L-threonine transaldolase mutant and application thereof in synthesis of chloramphenicol chiral intermediate

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an L-threonine transaldolase mutant and application thereof in synthesis of a chloramphenicol chiral intermediate. According to the method, 4-nitrobenzaldehyde, L-threonine and sodium formate are used as substrates, co-expression cells of L-threonine transaldolase mutants PmLTTA-Mu4, ethanol dehydrogenase and formate dehydrogenase or a crushing solution of the co-expression cells are used as a catalyst, and the (2S, 3R)-4-nitrophenyl serine is asymmetrically synthesized under mild conditions (25-35 DEG C, pH is 6.5-7.5). According to the method, 5% of 4-nitrobenzaldehyde can be completely converted within 5 h to generate the (2S, 3R)-4-nitrophenyl serine, and the space time yield is as high as 15 g / L / h and is the highest level of biological synthesis of the (2S, 3R)-4-nitrophenyl serine at present. The synthesis method has the advantages of mild conditions, simple operation, simple control, single product configuration, no substrate residue, total yield of 85%, high space time yield, convenient purification, green environmental protection and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an L-threonine aldolase mutant and application thereof in the synthesis of a chloramphenicol chiral intermediate. Background Art

[0002] Chloramphenicol can inhibit the ribosome 50S subunit of bacteria and affect the synthesis of bacterial proteins, thus exerting an antibacterial effect. In 1947, scientists first isolated chloramphenicol from the culture medium of soil microorganisms; in 1949, Rebstock and others successfully achieved the chemical synthesis of chloramphenicol. In clinical practice, chloramphenicol is the first choice for the treatment of typhoid fever and anaerobic bacterial infections; in addition, chloramphenicol can also be made into various external preparations such as eye ointments, eye drops, and ear drops.

[0003] The industrial synthesis of chloramphenicol involves 9 chemical reactions (such as Figure 1 As shown in the figure, chloramphenicol is synthesized from p-nitroacetophenone as a starting material through a series of chemical reaction steps (including bromination, amination, acetylation, aldol condensation, aluminum isopropoxide reduction, hydrolysis deprotection, chiral resolution, and dichloroacetylation reactions), with an overall yield of less than 40%. In addition, the reducing agent aluminum isopropoxide in this method is complicated to prepare, and the reduction product is a racemate that needs to be resolved, resulting in poor atom economy and high production cost of this route.

[0004] In chemical synthesis, (2S, 3R)-4-nitrophenylserinol is a key chiral precursor. Its optical purity and synthesis efficiency determine the product quality and synthesis cost of chloramphenicol. In order to reduce the synthesis cost of chloramphenicol, patent CN111662937 A proposes a new chloramphenicol synthesis route, the key of which is the simplification of the synthesis steps of (2S, 3R)-4-nitrophenylserinol. First, p-nitrobenzaldehyde and L-threonine are used as raw materials, and (2S, 3R)-4-nitrophenylserine is synthesized by transaldehyde enzyme. The latter is reduced to (2S, 3R)-4-nitrophenylserinol by a one-step chemical reaction, and further synthesized into chloramphenicol (such as attached) by dichloroacetylation. Figure 2 As shown in Figure 2 ). This route only requires 3 steps to achieve the synthesis of chloramphenicol. Compared with the original 9-step chemical synthesis method, it has the advantages of high yield, high atom economy, low cost and low pollution. Among them, the key to this route lies in the enzyme catalysis step, that is, the synthesis process of (2S,3R)-4-nitrophenylserine mediated by transaldolase. The highest substrate loading level reported so far for this step is 10%, but the reaction cycle is long (20h) and the space-time yield is only 7.5g / L / h. Summary of the invention

[0005] In response to the above technical problems, the present invention uses protein engineering technology to obtain an L-threonine aldolase mutant capable of efficiently asymmetric synthesis of (2S, 3R)-4-nitrophenylserine, and applies the mutant to the enzymatic synthesis of (2S, 3R)-4-nitrophenylserine, with a space-time yield of 15 g / L / h, which is twice the highest level previously reported.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides an L-threonine transaldolase mutant, which is a mutant (PmLTTA-Mu4) of PmLTTA (the amino acid sequence and DNA sequence of PmLTTA are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), which is obtained by mutating the amino acid residues at four positions of N69A / C91I / F93A / H103F on the amino acid sequence shown in SEQ ID NO.1. The amino acid residue mutation mode is represented by the form of XnY, X represents the original amino acid, n represents the mutation site, and Y represents the amino acid after mutation; for example: N69A represents that the amino acid N at position 69 in the amino acid sequence shown in SEQ ID NO.1 is mutated to A.

[0008] Specifically, the amino acid sequence and DNA sequence of the L-threonine transaldolase mutant are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0009] Based on the above, the present invention also provides a recombinant vector carrying a gene encoding the above L-threonine transaldolase mutant.

[0010] Specifically, the expression vector of the L-threonine aldolase mutant is obtained by circular PCR using pET-28a-pmltta plasmid as a template and mutant primers.

[0011] Based on the above, the present invention also provides a recombinant cell expressing the L-threonine transaldolase mutant or carrying the recombinant vector, wherein the recombinant cell uses Escherichia coli as an expression host.

[0012] The L-threonine aldolase mutant or recombinant vector or recombinant cell can be used to prepare the chloramphenicol chiral intermediate (2S, 3R)-4-nitrophenylserine or a product containing (2S, 3R)-4-nitrophenylserine.

[0013] Based on the above, the present invention also provides a method for enzymatically synthesizing (2S, 3R)-4-nitrophenylserine, comprising:

[0014] With 4-nitrobenzaldehyde and L-threonine as substrates, (2S,3R)-4-nitrophenylserine is synthesized by a multi-enzyme cascade under the catalysis of the L-threonine aldolase mutant, alcohol dehydrogenase and formate dehydrogenase.

[0015] In one embodiment of the present invention, sodium formate is added as a cosubstrate during the enzyme reaction.

[0016] In one embodiment of the present invention, in the enzyme-catalyzed synthesis of (2S, 3R)-4-nitrophenylserine, the acetaldehyde elimination system comprises alcohol dehydrogenase and formate dehydrogenase, wherein the amino acid sequence and DNA sequence of alcohol dehydrogenase are shown as SEQ ID NO.5 and SEQ ID NO.6, respectively; the amino acid sequence and DNA sequence of formate dehydrogenase are shown as SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0017] The coding genes of alcohol dehydrogenase and formate dehydrogenase are co-recombined on an expression vector to obtain a co-expression plasmid of the acetaldehyde elimination system. Preferably, the expression vector is a medium copy plasmid; more preferably, it is a pCDFDuet-1 plasmid.

[0018] Furthermore, the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase are derived from whole cells or cell fragments of recombinant cells that co-express the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase by genetic engineering, or are whole cells or cell fragments of recombinant cells that express the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase separately.

[0019] In one embodiment of the present invention, the co-expression plasmid of the acetaldehyde elimination system and the L-threonine transaldolase mutant expression plasmid are co-transformed into an expression host. Preferably, the expression host is Escherichia coli; more preferably, it is Escherichia coli BL21 (DE3). The co-expression whole cells are obtained by batch fed fermentation.

[0020] In one embodiment of the present invention, the reaction system for enzyme-catalyzed synthesis of (2S, 3R)-4-nitrophenylserine comprises: L-threonine, 4-nitrobenzaldehyde, sodium formate, nicotinamide adenine dinucleotide (NAD + ), pyridoxal phosphate (PLP) and the above co-expressed whole cells or their broken liquid. Stir at 30°C and pH 6.5-7.5 until the reaction is completed.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention utilizes protein engineering technology to obtain a highly catalytically active L-threonine transaldolase mutant, which can utilize an alcohol dehydrogenase-coupled formate dehydrogenase-mediated acetaldehyde elimination system to efficiently convert 5% 4-nitrobenzaldehyde into a target product (2S, 3R)-4-nitrophenylserine within 5 hours, with a conversion rate of >99%, a de value of >99%, and a space-time yield of up to 15 g / L / h, which is twice the highest level (7.5 g / L / h) previously reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a chemical synthesis route of chloramphenicol;

[0024] Figure 2 It is a synthetic route for synthesizing chloramphenicol by enzymatic-chemical coupling;

[0025] Figure 3 This is the HPLC detection spectrum of (2S,3R)-4-nitrophenylserine product;

[0026] Figure 4 To detect the optical purity of (2S,3R)-4-nitrophenylserine product;

[0027] Figure 5 This is the mass spectrum of the (2S,3R)-4-nitrophenylserine product. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The invention provides an L-threonine transaldolase mutant, which is a mutant of PmLTTA (the amino acid sequence and DNA sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), and is obtained by mutating the amino acid residues at four positions N69A / C91I / F93A / H103F on the amino acid sequence shown in SEQ ID NO.1.

[0030] The present invention uses 4-nitrobenzaldehyde, L-threonine and sodium formate as substrates, uses L-threonine transaldolase mutant (PmLTTA-Mu4), co-expressed cells of alcohol dehydrogenase and formate dehydrogenase or their broken liquid as catalysts, and asymmetric synthesis of (2S, 3R)-4-nitrophenylserine under mild conditions (25-35°C, pH 6.5-7.5). The method can completely convert 5% of 4-nitrobenzaldehyde to generate (2S, 3R)-4-nitrophenylserine within 5 hours, and the time-space yield is as high as 15g / L / h, which is the highest level of biological synthesis of (2S, 3R)-4-nitrophenylserine at present. The synthesis method has mild conditions, simple operation, simple control, single configuration of the product, no substrate residue, and a total yield of up to 85%. It has the advantages of high time-space yield, easy purification, and green environmental protection.

[0031] Example 1: Site-directed mutagenesis of PmLTTA

[0032] Using the recombinant plasmid pET28a-pmllta as a template, KOD one DNA polymerase was used to perform circular plasmid PCR amplification of the mutant expression plasmid. The primers used are shown in Table 1 below. The amplification program is: 98°C, 3min; 98°C, 10s; 60°C, 20s; 68°C, 40s; 30 cycles; 68°C, 2min. After the PCR is completed, 0.3U DMT is added to the reaction system, and the template is digested at 37°C for 1h. After the digestion is completed, 3μL of the digestion product is transferred into E.coli BL21 (DE3) competent cells and spread on LB plates containing 50mg / L kanamycin resistance. Single-point mutations are performed in each round of mutation. After sequencing confirms the successful mutation, the mutated plasmid is used as a template to mutate the next site, and so on, to complete the mutation of four sites.

[0033] The four-point mutant PmLLTA-N69A / C91I / F93A / H103F (i.e., PmLLTA-Mu4) was obtained, and the plasmid was extracted, namely the PmLLTA-Mu4 overexpression plasmid pET28a-pmllta-mu4.

[0034] Table 1 L-Threonine Aldolase Mutation Primer Information

[0035]

[0036] Example 2: Construction of acetaldehyde elimination system co-expression plasmid

[0037] Using the synthesized alcohol dehydrogenase gene and formate dehydrogenase gene as templates, primer pairs F1 / R1 and F2 / R2 were used to amplify scadh and cbfdh fragments, respectively. Using the obtained scadh and cbfdh fragments as templates, primer pair F1 / R2 was used for overlap extension to amplify the scadh-cbfdh fragment. Using pCDFDuet-1 as a template, the backbone fragment was amplified using primer pair F3 / R3, and the primers used were shown in Table 2 below. The backbone fragment and the scadh-cbfdh fragment were recombined using a one-step cloning kit and transformed into E.coli BL21 (DE3) competent cells and screened in LB medium containing 50 mg / L streptomycin sulfate. The transformants were picked and verified by colony PCR using primer pairs F1 / R2, and the positive clones were picked for DNA sequencing. After confirming that the coding reading frame was correct, the plasmid was extracted, which was the acetaldehyde elimination system co-expression plasmid pCDF-scadh-cbfdh.

[0038] Table 2 Primer information for the construction of acetaldehyde elimination system co-expression plasmid

[0039]

[0040] Example 3: Construction of co-expression strain

[0041] The PmLLTA-Mu4 overexpression plasmid pET28a-pmllta-mu4 and the acetaldehyde elimination system co-expression plasmid pCDF-scadh-cbfdh were co-transformed into E. coli BL21 (DE3) competent cells, spread in LB medium containing 50 mg / L streptomycin sulfate and 50 mg / L kanamycin sulfate, and cultured at 37°C for 12 h to obtain the PmLLTA-Mu4, alcohol dehydrogenase and formate dehydrogenase co-expression strain E. coli BL21 (DE3) (pET28a-pmllta-mu4 / pCDF-scadh-cbfdh).

[0042] Example 4: High-density fermentation of co-expression strains

[0043] The co-expression strain E. coli BL21 (DE3) (pET28a-pmllta-mu4 / pCDF-scadh-cbfdh) was activated on an LB plate containing 50 mg / L streptomycin sulfate and 50 mg / L kanamycin sulfate. After a single colony grew out, a single colony of the engineered bacteria was picked and inoculated into 150 mL LB medium containing streptomycin sulfate and kanamycin sulfate. The culture was cultured at 37 ° C and 200 rpm for 12 h as seed liquid, which was inoculated into a 5 L fermenter (liquid volume 3 L, glycerol 10 g / L, yeast extract 15 g / L, K2HPO4·3H2O 4 g / L, NaH2PO4·2H2O 2.24 g / L, NaCl 3 g / L, (NH4)2SO4 2.5 g / L, citric acid 2.1 g / L, glucose 2 g / L, MgSO4·7H2O 2. 0.49g / L, FeSO4 0.3g / L), and start the fermentation. The stirring speed is set to 300rpm, and the temperature is set to 37°C. During the fermentation process, the pH value is controlled at around 7.0 by automatically adding NH4OH (25%, v / v). In the early stage of feeding, as the bacteria proliferate, the dissolved oxygen slowly decreases. At this time, by increasing the stirring speed, the dissolved oxygen DO is maintained at around 30%, and the upper limit of stirring is 600rpm. When the nutrients are exhausted, the dissolved oxygen and pH soar. At this time, start feeding (110g / L yeast extract powder and 600g / L glycerol mixture), and maintain the dissolved oxygen at around 15% by adjusting the feeding speed. When OD 600 When the concentration of 50 μg / mL was about 60, IPTG was added to induce the expression of the target gene. After 28 h of fermentation, the fermentation broth was centrifuged (6000 rpm, 10 min) to obtain 550 g of wet cells.

[0044] Example 5: Asymmetric Synthesis of (2S, 3R)-4-Nitrophenylserine (3% substrate feed, whole cell reaction)

[0045] Take 3g 4-nitrobenzaldehyde, 2.85g L-threonine and 2g sodium formate, add 5g co-expression whole cells and 90mL water, stir and react at 30°C, and use 10% hydrochloric acid solution to adjust the pH to between 6.5-7.5. After 4h of reaction, the conversion rate detected by HPLC is >99%. Use concentrated sulfuric acid to adjust the pH of the reaction solution to below 4, filter out bacteria, use a 250Da roll ultrafiltration membrane to remove most of the soluble proteins and pigments, and then use a 100Da roll ultrafiltration membrane to remove impurities. The resulting clear liquid is decolorized with activated carbon and concentrated and crystallized. After filtration, 3.8g of light yellow powder is obtained, with a purity of >99%, de>99% (such as attached Figure 3 , Attachment Figure 4 and attached Figure 5 shown).

[0046] HPLC detection conditions for purity: chromatographic column: Agilent 18ZORBAX reversed-phase columns (250×4.6mm, 5μm); mobile phase: V (2.5mM sodium octane sulfonate solution, pH3.5): V (methanol) = 5:6; detection wavelength: 254nm; flow rate: 1mL / min; injection volume: 20μL; column temperature: 35℃.

[0047] Optical purity detection method: The reaction solution was mixed with OPA / NAC solution in a ratio of 1:4 and incubated at 30°C for 10 min for derivatization. The contents of (2S,3R)-4-nitrophenylserine and (2S,3S)-4-nitrophenylserine were detected after filtration through a 0.22 μm organic filter membrane. HPLC detection conditions were as follows: chromatographic column: Agilent 18ZORBAX reversed-phase columns (250×4.6 mm, 5 μm); mobile phase: V (acetonitrile): V (50 mM potassium dihydrogen phosphate solution) = 21:79; detection wavelength: 340 nm; flow rate: 1 mL / min; injection volume: 20 μL; column temperature: 30°C. The diastereomeric excess value de was calculated according to the following formula.

[0048]

[0049] Example 6: Asymmetric Synthesis of (2S, 3R)-4-Nitrophenylserine (10% substrate feed, whole cell reaction)

[0050] Take 10g 4-nitrobenzaldehyde, 9.5g L-threonine and 6.7g sodium formate, add 5g co-expression whole cells and 90mL water, stir and react at 30°C, and adjust the pH to 6.5-7.5 with 10% hydrochloric acid solution. After 10h of reaction, the conversion rate is >99% as detected by HPLC. Use concentrated sulfuric acid to adjust the pH of the reaction solution to below 4, filter and remove bacteria, use a 250Da roll ultrafiltration membrane to remove most of the soluble proteins and pigments, and then use a 100Da roll ultrafiltration membrane to remove impurities. The resulting clear solution is decolorized with activated carbon and concentrated and crystallized. After filtration, 13g of light yellow powder is obtained, with a purity of >99% and de>99%.

[0051] Example 7: Asymmetric Synthesis of (2S, 3R)-4-Nitrophenylserine (10% substrate feed, crude enzyme reaction)

[0052] Take 5g of co-expressed whole cells and resuspend them in 30mL of water, and crush them to obtain crude enzyme solution. Add 10g of 4-nitrobenzaldehyde, 9.5g of L-threonine, 6.7g of sodium formate and 60mL of water to the crude enzyme solution, stir and react at 30°C, and adjust the pH to between 6.5-7.5 with 10% hydrochloric acid solution. After 10h of reaction, the conversion rate was >99% as detected by HPLC. Use concentrated sulfuric acid to adjust the pH of the reaction solution to below 4, filter out denatured proteins, use a 250Da roll ultrafiltration membrane to remove most of the soluble proteins and pigments, and then use a 100Da roll ultrafiltration membrane to remove impurities. The resulting clear solution is decolorized with activated carbon and concentrated and crystallized. After filtration, 13g of light yellow powder is obtained, with a purity of >99% and de>99%.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A L-threonine transaldolase mutant, characterized in that: The L-threonine transaldolase mutant is a mutant of PmLTTA, which is obtained by mutating the amino acid residues at four positions of N69A / C91I / F93A / H103F in the amino acid sequence shown in SEQ ID NO.

1.

2. The L-threonine transaldolase mutant according to claim 1, characterized in that The amino acid sequence and DNA sequence of the L-threonine transaldolase mutant are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

3. A recombinant vector carrying a gene encoding the L-threonine transaldolase mutant according to claim 1 or 2.

4. A recombinant cell expressing the L-threonine transaldolase mutant of claim 1 or carrying the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell uses Escherichia coli as an expression host.

6. Use of the L-threonine transaldolase mutant according to claim 1 or 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4 or 5 in the preparation of a chloramphenicol chiral intermediate (2S, 3R)-4-nitrophenylserine or a product containing (2S, 3R)-4-nitrophenylserine.

7. A method for enzymatically synthesizing (2S, 3R)-4-nitrophenylserine, characterized in that: include: With 4-nitrobenzaldehyde and L-threonine as substrates, (2S, 3R)-4-nitrophenylserine is synthesized by multi-enzyme cascade under the catalysis of the L-threonine aldolase mutant as claimed in claim 1 or 2, alcohol dehydrogenase and formate dehydrogenase.

8. The method for enzymatically synthesizing (2S, 3R)-4-nitrophenylserine according to claim 7, characterized in that: During the enzyme reaction, sodium formate was added as a cosubstrate.

9. The method for enzymatically synthesizing (2S, 3R)-4-nitrophenylserine according to claim 7, characterized in that: The amino acid sequence and DNA sequence of the alcohol dehydrogenase are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively; the amino acid sequence and DNA sequence of the formate dehydrogenase are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

10. The method for enzymatically synthesizing (2S, 3R)-4-nitrophenylserine according to claim 7, characterized in that: The L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase are derived from whole cells or cell fragments of recombinant cells that co-express the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase by genetic engineering, or are whole cells or cell fragments of recombinant cells that express the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase separately.

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