CN106520715B - A kind of short-chain dehydrogenase and its gene, recombinant expression carrier, genetic engineering bacterium and its application in the synthesis of astaxanthin chiral intermediate - Google Patents

Abstract

The present invention provides a short-chain dehydrogenase, a gene thereof, a recombinant short-chain dehydrogenase, a recombinant expression vector containing the gene, a genetically engineered bacteria, a method for preparing the recombinant enzyme, and the short-chain dehydrogenase or Application of genetically engineered bacteria containing the enzyme in asymmetric reduction of latent chiral ketones to synthesize astaxanthin chiral intermediates. The short-chain dehydrogenase described in the present invention has significant advantages to prepare chiral alcohols by asymmetric reduction, and can synthesize astaxanthin chiral intermediates with high optical purity (ee>99%). The catalyst is easy to prepare, has mild reaction conditions, wide substrate adaptability, and is environmentally friendly, and its recombinant cells can efficiently catalyze the asymmetric reduction of latent chiral ketones in an isopropanol-containing reaction system without any coenzymes. Prospects for industrial application development.

Description

A short-chain dehydrogenase and its gene, recombinant expression vector, genetically engineered bacteria and its Application of Astaxanthin in the Synthesis of Chiral Intermediates

(1) Technical field

The invention belongs to the technical field of biochemical industry, in particular to a short-chain dehydrogenase and a gene thereof, as well as a recombinant expression vector and a recombinant expression transformant containing the gene, and the short-chain dehydrogenase or a recombinant cell containing the enzyme in Applications in the synthesis of astaxanthin chiral intermediates.

(2) Background technology

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-carotene) is a new type of carotenoid that exists widely in nature. Astaxanthin is one of the strongest natural antioxidants in the world. Its antioxidant capacity is more than 10 times that of other carotenoids and 300 to 500 times that of vitamin E. It has anti-tumor, anti-radiation, anti-aging, and immune-enhancing properties. Therefore, it is widely used in the production of health products, medicines, cosmetics, food and feed. Astaxanthin mainly has three different isomeric forms, and the strongest antioxidant activity is (3S,3'S)-configuration of astaxanthin.

At present, the main methods of obtaining astaxanthin include chemical synthesis, extraction from crustacean shells, and microbial fermentation. Among them, chemically synthesized astaxanthin was approved by the FDA in the 1980s as an additive for aquatic feed; however, the simultaneous existence of various isomers of chemically synthesized astaxanthin is not conducive to the digestion, absorption and body deposition of animals; in addition, People's doubts about the safety of chemically synthesized astaxanthin for vegetarian use have not been eliminated, and its use is limited to the feed additive industry. Compared with chemically synthesized astaxanthin, natural astaxanthin has the advantages of simpler production process, few non-functional isomers in the product, rich functions, easy absorption and strong antioxidant activity. The efficient production of the element has become an important choice. However, the extraction and reuse of crustacean astaxanthin are limited due to the high content of chitin and ash, and low concentrations of protein and other nutrients in crustacean carapaces. On the other hand, although the yield of astaxanthin of microorganisms (Faffia rhodochrous and Haematococcus pluvialis, etc.) can be improved through various ways such as strain breeding and optimization of fermentation conditions, due to the inferiority of synthetic astaxanthin The research on the metabolic mechanism is not in-depth enough, and the production of astaxanthin has not been improved by a breakthrough.

Biocatalysis has become one of the most attractive green synthesis technologies for pharmaceuticals and fine chemicals due to its high stereoselectivity, mild reaction conditions, and environmental friendliness. Therefore, it is of great significance to synthesize the important chiral intermediates of (3S,3'S)-astaxanthin with high stereoselectivity by biocatalysis, and then obtain the chiral astaxanthin monomer with the strongest antioxidant activity.

(3) Contents of the invention

The purpose of the present invention is to provide a Pseudomonas putida short-chain dehydrogenase gene and its recombinant short-chain dehydrogenase, a recombinant expression vector containing the gene, a recombinant expression transformant, and a method for preparing the recombinant enzyme, And the application of the short-chain dehydrogenase or the recombinant cell containing the enzyme in the synthesis of (3S,3'S)-astaxanthin chiral intermediate.

The technical scheme adopted in the present invention is:

A first aspect of the present invention provides a short-chain dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO: 2 in the sequence listing.

The short-chain dehydrogenase of the present invention is derived from Pseudomonas putida ATCC 12633.

Any deletion, insertion or substitution of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 and having short-chain dehydrogenase activity still falls within the protection scope of the present invention.

The second aspect of the present invention provides a short-chain dehydrogenase gene, whose (1) nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing; or (2) the encoded amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing protein shown in.

The gene was cloned from the genome of Pseudomonas putida ATCC12633 (Pseudomonas putida ATCC12633) by PCR technology. The specific preparation method is: design and synthesize primers according to the gene sequences of short-chain dehydrogenases recorded in Genbank, preferably, the upstream primers are: GCTGA GGATCC ATGGCTAATGCAAAAACCGC; the downstream primers are: GCATC CTCGAG TCACCAGACCAAGGGTTCGC; and then use Pseudomonas putida ATCC 12633 genomic DNA As a template, polymerase chain reaction (PCR) was used for gene amplification to obtain a short-chain dehydrogenase gene sequence with a full length of 687 bp. The nucleotide sequence of the short-chain dehydrogenase gene is shown in SEQ ID No: 1 in the sequence listing. The amino acid sequence of the protein encoded by the sequence is shown in SEQ ID No: 2 in the sequence listing.

As known to those skilled in the art, the nucleotide sequence of the short-chain dehydrogenase gene of the present invention can also be any other nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID No: 2 in the sequence listing.

Any nucleotide sequence obtained by the substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1, as long as it has more than 90% homology with nucleotides, All belong to the protection scope of the present invention.

The third aspect of the present invention provides a recombinant expression vector comprising the nucleotide sequence of the short-chain dehydrogenase gene of the present invention. These recombinant vectors can be constructed by linking the short-chain dehydrogenase nucleotide sequence of the present invention to various vectors by conventional methods in the art. The vector can be various conventional vectors in the field, such as various plasmids, phage or viral vectors, etc., preferably pET-30a. Preferably, the recombinant expression vector of the present invention can be obtained by the following method: the short-chain dehydrogenase gene product Ppysdr obtained by PCR amplification is connected with the carrier pET-30a to construct the short-chain dehydrogenase gene recombinant expression of the present invention. Plasmid pET30a-Ppysdr.

The fourth aspect of the present invention provides a genetically engineered bacterium expressing a recombinant short-chain dehydrogenase, which can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism can be any conventional host microorganism in the field, as long as the recombinant expression vector can stably replicate itself and the short-chain dehydrogenase gene of the present invention carried by it can be effectively expressed. In the present invention, E. coli is preferred, and E. coli BL21 (DE3) is more preferred. The recombinant plasmid pET30a-Ppysdr was transformed into E.coli BL21(DE3) to obtain engineering bacteria E.coli BL21(DE3)/pET30a-Ppysdr.

A fifth aspect of the present invention provides a method for preparing a recombinant short-chain dehydrogenase, comprising the steps of: culturing the recombinant expression transformant of the present invention, and inducing to obtain a recombinant short-chain dehydrogenase. Wherein, the medium used for culturing the recombinant expression transformant can be a medium in the art that can make the transformant grow and produce the short-chain dehydrogenase of the present invention, preferably LB medium: peptone 10g/L, yeast extract 5g /L, sodium chloride 10g/L, pH 7.2. The culture method and culture conditions are not particularly limited as long as the transformant can grow and produce short-chain dehydrogenase. The following method is preferred: inoculate the recombinant Escherichia coli E.coli BL21(DE3)/pET30a-Ppysdr involved in the present invention into LB medium containing kanamycin for culture, when the optical density OD ₆₀₀ of the culture solution reaches 0.5-0.7 When the final concentration is 0.1-1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant short-chain dehydrogenase of the present invention can be highly expressed.

The sixth aspect of the present invention provides the use of the short-chain dehydrogenase or its recombinant cell in asymmetrically catalyzing latent chiral ketone (I) to prepare (3S,3'S)-astaxanthin chiral intermediate (II).

Specifically, the application is as follows: using latent chiral ketone (I) as a substrate, using the short-chain dehydrogenase or its recombinant cell as a catalyst, using NADH or NADPH as a coenzyme, at 20 to 50° C. The reaction is carried out in a conversion reaction system a composed of a buffer solution with a pH of 5.5-10.5. After the reaction is completed, the reaction solution is separated and purified to obtain the corresponding product.

The reaction conditions can be selected according to conventional conditions used in the art.

Further, the initial concentration of the substrate in the transformation system a is 5-1000 mmol/L.

Further, the preferred concentration of the recombinant short-chain dehydrogenase pure enzyme in the reaction solution in the transformation system a is 0.1-2.0 mg/mL. The mass dosage of the bacterial cells in the transformation system a is 10-400 g/L in terms of the wet weight of the bacterial cells.

Further, the transformation system a also includes an organic solvent, and the transformation system b is composed of a substrate, a catalyst, an organic solvent and a buffer of pH 5.5 to 10.5, and the organic solvent accounts for 1 to 20% of the total volume of the transformation system b. The initial concentration of the substrate is 5-1000 mmol/L, and the mass and dosage of the bacteria is 10-400 g/L based on the wet weight of the bacteria.

Further, the reaction was carried out in a pH 7.5 buffer.

Further, 1-15% alcohol or sugar can be added to the reaction system as a co-substrate, which can significantly improve the activity of the reaction. The co-substrate includes but is not limited to one of the following: ① ethanol, ② isopropanol, ③ glucose, ④ sucrose, etc.

Further, the co-substrate in the reaction system is isopropanol.

Further, the concentration of isopropanol in the reaction system was 10%.

Further, the method for separation and purification of the transformation reaction solution is as follows: after the reaction is completed, the transformation reaction solution is centrifuged, the supernatant is extracted with an equal volume of ethyl acetate, the organic layer is the crude product containing the corresponding chiral alcohol, and the crude product is purified That is, the corresponding chiral alcohol is obtained. The method for purifying the crude product is a technique known in the art, usually organic solvent extraction, chromatographic separation, adsorption separation, and the like.

The transformation system a and transformation system b of the present invention are both transformation systems, and are named for the convenience of distinguishing the different compositions of transformation systems in different steps, and the letters themselves have no meaning.

The beneficial effects of the present invention are mainly reflected in: providing a short-chain dehydrogenase derived from Pseudomonas putida ATCC12633 and its gene, as well as recombinant expression vectors and recombinant expression transformants containing the gene, through the short-chain dehydrogenase or The asymmetric reduction of recombinant cells containing the enzyme can prepare astaxanthin chiral intermediates with high optical purity; the short-chain dehydrogenase described in the present invention or the recombinant cells containing the enzyme asymmetric reduction to prepare chiral alcohols have significant advantages, Able to synthesize high optical purity astaxanthin chiral intermediates (ee>99%). The catalyst is easy to prepare, has high stereoselectivity, mild reaction conditions, and is environmentally friendly, and has good application and development prospects.

(4) Description of drawings

Fig. 1 is the agarose gel electrophoresis picture of PCR amplification product of short-chain dehydrogenase gene;

Figure 2 shows the SDS-PAGE chart of the separation and purification of short-chain dehydrogenase.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1: Acquisition of Pseudomonas putida ATCC 12633 short-chain dehydrogenase gene, construction of recombinant plasmid and transformation of Escherichia coli

The whole genome DNA of Pseudomonas putida ATCC 12633 was extracted with a DNA extraction kit. Using the DNA as a template, the upstream primer (GCTGA GGATCC ATGGCTAATGCAAAAACCGC) and the downstream primer (GCATC CTCGAG TCACCAGACCAAGGGTTCGC) were used as the primers for PCR amplification reaction. The addition amount of each component of the PCR reaction system (total volume 50 μL): 5×PrimeSTAR ^TM HS DNA polymerase Buffer 10 μL, 10 mM dNTPmixture (2.5 mM each of dATP, dCTP, dGTP and dTTP) 4 μL, the upstream primer and the downstream primer each with a concentration of 50 μM 1 μL, genomic DNA 1 μL, PrimeSTAR ^™ HS DNA polymerase 0.5 μL, nucleic acid-free water 32.5 μL. The PCR reaction conditions were: pre-denaturation at 98°C for 1min, then entered a temperature cycle of 98°C for 10s, 56°C for 15s, 72°C for 1min, a total of 30 cycles, and finally extended at 72°C for 5min, with a termination temperature of 4°C. The results of agarose gel electrophoresis of the PCR amplification products of the short-chain dehydrogenase gene are shown in FIG. 1 . The results of sequencing analysis showed that the length of the nucleotide sequence amplified by the above process was 687 bp (the nucleotide sequence is shown in SEQ ID NO: 1), and the sequence encoded a complete open reading frame. Using software to analyze the gene sequence, it is inferred that the short-chain dehydrogenase gene encodes the amino acid sequence shown in SEQ ID NO: 2.

The PCR product was double digested by BamHI/XhoI, and the target fragment was recovered by agarose gel recovery kit, and then the fragment was ligated with the commercial vector pET-30a treated with the same restriction endonuclease by T4 ligase to construct Recombinant expression plasmid pET30a-Ppysdr.

The recombinant expression vector pET30a-Ppysdr constructed above was transformed into E. coli BL21(DE3) to obtain recombinant E. coli BL21(DE3)/pET30a-Ppysdr, which was spread on a plate containing kanamycin at 37°C Cultivated overnight, randomly picked clones for colony PCR identification, positive clones were sequenced and verified, the results showed that the recombinant expression vector pET30a-Ppysdr was successfully transformed into the expression host E.coli BL21(DE3), and the short-chain dehydrogenase gene had been successfully cloned into BamHI and XhoI sites of pET-30a.

Example 2: Inducible expression of recombinant short-chain dehydrogenase

The engineered bacteria E.coli BL21(DE3)/pET30a-Ppysdr constructed in Example 1 were inoculated into LB liquid medium containing 50 μg/mL kanamycin, cultured at 37°C overnight, and then at 1% inoculum (v/v ) was inoculated into 50 mL of LB medium containing 50 μg/mL kanamycin, cultivated at 37 °C, 200 rpm to a bacterial concentration of OD ₆₀₀ to about 0.6, added IPTG with a final concentration of 0.1 mM, and induced culture at 26 °C for 6 h. The cells were collected by centrifugation at 8000 rpm for 10 min and stored at -80 °C for later use.

Example 3: Isolation and purification of recombinant short-chain dehydrogenase

The bacterial cells collected in Example 2 were suspended in 10 mL of Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer (100 mM, pH 8.0), shaken and shaken well and then crushed under ultrasonic waves (effective time 8 min). The disrupted solution was centrifuged at 12,000 rpm for 10 min to remove cell debris, and the supernatant (crude enzyme solution) was collected for subsequent separation and purification of the enzyme. The purification column is Ni-NTA, and the packing volume is 5 mL. First, equilibrate the Ni-NTA column with loading equilibration buffer (20 mM sodium phosphate, 500 mM NaCl and 20 mM imidazole, pH 7.4), and load the crude enzyme at a rate of 5 mL/min. Then, eluted with loading equilibration buffer to remove unadsorbed protein, and finally eluted with elution buffer (20 mMT sodium phosphate, 500 mM NaCl and 500 mM imidazole, pH 7.4) to collect the target protein. The enzyme solution was desalted with a HiTrap desalting column, and the desalting buffer was Na ₂ HPO ₄ -NaH ₂ PO ₄ (100 mM, pH 7.5) buffer, and the obtained pure enzyme solution was stored at 4° C. for later use. The purified enzyme solution was analyzed by SDS-PAGE, and the SDS-PAGE electrophoresis was shown in Figure 2. The results showed that electrophoretic pure recombinant short-chain dehydrogenase PpYSDR was obtained by Ni-NTA affinity chromatography.

Example 4: Asymmetric reduction of latent chiral ketone (I) by recombinant short-chain dehydrogenase

Take 1 mL of the pure enzyme solution obtained in Example 3, and then add 10 mM latent chiral ketone (I) and 5 mM NADH as substrate and coenzyme, respectively. The reaction was carried out at 30°C with constant temperature shaking (200rpm) for 16h. The reaction solution was extracted with an equal volume of ethyl acetate, and the contents and enantiomeric excess (ee) of substrates and products were analyzed by chiral capillary gas chromatography. The optical purity of the product (3S,3'S)-astaxanthin chiral intermediate is >99%, and the conversion rate is 65%.

Example 5: Asymmetric reduction of latent chiral ketone (I) by recombinant short-chain dehydrogenase

Take 900 μL of the pure enzyme solution obtained in Example 3, add 100 μL of isopropanol, and then add 10 mM latent chiral ketone (I) and 5 mM NADH as substrate and coenzyme, respectively. The reaction was carried out at 30°C with constant temperature shaking (200rpm) for 16h. The reaction solution was extracted with an equal volume of ethyl acetate, and the contents and enantiomeric excess (ee) of substrates and products were analyzed by chiral capillary gas chromatography. The optical purity of the product (3S,3'S)-astaxanthin chiral intermediate (II) was >99%, and the conversion was 96.2%.

Example 6: Asymmetric reduction of latent chiral ketones (I) by recombinant Escherichia coli

In 900 μL, 100 mM Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer (pH 7.5), add 100 μL isopropanol, add 0.05 g of the wet bacterial cells obtained in Example 2, and then add 10 mM latent chiral ketone (I) as a substrate. The reaction was carried out at 30°C with constant temperature shaking (200rpm) for 12h. The reaction solution was centrifuged, the supernatant was taken, extracted with an equal volume of ethyl acetate, and the contents and enantiomeric excess (ee) of substrates and products were analyzed by chiral capillary gas chromatography. The optical purity of the product (3S,3'S)-astaxanthin chiral intermediate (II) was >99%, and the conversion rate was 95.8%.

Claims (3)

1. the application of a short-chain dehydrogenase or genetically engineered bacteria in asymmetric catalysis latent chiral ketone preparation (3S, 3'S)-astaxanthin chiral intermediate, it is characterized in that, described short-chain dehydrogenase The amino acid sequence is shown in SEQ ID NO: 2; the genetically engineered bacteria are represented by the nucleotide sequence comprising the short-chain dehydrogenase gene shown in SEQ ID NO: 1 or the amino acid sequence SEQ ID NO: 2. The recombinant expression vector showing the nucleotide sequence of the protein is transformed into the host microorganism to obtain; wherein the structural formula of the latent chiral ketone is shown in formula I, and the structural formula of the (3S,3'S)-astaxanthin chiral intermediate is shown in formula II. : The transformation system for preparing (3S,3'S)-astaxanthin chiral intermediates from asymmetrically catalyzed latent chiral ketones is transformation system a, in which the concentration of recombinant short-chain dehydrogenase in the reaction solution is 0.1-2.0 mg/mL, or the mass dosage of the genetically engineered bacteria in the transformation system a is 10-400 g/L based on the wet weight of the bacteria. 2. application according to claim 1, is characterized in that, in taking latent chiral ketone as substrate, taking short-chain dehydrogenase or its genetically engineered bacteria as catalyst, taking NADH or NADPH as coenzyme, 20～50 ℃ Then, the reaction is carried out in the conversion reaction system a composed of a buffer solution with pH 5.5 to 10.5. After the reaction is complete, the reaction solution is separated and purified to obtain the product (3S,3'S)-astaxanthin chiral intermediate. The reaction is as follows: 3. application according to claim 1 is characterized in that, reaction system can also add isopropanol as auxiliary substrate.