Proline dehydrogenase, genes and mutants and uses thereof

Abstract

The application provides proline dehydrogenase, genes and mutants and applications thereof. The enzyme is derived from Pseudomonas xantholysinigenes and is recombinantly expressed in Escherichia coli. The target gene is codon-optimized when constructing a plasmid. The optimized DNA sequence is shown in SEQ ID NO. 2. The proline dehydrogenase is subjected to mutation modification. The enzyme and the mutants thereof can be used for efficiently catalyzing L-proline degradation to complete biological resolution of DL-proline, and high-purity D-proline is obtained. The application has great economic value in the field of green production of D-proline.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a proline dehydrogenase, gene and mutant, and their applications. Background Technology

[0002] D-proline is a five-membered cyclic imine that is relatively rare in nature but plays several important roles. It can serve as a chiral intermediate in the synthesis of certain chiral drugs; for example, D-proline is a key chiral intermediate for eletriptan and a crucial precursor in the synthesis of various alkaloids. Furthermore, D-proline can act as a chiral catalyst in several asymmetric reactions.

[0003] Currently, there are two main methods for producing D-proline: chemical production and biological production. Chemical production mainly involves obtaining D-proline through a series of chemical reactions. For example, in the chemical asymmetric synthesis method (CN 107827802A), pyrrolidine-2-methanol is used as a raw material, which is reduced to D-proline alcohol under chiral metal catalysis, and then oxidized to obtain D-proline. However, this method uses expensive catalysts and has a low reaction ee value, resulting in low industrial value. On the other hand, the chemical asymmetric conversion method for synthesizing D-proline uses chemical reagents to achieve the separation of DL-proline, which has higher yield and efficiency, but higher production costs and causes significant environmental pollution, thus failing to meet the requirements of green production. The biological production method mainly involves enantioselective degradation. Currently, there is a patented study (CN111424060A) that uses Bacillus xylose-lysine-degrading to degrade L-proline in DL-proline to obtain D-proline, and produces the byproduct 1-pyrroline-5-carboxylic acid to improve industrial value. However, this method has low efficiency, long fermentation and conversion times, and a low total substrate concentration of only 40 g / L. It also requires batch feeding of 10 g / L DL-proline each time for the reaction, making the operation quite complicated. In total, it takes more than 168 hours to obtain 20 g / L D-proline. Therefore, this method cannot yet meet the needs of industrial production. Summary of the Invention

[0004] This invention addresses the problem of low D-proline production efficiency caused by insufficient catalytic activity of proline dehydrogenase in existing technologies by providing a proline dehydrogenase, its gene, a mutant, and their applications. The primary objective of this invention is to increase the substrate concentration in the conversion reaction, simplify the production process, and shorten the reaction time to improve production efficiency, making the overall reaction more suitable for industrial production.

[0005] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solutions.

[0006] A proline dehydrogenase derived from Pseudomonas xantholysinigenes, with an amino acid sequence as shown in SEQ ID NO.1, was used to optimize the codons of the target gene, and the optimized nucleotide sequence is shown in SEQ ID NO.2.

[0007] A mutant of proline dehydrogenase, wherein the amino acid sequence of the mutant is obtained by mutating one or more of the following sites in the amino acid sequence shown in SEQ ID NO.1: tyrosine at position 549, tyrosine at position 434, tyrosine at position 568, and alanine at position 368. The mutation includes at least one of the following mutation sites: Y434Q, Y434M, Y568I, Y549L, A368M, A368K, Y549L+Y434M, or A368M+Y434M; wherein the amino acid abbreviations are as follows: A-alanine, L-leucine, I-isoleucine, Y-tyrosine, K-lysine, Q-glutamine, and M-methionine.

[0008] An application of a proline dehydrogenase, comprising the following steps:

[0009] (1) Construction of recombinant Escherichia coli: The DNA fragment shown in SEQ ID NO.2 was cloned into the expression vector pET28a(+) using NheI and HindIII restriction sites to obtain the pET28a(+)-PxPutA recombinant plasmid, which was then transformed into Escherichia coli BL21(DE3) competent cells to obtain highly expressed recombinant Escherichia coli;

[0010] (2) Fermentation culture: Pick a single colony from step (1), inoculate it into a 5 mL LB tube (kanamycin sulfate resistant) and culture overnight. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation rate of 2%, culture at 37℃ for 2 h, add IPTG inducer to a final concentration of 0.2 mmol / L, continue to culture at 18℃ for 24 hours, end fermentation, and directly add the cells in the fermentation broth to the substrate for reaction without separation;

[0011] (3) Degradation of L-proline in DL-proline: The bacterial solution obtained from step (2) of fermentation was degraded by whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline was 30-50 g / L, the amount of recombinant Escherichia coli wet cell solution added was 20-30 g / L, the reaction temperature range was 20℃~40℃, and the degradation time was 24-36 h.

[0012] The application of a proline dehydrogenase mutant includes the following steps:

[0013] (1) Construction of recombinant Escherichia coli: Using the DNA fragment shown in SEQ ID NO.2 as a template, site mutation PCR was performed to construct a recombinant plasmid containing the coding gene of the mutation site. The mutant plasmid was transferred into competent cells of Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli with high expression.

[0014] (2) Fermentation culture: Pick a single colony from step (1), inoculate it into a 5 mL LB tube (kanamycin sulfate resistant) and culture overnight. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation rate of 2%, culture at 37℃ for 2 h, add IPTG inducer to a final concentration of 0.2 mmol / L, continue to culture at 18℃ for 24 hours, end fermentation, and directly add the cells in the fermentation broth to the substrate for reaction without separation;

[0015] (3) Degradation of L-proline in DL-proline: The bacterial solution obtained from step (2) of fermentation was degraded by whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline was 30-50 g / L, the amount of recombinant Escherichia coli wet cell solution added was 20-30 g / L, the reaction temperature range was 20℃~40℃, and the degradation time was 24-36 h.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) The amino acid sequence homology between the proline dehydrogenase provided by the present invention and PutA derived from Pseudomonas pseudoalcaligenes is 77.4%. The PxPutA provided by the present invention is a new proline dehydrogenase.

[0018] (2) This invention provides a proline dehydrogenase derived from Pseudomonas xantholysinigenes. By constructing a high-expression recombinant engineered bacterium, it was found that this enzyme can efficiently catalyze the dehydrogenation and degradation of L-proline, thereby producing high-purity D-proline.

[0019] (3) This invention provides an optimized mutant of proline dehydrogenase derived from Pseudomonas xantholysinigenes. By constructing a high-expression recombinant engineered bacterium, it was found that the enzyme has higher catalytic activity than the wild type, thereby greatly improving the reaction efficiency. Furthermore, the enzyme was applied to the preparation of D-proline and it was found to have the advantages of rapid reaction and good stability, overcoming the time-consuming defect of the biological resolution method of D-proline. It has important application value for the industrial preparation of D-proline. Detailed Implementation

[0020] The following examples are provided to better understand the present invention, but are not limited to it. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all performed in triplicate, and the results were averaged. For chromatograms of different reactions under the same conditions, the retention time of the target peak will have a certain error range; generally, a difference within 0.1 min can be considered as error and can be identified as the same target analyte.

[0021] The present invention will now be described in more detail with reference to specific embodiments.

[0022] In the example, the pre-column chiral derivatization-HPLC detection method for L-glutamic acid, D-proline, L-proline, and L-1-pyrroline-5-carboxylic acid used the following pre-column chiral derivatization-HPLC conditions: After centrifugation of the fermentation broth, the supernatant was diluted and then mixed with 4 g / L triethylamine / acetonitrile solution and 2 g / L 2,3,4,6-tetra-O-acetyl-β-D-glucopyranose isothiocyanate (GITC) / acetonitrile solution, and heated in a metal bath at 30°C for 30 min. After centrifugation, the derivatives of D-proline, L-proline, and L-glutamic acid, as well as L-1-pyrroline-5-carboxylic acid, in the supernatant were separated by HPLC. The concentrations were calculated using the internal standard method. Chromatographic column: C18 column; mobile phase: 0.1% trifluoroacetic acid aqueous solution / methanol (70:30, v / v), gradient elution, with mobile phase concentration changes shown in Table 1; flow rate: 1.0 mL / min; detection wavelength: 254 nm; column temperature: 30°C.

[0023] Example 1

[0024] Construction, culture, and induced expression of Escherichia coli genetically engineered bacteria with high expression of proline dehydrogenase PxPutA

[0025] (1) The protein sequence of Pseudomonas xantholysinigenes proline dehydrogenase was obtained from the NCBI database as shown in SEQ ID NO.1. After codon optimization, the optimized nucleotide sequence is SEQ ID NO.2, which was synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the NheI and HindIII restriction sites of the pET28a(+) vector to obtain the pET28a(+)-PxPutA recombinant plasmid.

[0026] (2) The obtained recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells.

[0027] (3) Pick a single colony and inoculate it into a 5 mL LB tube (kanamycin sulfate resistant) and culture it overnight. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation rate of 2%, culture it at 37°C for 2 h, add IPTG inducer to the final concentration of 0.2 mmol / L, continue to culture at 18°C ​​for 24 h, end the fermentation, and collect the bacterial solution for later use.

[0028] Example 2

[0029] Wild-type proline dehydrogenase PxPutA is used to prepare D-proline.

[0030] In a 10 ml system, 50 g / L of DL-proline was added to the fermentation broth containing 30 g / L wet cells (the bacterial culture prepared in Example 1). After conversion at 30 °C and shaking (220 rpm) for 24 h, the sample was taken and detected by HPLC. Since the reaction is essentially a specific dehydrogenation of L-proline, the final reaction result is expressed as the ee value of D-proline. The reaction results are shown in Table 1.

[0031] Example 3

[0032] Construction of the proline dehydrogenase PxPutA mutant plasmid

[0033] 1) Based on the amino acid sequence SEQ ID NO.1 of wild-type PxPutA, protein homology modeling analysis was performed on the alphafold3 website. The simulated structure was then molecularly docked with the substrate (AutoDock 4.2.6 software). Analysis of its substrate binding pocket revealed that sites Y434, E286, K326, A368, E369, R428, A433, Y434, L510, and Y549 are relatively close to the active site. It is speculated that these sites may affect the catalytic activity of PxPutA on the substrate.

[0034] 2) Using wild-type plasmid pET28a(+)-PxPutA as a template, a whole-plasmid single-point mutation PCR was performed to construct recombinant plasmids containing the encoding genes of PxPutA-Y434, A368, Y549, and Y568 sites mutated to Y434Q, Y434M, Y568I, Y549L, A368M, A368K, Y549L+Y434M, and A368M+Y434M (Y-tyrosine, A-alanine, Q-glutamine, M-methionine, I-isoleucine, L-leucine, and K-lysine), respectively. After confirming that the sequencing was correct, the plasmids were extracted and preserved.

[0035] Example 4

[0036] Construction, culture, and induced expression of Escherichia coli genetically engineered bacteria with high expression of proline dehydrogenase PxPutA mutant

[0037] The mutant plasmids obtained in Example 3 were transformed into competent E. coli BL21(DE3) cells, plated, and allowed to grow overnight. Single colonies were picked and inoculated into 5 mL LB tubes (kanamycin sulfate resistant) for overnight culture. The cells were then transferred at a 2% inoculum to TB medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C for 2 hours. IPTG was added to a final concentration of 0.2 mmol / L, and the culture was continued at 18°C ​​for 24 hours to terminate fermentation. The bacterial culture was collected for later use. (LB medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride; TB medium composition: 12 g / L tryptone, 24 g / L yeast extract, 4 g / L glycerol, 12.54 g / L K₂HPO₄, 2.31 g / L KH₂PO₄)

[0038] Example 5

[0039] The proline dehydrogenase PxPutA mutant was used to prepare D-proline.

[0040] In a 10 ml system, 50 g / L of DL-proline was added to the fermentation broth containing 30 g / L wet cells (the bacterial culture prepared in Example 4). After conversion at 30 °C and shaking (220 rpm) for 24 h, a sample was taken. Since the reaction is essentially a specific dehydrogenation of L-proline in DL-proline, the final reaction result is expressed as the ee value of D-proline. The ee values ​​of D-proline produced by wild-type proline dehydrogenase PxPutA (WT) and proline dehydrogenase PxPutA mutant are shown in Table 1 below.

[0041] Table 1. Summary of ee values ​​for D-proline production by wild-type proline dehydrogenase PxPutA and mutants.

[0042]

[0043]

[0044] Example 6

[0045] The proline dehydrogenase PxPutA mutant Y434M was used to prepare D-proline (by changing the DL-proline concentration). In a 10 ml system, 30 g / L of DL-proline was added to a fermentation broth containing 30 g / L Y434M wet cells (the bacterial broth prepared in Example 4). After transformation at 30 °C and shaking (220 rpm) for 24 h, a sample was taken and analyzed by HPLC. The L-proline was completely dehydrogenated, the D-proline ee value was 100%, and the D-proline concentration was 10.5 g / L.

[0046] Example 7

[0047] The proline dehydrogenase PxPutA mutant Y434M was used to prepare D-proline (by altering the conversion time).

[0048] In a 10 ml system, 50 g / L of DL-proline was added to the fermentation broth containing 30 g / L Y434M wet cells. The mixture was then shaken on a shaker (220 rpm) at 30 °C for 36 h. The L-proline was completely dehydrogenated, the D-proline ee value was 100%, and the D-proline concentration was 17.5 g / L.

Claims

1. A mutant of proline dehydrogenase, characterized in that, The amino acid sequence of the mutant is obtained by performing any one of the following mutations on the amino acid sequence shown in SEQ ID NO.1: Y434M, Y549L+Y434M, or A368M+Y434M; where the amino acid abbreviations mean the following: A-alanine, L-leucine, Y-tyrosine, and M-methionine.

2. The application of the proline dehydrogenase mutant as described in claim 1 in the degradation of L-proline, characterized in that, Includes the following steps: (1) Construction of recombinant Escherichia coli: Using the DNA fragment shown in SEQ ID NO. 2 as a template, site mutation PCR was performed to construct a recombinant plasmid containing the coding gene of the mutant described in claim 1. The plasmid containing the mutant was transferred into competent cells of Escherichia coli BL21(DE3) to obtain highly expressed recombinant Escherichia coli. (2) Fermentation culture: Pick a single colony from step (1), inoculate it into a test tube containing kanamycin sulfate in LB medium and culture overnight. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation rate of 2%, culture at 37°C for 2 hours, add IPTG inducer to a final concentration of 0.2 mmol / L, continue to culture at 18°C ​​for 24 hours, end fermentation, and directly add the cells in the fermentation broth to the substrate for reaction without separation; (3) Degradation of L-proline in DL-proline: The bacterial solution obtained from step (2) of fermentation was degraded by whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline was 30-50 g / L, the amount of recombinant Escherichia coli wet cell solution added was 20-30 g / L, the reaction temperature range was 20℃~40℃, and the degradation time was 24-36 h.

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