CN117778342A - Carbonyl reductase mutant and application thereof in synthesis of 11 beta-hydroxy steroid compounds - Google Patents

Abstract

The invention provides a carbonyl reductase mutant and application thereof in 11 beta-hydroxy steroid synthesis. The mutein provided by the invention has the advantage of obviously improving the activity of catalyzing 11-site carbonyl steroid to reduce to 11 beta-hydroxy steroid. The most suitable carbonyl reductase mutant strain is applied to the production process of carbonyl steroid reduction, can improve the carbonyl reduction capacity of the carbonyl reductase mutant strain, and can convert 20g/L of cortisone and 70g/L of prednisone to generate hydrocortisone and prednisolone.

Description

Carbonyl reductase mutant and application thereof in synthesis of 11 beta-hydroxy steroid compounds

Technical Field

The invention belongs to the fields of molecular biology and enzyme engineering, relates to a carbonyl reductase mutant and application thereof in 11 beta-hydroxy steroid synthesis, and in particular relates to a carbonyl reductase mutant, a recombinant host cell, a cell culture and a method for synthesizing the 11 beta-hydroxy steroid by an enzyme method.

Background

11 beta-hydroxy steroid compounds such as hydrocortisone and prednisolone belong to adrenocortical hormone drugs, and can be used for diseases caused by adrenal insufficiency, rheumatoid arthritis, rheumatic fever, gout, bronchial asthma and the like, and also can be used for allergic dermatitis, seborrheic dermatitis, pruritus and the like. The biological technology is adopted to directly produce steroid hormone drug bulk drugs, and the technology is still in a development stage, xue Weiying and the like, and ARTP-LiCl composite mutagenesis is carried out on a production strain blue Absidia of hydrocortisone, so that a strain AL-172 with better genetic stability is obtained, and when the substrate concentration is 3.5 g/L, the conversion rate of Hydrocortisone (HC) reaches 72.52 percent (Xue Weiying, the high conversion rate strain of hydrocortisone is selected by composite mutagenesis, shandong university of agriculture report (Nature science edition), 2018,49 (3): 396-401).

Highly regioselective and stereoselective 11 beta-hydroxysteroid dehydrogenase (11 beta-HSDH) provides a potential biocatalyst for asymmetric reduction of cortisone to optically pure HC, avoiding non-selective hydroxylation reactions. The soluble expression of 11 beta-HSDH derived from guinea pigs was improved in the early stage of the study group, and then the recombinant expression was co-expressed with glucose dehydrogenase, so that highly selective biosynthesis of hydrocortisone (Zhang et al, engineering a hydroxysteroid dehydrogenase to improve its soluble expression for the asymmetric reduction of cortisone to, 11 beta-hydroortisine) could be achieved.Applied Microbiology and Biotechnology 2014, 98(21) 8879-8886.), the conversion concentration reached 10g/L.

Disclosure of Invention

Based on the results of the previous study, we excavate a strain derived fromCricetulus griseusThe 11 beta-HSDH is modified, so that the catalytic activity of the enzyme is improved, and the efficient biosynthesis of hydrocortisone and prednisolone is realized. And a production strain suitable for synthesizing the steroid medicine bulk drug is constructed, the chemical synthesis difficulty is reduced, the synthesis steps are simplified, the yield of the bulk drug is improved, the pollutant emission in the synthesis process is reduced, and a new environment-friendly synthesis process is established.

Therefore, the invention provides a carbonyl reductase mutant, a coding gene and application thereof, and the carbonyl reductase mutant can improve enzyme activity, substrate tolerance and the like, so that the carbonyl reductase mutant meets the industrial use requirements.

The invention is derived fromCricetulus griseusThe carbonyl reductase (Cg-11 beta-HSDH, NCBI: XP_ 003498616.2) of the plant is subjected to directed evolution to obtain mutants with improved enzyme activity and substrate tolerance, and the mutants catalyze cortisone to generate hydrocortisone and prednisone to generate prednisolone.

The present invention first provides a carbonyl reductase mutant, wherein the mutant is selected from any one of the following groups (I) - (IV):

(I) The carbonyl reductase mutant comprises a mutation at one or more positions Y103, M106, M151, H157, V160, L197, L211, L264 and T265 corresponding to the sequence shown in SEQ ID NO.1, compared with the sequence shown in SEQ ID NO. 1;

(II) a mutant which has at least 98% sequence identity with the sequence shown in (I) and does not include the sequence shown in SEQ ID NO. 1;

(III) a mutant encoded by a polynucleotide which hybridizes under very high stringency conditions with a polynucleotide as set forth in (a) or (b):

(a) A polynucleotide encoding a mutant of the amino acid sequence as shown in (I);

(b) The full-length complementary polynucleotide of (a);

(IV) a fragment of the mutant as set forth in any one of (I), (II) or (III), and which fragment still has carbonyl reductase activity.

Preferably, the mutant corresponds to a mutation of tyrosine (Y) at position 103 of the sequence shown in SEQ ID NO.1 into alanine (A), glycine (G), glutamine (Q), and/or

The mutant is mutated into serine (S), alanine (A), glycine (G) and/or (L) corresponding to methionine (M) at position 106 of the sequence shown in SEQ ID NO.1,

the mutant corresponds to the 151 th methionine (M) mutation of the sequence shown in SEQ ID NO.1 into valine (V), asparagine (N) and leucine (L), and/or,

the mutant corresponds to the 157 th histidine (H) mutation of the sequence shown in SEQ ID NO.1 into alanine (A), aspartic acid (D), glycine (G), and/or,

the mutant is mutated into isoleucine (I) and leucine (L) corresponding to valine (V) at position 160 of the sequence shown in SEQ ID NO.1, and/or,

the mutant is mutated from leucine (L) at 197 th position of a sequence shown in SEQ ID NO.1 into alanine (A) and glycine (G); leucine (L) at position 211 to alanine (A), glycine (G), and/or,

the mutant is mutated into alanine (A), glycine (G) and/or (L) corresponding to leucine (L) at position 211 of the sequence shown in SEQ ID NO.1,

the mutant is mutated into isoleucine (I), glycine (G) and/or corresponding to leucine (L) at position 264 of the sequence shown in SEQ ID NO.1,

the mutant is mutated into arginine (R) and lysine (K) corresponding to threonine (T) at 265 th position of a sequence shown in SEQ ID NO. 1.

Further, the carbonyl reductase mutant is a mutant comprising a mutation as shown in at least one of the following (c) to (k):

(c) The mutant is mutated into glycine (G) corresponding to tyrosine (Y) at 103 rd position of a sequence shown in SEQ ID NO. 1;

(d) The mutant is mutated into alanine (A) corresponding to methionine (M) at position 106 of the sequence shown in SEQ ID NO. 1;

(e) The mutant is mutated into valine (V) corresponding to methionine (M) at position 151 of the sequence shown in SEQ ID NO. 1;

(f) The mutant is mutated into aspartic acid (D) corresponding to histidine (H) at 157 th position of the sequence shown in SEQ ID NO. 1;

(g) The mutant is mutated into leucine (L) corresponding to valine (V) at position 160 of a sequence shown in SEQ ID NO. 1;

(h) The mutant is mutated into alanine (A) corresponding to leucine (L) at position 197 of the sequence shown in SEQ ID NO. 1;

(i) The mutant is mutated into alanine (A) corresponding to leucine (L) at position 211 of a sequence shown in SEQ ID NO. 1;

(j) The mutant is mutated into isoleucine (I) corresponding to leucine (L) 264 of the sequence shown in SEQ ID NO. 1.

(k) The mutant is mutated into arginine (R) corresponding to threonine (T) at 265 th position of a sequence shown in SEQ ID NO.1

In addition, the mutant includes deletion or addition of at least one amino acid residue at the N-terminal or C-terminal position of the above mutant.

The invention also provides a recombinant polypeptide, wherein the recombinant polypeptide comprises the carbonyl reductase mutant and an exogenous polypeptide fused with the mutant.

The invention further provides an isolated polynucleotide, wherein the polynucleotide comprises a nucleotide sequence encoding the carbonyl reductase mutant, or comprises a nucleotide sequence of the recombinant polypeptide.

The invention also provides a nucleic acid construct comprising said isolated polynucleotide, optionally operably linked to one or more regulatory sequences, the regulatory sequences being nucleotide sequences comprising a promoter and/or a ribosome binding site, which direct the expression of the mutant gene in a host cell and synthesis of a mutant enzyme.

The invention also provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated polynucleotide, or the nucleic acid construct.

The invention provides a recombinant host cell or recombinant genetically engineered bacterium, wherein the recombinant host cell or recombinant genetically engineered bacterium comprises the carbonyl reductase mutant, the recombinant polypeptide, the isolated polynucleotide, the nucleic acid construct, or the recombinant expression vector.

Preferably, the recombinant host cell or genetically engineered bacterium is Escherichia genusEscherichia) Genus ErwiniaErwinia) Serratia genusSerratia) Provedsia species @Providencia) The intestinal bacteria genusEnterobacteria) Salmonella genusSalmonella) Streptomyces genusStreptomyces) Genus PseudomonasPseudomonas) Genus BrevibacteriumBrevibacterium) Bacillus genusBacillus) Or corynebacterium genusCorynebacterium) Microorganisms of (a);

preferably, the recombinant host cell or genetically engineered bacterium is E.coli @Escherichia coli) Corynebacterium glutamicumCorynebacterium glutamicum) Or bacillus subtilis @Bacillus subtilis)；

More preferably, the recombinant host cell or genetically engineered bacterium is derived from E.coli @Escherichia coli)。

The invention further provides a cell culture comprising the recombinant host cell or recombinant genetically engineered bacterium.

The invention also provides application of the carbonyl reductase mutant, the recombinant polypeptide, the isolated polynucleotide, the nucleic acid construct, the recombinant expression vector, the recombinant host cell or recombinant genetically engineered bacterium or the cell culture in synthesizing 11 beta-hydroxy steroid compounds. Alternatively, cortisone is used as a substrate.

The present invention provides, inter alia, a method of synthesizing an 11 beta-hydroxysteroid compound, said method comprising the step of synthesizing an 11 beta-hydroxysteroid compound using said carbonyl reductase mutant, said recombinant polypeptide, said isolated polynucleotide, said nucleic acid construct, said recombinant expression vector, said recombinant host cell or recombinant genetically engineered bacterium, or said cell culture;

the method generates 11 beta-hydroxy steroid compounds under the action of carbonyl reductase.

Preferably, the concentration of the substrate is 10-70g/L;

optionally, in the step of synthesizing the 11 beta-hydroxysteroid compound, the pH is 6.0-9.0, the reaction temperature is 20-40 ℃, and the reaction time is more than 20 hours.

The present invention also provides a method for producing the mutant, which comprises the steps of culturing a recombinant host cell or recombinant genetically engineered bacterium containing the mutant, and recovering the carbonyl reductase mutant from the recombinant host cell, recombinant genetically engineered bacterium or a culture thereof.

The mutein provided by the invention has the advantage of obviously improving the activity of catalyzing 11-site carbonyl steroid to reduce to 11 beta-hydroxy steroid. The most suitable carbonyl reductase mutant strain is applied to the production process of carbonyl steroid reduction, can improve the carbonyl reduction capacity of the carbonyl reductase mutant strain, and can convert 20g/L of cortisone and 70g/L of prednisone to generate hydrocortisone and prednisolone.

Drawings

Figure 1A shows a cortisone pattern.

FIG. 1B shows a liquid-phase diagram of hydrocortisone.

Fig. 1C shows a prednisone liquid phase profile.

Fig. 1D shows a prednisolone liquid phase profile.

FIG. 2 shows an electrophoretogram of Cg-11. Beta. -HSDH wild-type and mutant proteins.

Detailed Description

The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, such as those not specified in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.

Example 1: construction of carbonyl reductase mutant library and screening of mutants

1. First round vitality reconstruction

Searching in a PDB database by taking Cg-11 beta-HSDH (the amino acid sequence is shown as SEQ ID NO: 1) as a template, carrying out homologous modeling by taking 2rbe, 3gmd, 3pdj and 4c7j which are high in similarity with target proteins in the PDB database as templates and utilizing DS software to obtain a simulated configuration, and then butting substrate molecules and selecting points.

The 10A range of spots and the substrate channel sites were analyzed centered on the substrate, where S150/Y163/K167 is the catalytic triplet. Analyzing the structure, and selecting the following sites from the structure to establish a mutation library: i101, Y103, T104, M106, 148, 149, M151, A152, 153, H157, V160, L192, L197, I198, 195, 196, T200, T202, A206, T207, L211, V261, K262, S263, L264, T265 were each subjected to saturation mutagenesis, and degenerate codon NNK was used to design mutation primers, with pET21 a-Cg-11. Beta. -HSDH as template. PCR was performed using a two-step method, using a high fidelity polymerase Pfx.

1) Construction of pET21a-Cg-11 beta-HSDH plasmid

The wild type amino acid sequence of the carbonyl reductase is shown as SEQ ID NO. 1. The corresponding nucleotide sequence is fully synthesized and cloned between restriction enzyme sites NdeI and XhoI of a pET-21a vector to obtain a recombinant plasmid pET21a-Cg-11 beta-HSDH, and the recombinant plasmid pET21a-Cg-11 beta-HSDH is further transformed into an expression hostE.coli BL21 (DE 3), and selecting positive clone to obtain recombinant expression transformantE.coli BL21(DE3)/pET21a-Cg-11β-HSDH。

2) Construction of carbonyl reductase single-point mutant library

The PCR was performed using pET21 a-Cg-11. Beta. -HSDH as a template and the primers shown in Table 1, and the forward primers I101 to I198 were combined with the reverse primers 1 to 260-R, respectively, and the forward primers 2 to 150-F were combined with the reverse primers 195-R to T265-R, respectively, and the PCR reaction system and reaction conditions were as follows:

a. first-step PCR reaction system and reaction conditions

Round 1: to a PCR reaction system having a total volume of 25. Mu.L, 15 ng of the template, 12.5. Mu.L of 2 XPfx MIX, 0.5. Mu.L (10. Mu.M) of each of a pair of mutation primers was added, and distilled water was added to 25. Mu.L after sterilization.

First step PCR reaction procedure: (1) the steps (2) to (4) were carried out in total for 30 cycles, wherein the steps (1) were performed at 98℃for 2min, (2) were performed at 98℃for 30 sec, (3) were performed at Tm-5℃for 30 sec, and (4) were performed at 72℃for 30 sec, and (5) were performed at 72℃for 5 min.

b. Second step PCR reaction system and reaction condition

Round 2: to a PCR reaction system with a total volume of 50. Mu.L, 30 ng of template, 25. Mu.L of 2 XPfx MIX, 1. Mu.L of primer as the first round PCR product fragment, and distilled water to 50. Mu.L were added.

Second step PCR reaction procedure: (1) pre-denaturation at 98℃for 2min, (2) denaturation at 98℃for 30 sec, (3) annealing at 55℃for 30 sec, (4) extension at 72℃for 3min, and (5) final extension at 72℃for 10min, steps (2) to (4) were performed for 25 cycles in total.

After the PCR products obtained in the above steps were confirmed by agarose gel electrophoresis analysis (FIG. 2), the restriction enzyme DpnI was added and digested at 37℃for 2 hours. Transferring the digested product intoE .coliBL21 (DE 3) competent cells were plated on plates containing 10. Mu.g/mL of ampicillin, and cultured in an incubator at 37℃for about 12 hours to give single colonies, thereby obtaining a carbonyl reductase mutant library.

Meanwhile, pET21a-Cg-11 beta-HSDH mutant library plasmid is transferredE.coliBL21 (DE 3) competent cells were plated on plates containing 100mg/mL of ampicillin, and cultured in an incubator at 37℃for about 12 hours to give single colonies, thereby obtaining a strain expressing a carbonyl reductase mutant.

TABLE 1 first round of modified primer sequences

3) Inducible expression of carbonyl reductase mutants

The monoclonal colonies obtained after the culture in the step 2 were picked up to give a culture solution containing 4mL ampicillin-containing (100 mg/L) LB liquid medium (peptone 10g/L, yeast powder 5g/L, naCl 10 g/L) and cultured overnight at 37℃and 200 rpm. Inoculating the culture solution to fermentation medium (LB liquid medium) according to 1% (v/v) inoculum size, shake culturing at 37deg.C and 200rpm to OD ₆₀₀ IPTG was added to a final concentration of 0.1mM at 0.6-0.8 and induced for 8-12h at 25℃with a shaker at 200 rpm. And collecting thalli by using 6000 g centrifugal culture solution, crushing under high pressure to obtain crude enzyme liquid of carbonyl reductase, and performing subsequent enzyme activity detection.

4) Screening of carbonyl reductase mutants

The screening method is to detect a decrease in NADPH at 340 nm. The specific method of the reaction is as follows: cortisone 10 mM,NADPH 0.25mg/mL, crude enzyme solution 50. Mu.L, potassium phosphate buffer was made up to 200. Mu.L. NADPH has characteristic absorbance at 340nm, and the decrease of absorbance at 340nm is detected by using an enzyme-labeled instrument, if the enzyme activity is relatively high, the consumption of NADPH is rapid, and the slope of the decrease curve is large. The useful mutation sites for which the enzyme activity was improved by screening were 103, 106, 151, 157, 160, 197, 211, 264 and 265.

2. Second round of vitality reconstruction

And constructing a combined mutant of a single mutation site according to a saturated mutation result, picking the obtained mutant into a test tube containing 4mL of LB culture medium for culture, and detecting the activity of the expressed protein, wherein the results are shown in Table 2, and the activity of mutants 21 and 22 is highest and is improved by 8.8 and 6.3 times compared with that of a wild type.

TABLE 2 relative vitality of Cg-11. Beta. -HSDH and mutants thereof on substrates

EXAMPLE 2 Whole cell catalytic synthesis of 11 beta-hydroxysteroids Using mutant 21

Mutant 21 was obtained by inducing expression in the same manner as in example 1, and the cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The strain was resuspended in 20 mL potassium phosphate buffer (pH 6.0, 100 mM), 20g/L cortisone, 200 mM glucose, 0.5 g/L NADP ⁺ Whole cells, 3U/mLGDH, 30 mg/mL, were reacted at 30℃for 24 hours.

After the reaction was completed, the conversion by HPLC analysis (FIG. 1A and FIG. 1B) was 95%, and the isolation yield was 90%.

Example 3 catalytic Synthesis of 11 beta-hydroxysteroids Using crude enzyme solution of mutant 21

Mutant 21 was obtained by inducing expression in the same manner as in example 1, and the cells were collected by centrifugation (6000 rpm) and the crude enzyme solution after the sterilization was used as a biocatalyst. The other conditions were the same as in example 2, and the conversion was 97% and the isolation yield was 94%.

EXAMPLE 4 Whole cell catalytic synthesis of 11 beta-hydroxysteroids Using mutant 21

Mutant 21 was obtained by inducing expression in the same manner as in example 1, and the cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The strain was resuspended in 20 mL potassium phosphate buffer (pH 6.0, 100 mM), 70g/L prednisone, 300 mM glucose, 0.5 g/L NADP ⁺ Whole cells, 3U/mLGDH, 50 mg/mL, were reacted at 30℃for 30 hours.

After the reaction was completed, the conversion by HPLC analysis (FIG. 1C and FIG. 1D) was 95%, and the isolation yield was 90%.

Claims (13)

1. A mutant of a carbonyl reductase, characterized in that the mutant has a protein sequence identical to SEQ ID NO:1 and the mutant has activity in reducing carbonyl steroid at position 11.

2. The mutant of claim 1, wherein the position corresponds to SEQ ID NO:1, Y103, M106, M151, H157, V160, L197, L211, L264, T265.

3. The mutant of claim 2, which is comprised in a sequence corresponding to SEQ ID NO:1, a mutation at any one of the following sites:

a mutation at position Y103;

a mutation at position M106;

a mutation at position M151;

a mutation at position H157;

a mutation at position V160;

a mutation at position L197;

a mutation at position L211;

a mutation at position L264;

mutation at position T265.

4. A mutant according to claim 3, which corresponds to SEQ ID NO:1 to alanine, glycine, glutamine; methionine at position 106 is mutated to serine, alanine, glycine; methionine at position 151 to valine, asparagine and leucine; histidine 157 is mutated to alanine, aspartic acid, glycine; valine at position 160 to isoleucine and leucine; leucine 197 to alanine, glycine; leucine 211 to alanine, glycine; leucine 264 is mutated to isoleucine, glycine; threonine at position 265 is mutated to arginine, lysine.

5. The mutant of claim 2, comprising a sequence corresponding to SEQ ID NO:1, Y103, M106, M151, H157, V160, L197, L211, L264, T265.

6. The mutant of claim 5, wherein the sequence corresponding to SEQ ID NO:1, a mutation at any one of the following sites:

a combined mutation at positions 103 and 151;

a combined mutation at positions 157 and 160;

a combined mutation at positions 264 and 265;

a combined mutation at positions 103 and 106 and 160;

combined mutations at positions 103 and 151 and 157;

combined mutations at positions 151 and 160 and 265;

a combined mutation at positions 103 and 151 and 157 and 265;

combined mutations at positions 151 and 157 and 160 and 265.

7. The mutant of claim 6, which corresponds to SEQ ID NO:1 to alanine, glycine, glutamine; methionine at position 106 is mutated to serine (S), alanine, glycine; methionine at position 151 to valine, asparagine, leucine; histidine 157 is mutated to alanine, aspartic acid, glycine; valine at position 160 to isoleucine and leucine; leucine 197 to alanine, glycine; leucine 211 to alanine, glycine; leucine 264 is mutated to isoleucine, glycine; threonine at position 265 is mutated to arginine, lysine.

8. An isolated polynucleotide comprising a nucleotide sequence encoding a mutant of the carbonyl reductase of any one of claims 1 to 7.

9. A recombinant expression vector comprising the isolated polynucleotide of claim 8.

10. A recombinant host cell or recombinant genetically engineered bacterium comprising the isolated polynucleotide of claim 8 or the recombinant expression vector of claim 9;

the recombinant host cell or genetically engineered bacterium is Escherichia genusEscherichia) Genus ErwiniaErwinia) Serratia genusSerratia) Provedsia species @Providencia) The intestinal bacteria genusEnterobacteria) Salmonella genusSalmonella) Streptomyces genusStreptomyces) Genus PseudomonasPseudomonas) Genus BrevibacteriumBrevibacterium) Bacillus genusBacillus) Or corynebacterium genusCorynebacterium) Is a microorganism of the genus (A).

11. A method for preparing an 11 beta-hydroxysteroid compound by reducing a carbonyl steroid at the 11-position, comprising the steps of:

contacting a mutant of the carbonyl reductase according to any one of claims 1-7 with a reaction substrate, cortisone or prednisone, and adding a coenzyme circulation system to perform a catalytic reaction, thereby obtaining the hydrocortisone or prednisolone.

12. The method of claim 11, wherein the catalytic reaction system has a pH of 6.0 to 8.0; the reaction time is 24-48 hours.

13. The method of claim 11, further comprising the step of isolating and purifying the 11 β -hydroxysteroid compound.