L-Threonine Aldolase Mutants and Their Application in the Synthesis of Chloramphenicol Chiral Intermediates

Abstract

The present invention belongs to the field of biomedicine, and in particular to a kind of L-threonine transaldolase mutant and its application in the synthesis of chloramphenicol chiral intermediate.The present invention is with 4-nitrobenzaldehyde, L-threonine and sodium formate as substrates, using the co-expression cells of L-threonine transaldolase mutant PmLTTA-Mu4, alcohol dehydrogenase and formate dehydrogenase or its broken liquid as catalyst, under mild conditions (25-35 DEG C, pH6.5-7.5) asymmetric synthesis (2S, 3R)-4-nitrophenylserine.The method can completely convert 5% 4-nitrobenzaldehyde into (2S, 3R)-4-nitrophenylserine in 5h, and space-time yield is up to 15g / L / h, which is the highest level of current (2S, 3R)-4-nitrophenylserine biological synthesis. This synthesis method has mild conditions, simple operation, simple control, a single product configuration, 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.

Description

Technical Field

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

[0002] Chloramphenicol inhibits bacterial ribosomal 50S subunits, affecting bacterial protein synthesis and thus exerting its antibacterial effects. In 1947, scientists first isolated chloramphenicol from a culture medium of soil microorganisms; in 1949, Rebstock et al. successfully chemically synthesized chloramphenicol. Clinically, chloramphenicol is the drug of choice for treating typhoid fever and anaerobic infections. Chloramphenicol can also be formulated into various topical preparations, including eye ointments, eye drops, and ear drops.

[0003] The industrial synthesis of chloramphenicol involves 9 chemical steps (such as Figure 1 , using p-nitroacetophenone as the starting material, chloramphenicol is synthesized through a series of chemical reaction steps (including bromination, amination, acetylation, aldol condensation, aluminum isopropoxide reduction, hydrolysis and deprotection, chiral resolution, and dichloroacetylation) with an overall yield of less than 40%. Furthermore, the reducing agent, aluminum isopropoxide, is complex to prepare, and the reduction product is a racemate that requires resolution, resulting in poor atom economy and high production costs.

[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 lies in 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 transaldolase. The latter is reduced to (2S,3R)-4-nitrophenylserinol through a one-step chemical reaction, and further synthesized into chloramphenicol (such as the attached) through dichloroacetylation. Figure 2 This route requires only three steps to synthesize chloramphenicol. Compared to the traditional nine-step chemical synthesis, it offers advantages such as high yield, atom economy, low cost, and minimal pollution. The key to this route lies in the enzymatic step, the transaldolase-mediated synthesis of (2S,3R)-4-nitrophenylserine. The highest reported substrate loading for this step to date has reached 10%, but the reaction cycle is long (20 hours) and the space-time yield is only 7.5 g / L / h. Summary of the Invention

[0005] To address the above technical problems, the present invention utilizes protein engineering technology to obtain an L-threonine transaldolase mutant capable of efficiently asymmetric synthesis of (2S,3R)-4-nitrophenylserine. The mutant is applied 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 solutions:

[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), obtained by mutating the amino acid residues at four positions, N69A, C91I, F93A, and H103F, in the amino acid sequence shown in SEQ ID NO. 1. The amino acid residue mutation pattern is represented by the form XnY, where X represents the original amino acid, n represents the mutation site, and Y represents the mutated amino acid; for example, N69A indicates 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 as 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 transaldolase 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 further provides a recombinant cell expressing the aforementioned L-threonine transaldolase mutant or carrying the aforementioned recombinant vector, wherein the recombinant cell uses Escherichia coli as an expression host.

[0012] The L-threonine transaldolase mutant, recombinant vector or recombinant cell can be used to prepare the chloramphenicol chiral intermediate (2S, 3R)-4-nitrophenylserine or products 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] Using 4-nitrobenzaldehyde and L-threonine as substrates, (2S,3R)-4-nitrophenylserine was synthesized by a multi-enzyme cascade under the catalysis of the aforementioned L-threonine transaldolase 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 enzymatic 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 genes encoding alcohol dehydrogenase and formate dehydrogenase are co-recombined into an expression vector to obtain a co-expression plasmid for the acetaldehyde elimination system. Preferably, the expression vector is a medium-copy plasmid; more preferably, it is the pCDFDuet-1 plasmid.

[0018] Furthermore, the L-threonine transaldolase mutant, alcohol dehydrogenase, and formate dehydrogenase are derived from whole cells or cell lysates of recombinant cells that co-express the L-threonine transaldolase mutant, alcohol dehydrogenase, and formate dehydrogenase using genetic engineering methods, or are whole cells or cell lysates of recombinant cells that individually express the L-threonine transaldolase mutant, alcohol dehydrogenase, and formate dehydrogenase.

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

[0020] In one embodiment of the present invention, the reaction system for enzymatic 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 fragments. Stir at 30°C, pH 6.5-7.5 until the reaction is complete.

[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. The mutant can utilize an alcohol dehydrogenase-coupled formate dehydrogenase-mediated acetaldehyde elimination system to efficiently convert 5% 4-nitrobenzaldehyde into the target product (2S,3R)-4-nitrophenylserine within 5 hours. The conversion rate is >99%, the de value is >99%, and the space-time yield is as high as 15 g / L / h, which is twice the previously reported highest level (7.5 g / L / h). 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 method;

[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] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] The present 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, in the amino acid sequence shown in SEQ ID NO.1.

[0030] The present invention uses 4-nitrobenzaldehyde, L-threonine, and sodium formate as substrates, and utilizes a mutant L-threonine transaldolase (PmLTTA-Mu4), co-expressing cells of alcohol dehydrogenase and formate dehydrogenase, or their fragmented liquid, as catalysts to asymmetric synthesize (2S,3R)-4-nitrophenylserine under mild conditions (25-35°C, pH 6.5-7.5). This method can completely convert 5% of 4-nitrobenzaldehyde to (2S,3R)-4-nitrophenylserine within 5 hours, with a space-time yield of up to 15 g / L / h, representing the highest level of biosynthesis of (2S,3R)-4-nitrophenylserine to date. This synthesis method features mild conditions, simple operation, and easy control. The product has a single configuration, no substrate residue, and an overall yield of up to 85%. It offers advantages such as high space-time yield, ease of purification, and environmental friendliness.

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

[0032] Using the recombinant plasmid pET28a-pmllta as a template, circular plasmid PCR was performed using KOD one DNA polymerase. The primers used are listed in Table 1. The amplification program was as follows: 98°C for 3 minutes; 98°C for 10 seconds; 60°C for 20 seconds; 68°C for 40 seconds; 30 cycles; and 68°C for 2 minutes. After the PCR, 0.3 U of DMT was added to the reaction system, and the template was digested by incubation at 37°C for 1 hour. After digestion, 3 μL of the digestion product was transformed into E. coli BL21 (DE3) competent cells and plated on LB plates containing 50 mg / L kanamycin. Each round of mutagenesis involved a single point mutation. After successful mutation was confirmed by sequencing, the mutated plasmid was used as a template for mutation at the next site, and so on, until all four sites were mutated.

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

[0034] Table 1 L-threonine transaldolase mutation primer information

[0035]

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

[0037] Using the synthesized alcohol dehydrogenase and formate dehydrogenase genes as templates, the scadh and cbfdh fragments were amplified using primer pairs F1 / R1 and F2 / R2, respectively. Using the obtained scadh and cbfdh fragments as templates, overlap extension was performed using primer pair F1 / R2 to amplify the scadh-cbfdh fragment. Using pCDFDuet-1 as a template, the backbone fragment was amplified using primer pair F3 / R3. The primers used are shown in Table 2 below. The backbone and scadh-cbfdh fragments were recombined using a one-step cloning kit and transformed into E. coli BL21(DE3) competent cells. The cells were screened in LB medium supplemented with 50 mg / L streptomycin sulfate. Transformants were selected for colony PCR verification using primer pair F1 / R2. Positive clones were selected for DNA sequencing. After confirming the correct coding reading frame, the plasmid was extracted to obtain 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 on 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 LB plates containing 50 mg / L streptomycin sulfate and 50 mg / L kanamycin sulfate. After a single colony grew, 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 incubated at 37°C and 200 rpm for 12 h as seed liquid, which was then inoculated into a 5 L fermentor (3 L liquid volume, 10 g / L glycerol, 15 g / L yeast extract, 4 g / L K2HPO4·3H2O, 2.24 g / L NaH2PO4·2H2O, 3 g / L NaCl, 2.5 g / L (NH4)2SO4, 2.1 g / L citric acid, 2 g / L glucose, MgSO4·7H2O). 0.49g / L, FeSO4 0.3g / L), start fermentation. Set the stirring speed to 300rpm, the temperature to 37℃, and automatically add NH4OH (25%, v / v) to control the pH value at around 7.0 during the fermentation process. 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 (a mixture of 110g / L yeast extract and 600g / L glycerol), and maintain the dissolved oxygen at around 15% by adjusting the feeding speed. When OD 600 When the C-value reached about 60, IPTG was added at a final concentration of 0.2 mM 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 between 6.5-7.5. After 4 hours of reaction, the conversion rate was detected by HPLC>99%. Use concentrated sulfuric acid to adjust the pH of the reaction solution to below 4, filter to remove bacteria, use a 250Da pore size 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>99% and de>99% (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 was determined by mixing the reaction mixture with the OPA / NAC solution in a 1:4 ratio and incubating at 30°C for 10 minutes for derivatization. The mixture was filtered through a 0.22 μm organic filter membrane and the contents of (2S,3R)-4-nitrophenylserine and (2S,3S)-4-nitrophenylserine were determined. HPLC conditions were as follows: Agilent 18 ZORBAX 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. Diastereomeric excess (d) was calculated according to the following formula.

[0048]

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

[0050] 10g of 4-nitrobenzaldehyde, 9.5g of L-threonine, and 6.7g of sodium formate were added to 5g of co-expressed whole cells and 90mL of water. The reaction was stirred at 30°C and the pH was adjusted to between 6.5 and 7.5 using 10% hydrochloric acid solution. After 10 hours of reaction, the conversion rate was >99% as determined by HPLC. The reaction solution was adjusted to a pH below 4 with concentrated sulfuric acid, filtered to remove bacteria, and most soluble proteins and pigments were removed using a 250Da pore size spiral ultrafiltration membrane. Impurities were then removed using a 100Da spiral ultrafiltration membrane. The resulting clear solution was decolorized with activated carbon and concentrated for crystallization. After filtration, 13g of a light yellow powder was obtained with a purity >99% and a desaturation >99%.

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

[0052] 5g of co-expressed whole cells were resuspended in 30mL of water and disrupted to obtain a crude enzyme solution. 10g of 4-nitrobenzaldehyde, 9.5g of L-threonine, 6.7g of sodium formate, and 60mL of water were added to the crude enzyme solution and stirred at 30°C. The pH was adjusted to between 6.5 and 7.5 using 10% hydrochloric acid solution. After 10 hours of reaction, the conversion rate was >99% as determined by HPLC. The pH of the reaction solution was adjusted to below 4 with concentrated sulfuric acid, and denatured proteins were removed by filtration. Most soluble proteins and pigments were removed using a 250Da spiral wound ultrafiltration membrane, and impurities were removed using a 100Da spiral wound ultrafiltration membrane. The resulting clear solution was decolorized with activated carbon and concentrated for crystallization. After filtration, 13g of a pale yellow powder was obtained with a purity >99% and a desaturation >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 they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. 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 various embodiments of the present invention.

Claims

1. An 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: 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 according to claim 1 or carrying the recombinant vector according to 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 a multi-enzyme cascade under the catalysis of the L-threonine transaldolase mutant according to 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 lysates of recombinant cells that co-express the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase using genetic engineering methods, or are whole cells or cell lysates of recombinant cells that individually express the L-threonine aldolase mutant, alcohol dehydrogenase, and formate dehydrogenase.

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