular recited value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "containing" or "including" may be open, semi-closed, and closed. In other words, the terms also include "consisting essentially of," or "consisting of."

The following terms used in the specification and claims have the meanings commonly understood by those skilled in the art, unless otherwise defined. All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety herein in their entirety herein

The above description and the following detailed description are to be considered as illustrative and not restrictive. In the present application, the use of the singular includes the plural unless otherwise specified. It must be noted that the singular forms used in the specification and claims are in the It should also be noted that "or" or "or" is used to mean "and/or" unless otherwise indicated. Furthermore, the terms "comprising" or "including" may be open, semi-closed, and closed. In other words, the terms also include "consisting essentially of," or "consisting of."

The definition of standard chemical terms can be found in references (including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED." Vols. A (2000) and B (2001), Plenum Press, New York. Conventional methods within the skill of the art, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy and pharmacological methods, are employed unless otherwise indicated. Unless specifically defined, the terms used herein in the descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and pharmaceutical chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. For example, the reaction can be carried out and purified using the manufacturer's instructions for use of the kit, or in a manner well known in the art or as described in the present invention. The above techniques and methods can generally be carried out according to conventional methods well known in the art, as described in the various summaries and more specific references cited and discussed in this specification. In the present specification, the group and its substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.

When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left. For example, -CH ₂ O- is equivalent to -OCH ₂ -.

The section headings used herein are for the purpose of organizing articles only and are not to be construed as limiting the subject matter. All documents or parts of the literature cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals and papers, are hereby incorporated by reference in their entirety.

Certain chemical groups defined herein are preceded by a simplified symbol to indicate the total number of carbon atoms present in the group. For example, C1-C6 alkyl refers to an alkyl group as defined below having a total of from 1 to 6 carbon atoms. The total number of carbon atoms in the simplified symbol does not include carbon that may be present in the substituents of the group.

In addition to the foregoing, when used in the specification and claims of the present application, the following terms have the meanings indicated below unless otherwise specifically indicated.

In the present application, the term "halogen" means fluoro, chloro, bromo or iodo.

"Hydroxy" means an -OH group.

In the present application, as a group or part of another group (for example, in a group such as a halogen-substituted alkyl group), the term "alkyl" means a fully saturated straight or branched hydrocarbon chain group, It consists only of carbon atoms and hydrogen atoms, has, for example, 1 to 7 carbon atoms, and is linked to the rest of the molecule by a single bond, including, for example, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl Base, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, heptyl and the like.

Enantiomeric excess (ee) is generally used to characterize the excess value of one enantiomer relative to the other enantiomer in a chiral molecule.

Peptide

The terms "polypeptide" or "polypeptide of the invention" or "polypeptide of the invention" or "D-lactate dehydrogenase" or "stereoselective lactate dehydrogenase" as used herein have the same meaning and are used interchangeably herein. , both refer to a protein having a catalytically active compound which produces a compound of formula A. This polypeptide is naturally absent from E. coli and is an exogenous protein.

Based on the knowledge of the prior art, it will be readily apparent to one of ordinary skill in the art that alteration of a few amino acid residues in certain regions of the polypeptide, such as non-significant regions, does not substantially alter biological activity, for example, sequences that are suitably substituted for certain amino acids. It does not affect its activity (see Watson et al, Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224). Thus, one of ordinary skill in the art would be able to implement such substitutions and ensure that the resulting molecules still possess the desired biological activity.

Thus, in a specific embodiment, the polypeptide of the present invention may be: (a1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; or (b1) passing one or more of the amino acid sequence shown in SEQ ID NO: 1. Substituents, deletions or additions of preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 amino acid residues A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1).

In a preferred embodiment, the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.

In a preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.

In a preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO:3.

In a specific embodiment, the polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 1, the coding sequence of which is set forth in SEQ ID NO: 5.

In a specific embodiment, the polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 3, the coding sequence of which is set forth in SEQ ID NO: 7.

In the present invention, the polypeptide of the present invention comprises up to 20, preferably up to 10, preferably up to 8, and preferably up to 3, compared to the polypeptide of the amino acid sequence of SEQ ID NO: 1. More preferably up to two, preferably up to one amino acid is replaced by a similar or similar amino acid. Mutants of these conservative variations can be produced according to, for example, amino acid substitutions as shown in the table below.

Initial residue	Representative substituted residues	Preferred substituted residue
Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
Asn(N)	Gln;His;Lys;Arg	Gln
Asp(D)	Glu	Glu
Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
Glu(E)	Asp	Asp
Gly(G)	Pro; Ala	Ala
His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu;Val;Met;Ala;Phe	Leu
Leu(L)	Ile;Val;Met;Ala;Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg

Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Leu;Val;Ile;Ala;Tyr	Leu
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr;Phe	Tyr
Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala	Leu

The invention also provides polynucleotides encoding the polypeptides of the invention. The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.

Therefore, as used herein, "includes", "includes" or "includes" includes "includes", "consisting essentially of", "consisting essentially of", and "consisting of"; "Consisting", "consisting essentially of" and "consisting of" belong to the subordinate concept of "contains," "has," or "includes."

In a specific embodiment, the homology or sequence identity may be 80% or more, preferably 90% or more, more preferably 95% to 98%, and most preferably 99% or more.

Methods for determining sequence homology or identity as known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. M. Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). The preferred method of determining identity is to obtain the largest match between the sequences tested. The method of determining identity is compiled in a publicly available computer program. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990). The BLASTX program is available to the public from NCBI and other sources (BLAST Handbook, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Use of the polypeptide

The present inventors have unexpectedly discovered that the polypeptide of the present invention has D-lactate dehydrogenase activity and can be used to produce a compound of formula A or a downstream product of a compound of formula A as a precursor.

In a specific embodiment, the polypeptide is:

(a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

(b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- a polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) formed by substitution, deletion or addition of three, most preferably one amino acid residues;

Wherein R ₁ and R ₂ are as defined above.

In a preferred embodiment, the compound of formula A is formed by asymmetric reduction of a prochiral carbonyl acid compound.

Typically, the prochiral carbonyl acid compound is a compound of formula B:

Wherein R ₁ and R ₂ are as defined above.

Typically, the prochiral carbonyl acid compound comprises the following compound or a pharmaceutically acceptable salt thereof:

In a preferred embodiment, the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.

Typically, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

In a specific embodiment, the polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 1, the coding sequence of which is set forth in SEQ ID NO: 5.

In a specific embodiment, the polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 3, the coding sequence of which is set forth in SEQ ID NO: 7.

In a preferred embodiment, the compound of formula A comprises R-HPBA or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of formula A comprises (R)-(E)-2-hydroxy-4-phenyl-3-butenoic acid or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the downstream products of the compound of formula A include: enalapril, benazepril, ramipril, cilazapril, cepril, sulpiride.

Compound A production strain

The present inventors have unexpectedly discovered that a strain expressing a polypeptide of the present invention is capable of stereoselectively catalyzing a reaction of the formula I, such as OPBA, E-2-oxo-4-phenyl-3- Butenoic acid) is efficiently converted to a compound of formula A (eg R-HPBA, (R)-(E)-2-hydroxy-4-phenyl-3-butenoic acid), conversion ≥98%, chiral ee The value is ≥99%.

In a specific embodiment, the strain expresses the following polypeptide:

(a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

(b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), which is formed by substitution, deletion or addition of three, most preferably one amino acid residues.

In another preferred embodiment, the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10 at either end of the amino acid sequence set forth in SEQ ID NO:1. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), formed by addition of 1-8, more preferably 1-3, most preferably 1 amino acid residues Derived polypeptide.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% of the amino acid sequence set forth in SEQ ID NO:1. 96%, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.

In another preferred embodiment, the amino acid sequence of the polypeptide is set forth in SEQ ID NO:3.

In another preferred embodiment, the production strain is a bacterium, preferably E. coli (e.g. E. coli BL21 (DE3), E. coli C2566).

Method for producing a compound of formula A

In the present invention, the stereoselective lactate dehydrogenase (D-lactate dehydrogenase) can be used in various forms. For example, resting cells or wet cells expressing the stereoselective lactate dehydrogenase of the present invention may be used, or various forms such as a crude enzyme solution, a pure enzyme or a crude enzyme powder may be used, or an immobilized enzyme may be used.

In a preferred embodiment, the method of producing a compound of formula A comprises:

(a) in the liquid reaction system, using the compound of the formula B as a substrate, in the presence of a coenzyme, under the catalysis of a stereoselective lactate dehydrogenase (D-lactate dehydrogenase), the reaction shown in the reaction formula I is carried out. Thereby forming a compound of formula A;

In Reaction Scheme I, R ₁ and R ₂ are as defined above;

(b) optionally isolating the compound of formula A from the reaction system after the reaction of the previous step.

In another preferred embodiment, the method of producing a compound of formula A comprises:

1) cultivating the compound of the formula A of the present invention to produce a compound of the formula A according to the production conditions;

2) Optionally, the compound of formula A is isolated from the culture system of 1).

Method for constructing compound of formula A production

The inventors have unexpectedly discovered that a compound producing strain of the formula A having high conversion can be constructed by including the strain comprising an expression vector expressing the polypeptide of the present invention or by integrating a gene expressing the polypeptide of the present invention into the genome of the strain.

In another specific embodiment, the method further comprises determining the conversion of the resulting strain and/or the yield of the compound of formula A to verify the resulting strain.

The main advantages of the invention:

(1) using a cheap and readily available raw material carbonyl acid compound as a starting material;

(2) obtaining a chiral pure hydroxy acid compound by using the lactate dehydrogenase of the present invention, for example, OPBA can obtain R-HPBA under the catalysis of the lactate dehydrogenase of the present invention, and can obtain a higher yield. Obtaining the final product, and the ee value of the product is >98%;

(3) The reduction reaction using the lactate dehydrogenase of the present invention is low in cost and easy to enlarge, and is suitable for large-scale production.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

The reagents and starting materials used in the present invention are commercially available.

Example 1 Acquisition of lactate dehydrogenase gene

The genomes of Staphylococcus epidermidis ATCC 12228 and Staphylococcus epidermidis strain C10C retrieved from the NCBI database have been sequenced, and the genebank accession numbers are NC_004461 and NZ_JQHC01000048.1, respectively. From the genomic sequence, lactate dehydrogenase was found and found to have four D-lactate dehydrogenases SEQ ID NO: 1-4 (accession numbers are NP_765629.1 and NP_765676.1; WP_002484424.1 and WP_002456698.1, respectively). Primers were designed according to the DNA sequences SEQ ID NO: 5-6, see SEQ ID NOs: 9-12:

Table 1:

PCR was carried out using genomic DNA as a template. The experimental conditions were as follows:

After purification and recovery of the PCR product, the cloning enzyme was ligated into the NdeI&HindIII site of the expression vector pET28a to obtain two expression plasmids.

The gene sequences SEQ ID NO: 7 and SEQ ID NO: 8 were obtained by gene synthesis.

SEQ ID NO: 1

SEQ ID NO: 5

SEQ ID NO: 2

SEQ ID NO: 6

SEQ ID NO: 3

SEQ ID NO: 7

SEQ ID NO: 4

SEQ ID NO: 8

Example 2 Plasmid Transformation Screening and Bacterial Fermentation

The recombinant expression plasmid of Example 1 was transformed into E. coli BL21 (DE3) competent cells in E. coli BL21 (DE3) competent cells under the conditions of 42 ° C, heat shock for 90 seconds, and the positive recombinants were plated on a plate containing kanamycin antibiotics. Screening, picking monoclonal, colony PCR to verify positive clones. The recombinant recombinant strain was cultured to obtain the positive recombinant transformant E.coli BL21(DE3)/pET28a-LDH1(NP_765629.1), E.coli BL21(DE3)/pET28a-LDH2(NP_765676.1), E.coli BL21(DE3 ) / pET28a-LDH3 (WP_002484424.1), and E. coli BL21 (DE3) / pET28a - LDH4 (WP_002456698.1).

High-density fermentation of LDH: The above recombinant Escherichia coli was inoculated into 200 mL of LB medium containing 50 ug/mL kanamycin, and cultured at 37 ° C, 180-220 rpm for 10-16 h. The cultured seeds were inoculated in a 3 L upper tank fermentation medium (M9) at a ratio of 10% (v/v) (glucose 4 g/L, disodium hydrogen phosphate 12.8 g/L, potassium dihydrogen phosphate 3 g/L). , ammonium chloride 1g / L, sodium sulfate 0.5g / L, calcium chloride 0.0152g / L, magnesium chloride hexahydrate 0.41g / L), at 25-35 ° C, 300-800rpm, air flow 2-6L / min Culture under conditions. After 6-10 hours of culture, a feed medium containing 60% glycerol was fed at a rate of 5-20 mL/h until the end of the fermentation. When the feed medium was fed for several hours until the OD600 reached 20-40, induction was started by adding 0.1-1 mM IPTG. After induction for 5-15 hours, the cells were placed in a can, and the cells were collected by centrifugation at 5000 rpm.

Example 3 Enzyme activity assay

100 g of the cells were taken, 400 mL of PBS buffer was added, and the mixture was disrupted three times (800-1000 Pa) by high pressure, and the fragments were removed by centrifugation at 12000 rpm to obtain an enzyme solution. The enzyme solution was lyophilized to obtain 8 g of lyophilized powder, which was stored at -80 °C.

The enzyme activity (U) is defined as the amount of enzyme required to consume 1 μmol of NADH per minute.

The enzyme activity (U) was measured by measuring the NADH consumption rate at a wavelength of 340 nm using a spectrophotometer at 50 ° C in a 50 mM phosphate buffer, an OPBA concentration of 1 mM, and a NADH concentration of 1 mM.

Enzyme activity list

Serial number	Specific enzyme activity (U/mg)
LDH1	8.2
LDH2	1.9
LDH3	7.5
LDH4	1.3

Examples 4-7 Asymmetric Reduction of OPBA

In the catalytic reaction system, the concentration of OPBA is 0.1M, the concentration of NAD+ is 0.02 mM, D-lactate dehydrogenase is 1 to 10 g/L, and the glucose dehydrogenase GDH is 0.1 to 3 g/L. Reaction temperature: 20 to 35 ° C, reaction time: 1 h, pH between 6.5 and 7.5 was controlled with 1 M NaOH or saturated Na ₂ CO ₃ . After the end of the reaction, the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain HPBA. The reaction conversion rate was determined by HPLC, and the ee value was detected by chiral HPLC.

Example	sequence	Conversion rate	R:S
4	1, D-LDH1	100%	100:0
5	2, D-LDH2	10%	60:40
6	3, D-LDH3	99%	99:1
7	4, D-LDH4	5%	55:45

Example 8 One-time feeding

In the 1 L reaction system, OPBA 50 g, NAD+ concentration was 0.1 g, D-lactate dehydrogenase (sequence 1) 30000 U, formate dehydrogenase 50000 U, and sodium formate 50 g. Reaction temperature: 20-30 ° C, reaction time: 8 h, pH value 6.5-7.5 was controlled with 1 M NaOH. After the completion of the reaction, the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain crude R-HPBA. The reaction conversion rate was 99% and the ee value was 99.9% by HPLC.

Example 9 Batch feeding

In the 1 L reaction system, OPBA 50 g was added, the concentration of NAD+ was 0.1 g, D-lactate dehydrogenase (sequence 1) was 30000 U, glucose dehydrogenase was 40000 U, and glucose was 50 g. Reaction temperature: 20 ~ 30 ° C, with 1M NaOH control pH between 7.5 ~ 8.0. The progress of the reaction was monitored according to the amount of NaOH. After the pH was stabilized, 30 g of OPBA was added and the reaction was continued for 10 hours. After the reaction was completed, the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain crude R-HPBA. The reaction conversion rate was 100% and the ee value was 99.9% by HPLC.

Example 10 One-time feeding

In a 1 L reaction system, 50 g of E-2-oxo-4-phenyl-3-butenoic acid was added, the concentration of NAD+ was 0.1 g, D-lactate dehydrogenase (sequence 1) was 30000 U, and formate dehydrogenase was 40000 U. Sodium formate 50g. Reaction temperature: 20-30 ° C, reaction time: 24 h, pH value 7.5-8.0 was controlled with 1 M NaOH. After the reaction, the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain (R)-(E)-2-hydroxy-4-phenyl-3-butyl. Crude olefinic acid. The reaction conversion rate was 98% and the ee value was 99% by HPLC.

Example 11 R-HPBA Esterification Synthesis of R-HPBE

18 g of R-HPBA was dissolved in 100 mL of ethanol, and 14. mL of SOCl ₂ was added dropwise in an ice water bath. After the completion of the dropwise addition, the temperature was raised to 80 ° C and refluxed for 4 to 8 hours. After the TLC reaction was completed, the solvent was distilled off under reduced pressure. 19.8 g of a pale yellow oil was obtained, the yield was 95.2%, the purity of GC was >99.5%, and the ee was 99.9%.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

Claims (14)

1. Use of a polypeptide for the production of a compound of formula A or a downstream product of a compound of formula A, wherein the polypeptide is:

   (a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

   (b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- a polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) formed by substitution, deletion or addition of three, most preferably one amino acid residues;

   

   In the formula,

   R ₁ represents hydrogen, or 1, 2, 3 or 4 substituents selected from the group consisting of halogen, -OH, substituted or unsubstituted C ₁ -C ₈ alkyl, substituted or unsubstituted C ₃ -C ₈ a cycloalkyl, substituted or unsubstituted C ₁ -C ₈ alkoxy group, or a substituted or unsubstituted C ₃ -C ₈ cycloalkoxy group, wherein said substitution has one or more selected from the group consisting of Substituents: halogen, -OH, -NH ₂ , -CN, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, -NH(C ₁ -C ₃ alkyl), -N(C ₁ -C ₃ alkyl) ₂ ;

   R ₂ is a substituted or unsubstituted C ₁ -C ₁₀ linear alkylene group, a substituted or unsubstituted C ₃ -C ₈ cycloalkyl group, a substituted or unsubstituted C ₂ -C ₆ straight or branched alkenyl group And -(CH ₂ ) _n -, -(CH=CH) _p - and a substituted or unsubstituted C ₂ -C ₁₀ linear or branched alkynyl group, wherein n is selected from any integer from 0 to 10, p is A positive integer of 1-5.
2. The use according to claim 1, wherein the polypeptide is one or more, preferably 1-20, more preferably 1-15, more at either end of the amino acid sequence shown in SEQ ID NO: 1. Preferably, the addition of 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residues is provided by SEQ ID NO: 1 having the function of the polypeptide of (a1) A polypeptide derived from a polypeptide of an amino acid sequence.
3. The use according to claim 1, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 3.
4. The use according to any one of claims 1 to 3, wherein the compound of the formula A comprises the following compound or a pharmaceutically acceptable salt thereof:

   
5. The use according to any one of claims 1 to 3, wherein the downstream product of the compound of formula A comprises: enalapril, benazepril, ramipril, sira Puli, 吲哚普利, 螺普利.
6. A compound producing strain of the formula A, characterized in that the strain expresses the following polypeptide:

   (a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

   (b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), which is formed by substitution, deletion or addition of three, most preferably one amino acid residues.
7. The production strain according to claim 6, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 3.
8. A method of producing a compound of formula A, characterized in that the method comprises:

   (a) in a liquid reaction system, using a compound of formula B as a substrate, in the presence of a coenzyme, under the catalysis of stereoselective lactate dehydrogenase, the reaction of formula I is carried out to form a compound of formula A;

   

   In Reaction Scheme I, R ₁ and R ₂ are as defined above;

   (b) optionally isolating the compound of formula A from the reaction system after the reaction of the previous step.
9. The method according to claim 8, wherein a coenzyme is further present in said reaction system.
10. The method according to claim 8, wherein in the reaction system, an enzyme for coenzyme regeneration is further present.
11. A method of producing a compound of formula A, characterized in that the method comprises:

    1) cultivating the production strain of claim 6 under production conditions, thereby obtaining a compound of formula A;

    2) Optionally, the compound of formula A is isolated from the culture system of 1).
12. A method for constructing a strain of a compound of formula A, characterized in that the method comprises:

    The strain is made to comprise an expression vector expressing a polypeptide or such that a gene expressing the following polypeptide is integrated into the genome of the strain, the polypeptide being:

    (a1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or

    (b1) The amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1), which is formed by substitution, deletion or addition of three, most preferably one amino acid residues.
13. The method of claim 12, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 3.
14. The method according to claim 12, wherein the sequence of the gene is selected from the group consisting of:

    (i) the sequence set forth in SEQ ID NO. 5 or SEQ ID NO: 7;

    (ii) a polynucleotide complementary to any of the sequences defined in (i); or

    (iii) having at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more of any of the sequences defined by (i) Any polynucleotide or complementary sequence of sequence identity.