CN114457128A - Recombinant engineering bacterium and construction method and application thereof - Google Patents
Recombinant engineering bacterium and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to a recombinant engineering bacterium, a construction method and application thereof, wherein the recombinant engineering bacterium is induced to efficiently express L-pantolactone dehydrogenase, ketopantolactone reductase, formate dehydrogenase and D-pantolactone hydrolase, and efficiently convert DL-pantolactone to generate D-pantolactone. The recombinant engineering bacteria of the invention improve the selectivity of L-pantolactone dehydrogenase, obviously improve the purity and quality of D-pantolactone, the reaction efficiency and the resource utilization rate, and have the advantages of simple operation, environmental protection, higher cost and the like.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium, a construction method and application thereof.
Background
D-pantoic acid lactone is a medical intermediate for synthesizing vitamin medicine D-panthenol and neurotrophic medicine D-calcium homopantothenate, is used as a synthetic precursor of feed additives and daily chemical products, and has annual output of more than ten thousand tons.
DL-pantoic acid lactone is usually prepared by a chemical synthesis method, and then the prepared DL-pantoic acid lactone is converted into D-pantoic acid lactone. The method for obtaining the D-pantolactone by resolving the DL-pantolactone by a microbial enzyme method comprises the following steps: 1) d-pantoic acid lactone or L-pantoic acid lactone in the DL-pantoic acid lactone is selectively hydrolyzed by microbial enzyme to obtain L-pantoic acid lactone and D-pantoic acid or L-pantoic acid and D-pantoic acid lactone; 2) extracting the mixed solution by using an organic reagent to realize the separation of L-pantolactone and D-pantoic acid; 3) d-pantoic acid is subjected to lactonization to obtain D-pantoic acid lactone, and L-pantoic acid lactone is subjected to chemical racemization to be changed into DL-pantoic acid lactone again and then is split. This method has the following drawbacks: firstly, the separation steps are multiple, and the separation efficiency is low; secondly, a great amount of pigment and other impurities are generated in the chemical racemization process, and the D-pantoic acid lactone prepared by splitting has darker color and poorer appearance quality; thirdly, chemical racemization can generate a large amount of sulfate, form solid waste and cause environmental pollution.
CN110423717A discloses a multienzyme recombinant cell and a method for synthesizing D-pantolactone by multienzyme cascade catalysis, which converts D, L-pantolactone into ketopantolactone and further generates D-pantolactone by expressing L-pantolactone dehydrogenase, ketopantolactone reductase and glucose dehydrogenase by the recombinant cell. This method has the following drawbacks: the L-pantolactone dehydrogenase has poor selectivity on L-pantolactone and catalytic reaction thereof, and can catalyze the L-pantolactone to generate ketopantolactone and D-pantolactone to generate ketopantolactone, wherein the L-pantolactone and the D-pantolactone compete to combine with an active site of the L-pantolactone dehydrogenase simultaneously, and the D-pantolactone can enter into a cyclic reaction, so that the enzyme activity is reduced; when the content of L-pantolactone in a substrate is low, the L-pantolactone dehydrogenase cannot be effectively contacted with the L-pantolactone dehydrogenase, complete conversion is difficult, so that the prepared product contains the L-pantolactone, the purification difficulty is increased, and the product quality is difficult to ensure.
Therefore, how to develop a recombinant vector and engineering bacteria thereof which have high selectivity and are beneficial to improving the purity and quality of D-pantoic acid lactone, the reaction efficiency, the resource utilization rate and the like becomes an aspect which needs to be solved urgently in the field.
Disclosure of Invention
The invention aims to provide a recombinant vector which contains a recombinant vector, wherein the recombinant vector contains a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence shown as SEQ ID No.1, a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence shown as SEQ ID No.2, a nucleotide sequence of a formate dehydrogenase modified gene sequence shown as SEQ ID No.3 and a nucleotide sequence of a D-pantoate lactone hydrolase modified gene sequence shown as SEQ ID No. 4.
In a preferred technical scheme of the invention, a nucleotide sequence of an L-pantoate lactone dehydrogenase modified gene sequence is shown as SEQ ID NO.1, a nucleotide sequence of a ketopantoate lactone reductase modified gene sequence is shown as SEQ ID NO.2, a nucleotide sequence of a formate dehydrogenase modified gene sequence is shown as SEQ ID NO.3, and a nucleotide sequence of a D-pantoate lactone hydrolase modified gene sequence is shown as SEQ ID NO.4 on a first recombinant vector, a second recombinant vector, a third recombinant vector and a fourth recombinant vector respectively.
In a preferred technical scheme of the present invention, the recombinant vector optionally includes any one of a fifth recombinant vector or a sixth recombinant vector, wherein the fifth recombinant vector comprises a nucleotide sequence of an L-pantoate lactone dehydrogenase-modified gene sequence shown in SEQ ID No.1 and a nucleotide sequence of a ketopantoate lactone reductase-modified gene sequence shown in SEQ ID No.2, the sixth recombinant vector comprises a nucleotide sequence of an L-pantoate lactone dehydrogenase-modified gene sequence shown in SEQ ID No.1, a nucleotide sequence of a ketopantoate lactone reductase-modified gene sequence shown in SEQ ID No.2, a nucleotide sequence of a formate dehydrogenase-modified gene sequence shown in SEQ ID No.3, and a nucleotide sequence of a D-pantoate lactone hydrolase-modified gene sequence shown in SEQ ID No. 4.
In the preferable technical scheme of the invention, the recombinant vector is used for preparing D-pantolactone from DL-pantolactone.
In the preferable technical scheme of the invention, the L-pantoate lactone dehydrogenase encoding gene is subjected to codon optimization to obtain an L-pantoate lactone dehydrogenase modified gene sequence.
In a preferred technical scheme of the invention, the coding gene of the L-pantoate lactone dehydrogenase is derived from any one of rhodococcus erythropolis, mycobacterium, nocardia, streptomycete and actinomycetes.
In the preferable technical scheme of the invention, the L-pantoate lactone dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 1, and the nucleotide sequence of the target gene 1 is shown as SEQ ID NO. 1.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase encoding gene is subjected to codon optimization to obtain a ketopantoate lactone reductase modified gene sequence.
In a preferred technical scheme of the invention, the ketopantoate lactone reductase coding gene is derived from any one of candida magnoliavine, micromonospora and streptomyces.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 2, and the nucleotide sequence of the target gene 2 is shown as SEQ ID NO. 2.
In a preferred embodiment of the present invention, the cleavage site is selected from SacI and NotI, or a combination thereof.
In the preferable technical scheme of the invention, the formate dehydrogenase encoding gene is subjected to codon optimization to obtain a formate dehydrogenase modified gene sequence.
In a preferred embodiment of the present invention, the formate dehydrogenase-encoding gene is derived from any one of Burkholderia, Escherichia coli and Aeromonas.
In the preferred technical scheme of the invention, the formate dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 3, and the nucleotide sequence of the target gene 3 is shown as SEQ ID NO. 3.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In the preferred technical scheme of the invention, the D-pantolactone hydrolase coding gene is subjected to codon optimization to obtain a D-pantolactone modified gene sequence.
In a preferred technical scheme of the invention, the D-pantoate lactone hydrolase coding gene is derived from any one of fusarium moniliforme, fusarium solani and fusarium.
In the preferred technical scheme of the invention, the D-pantoic acid lactone hydrolase modification gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 4, and the nucleotide sequence of the target gene 4 is shown as SEQ ID NO. 4.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In a preferred embodiment of the present invention, the codon optimization is performed according to the codon preference of E.coli.
In a preferred embodiment of the present invention, the vector for recombination is selected from any one of pET-21a plasmid, pET-28a plasmid, pRSFDuet-I plasmid, or a combination thereof.
In a preferred embodiment of the present invention, the method for obtaining the first recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 into a first vector to obtain a first recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the second recombinant vector comprises the following steps: the ketopantoate lactone reductase is modified into a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 into a second vector to obtain a second recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the third recombinant vector comprises the following steps: modifying a gene sequence of formate dehydrogenase or a target gene 3 or a gene sequence shown as SEQ ID NO: 3 into a third vector to obtain a third recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: modifying a D-pantoic acid lactone hydrolase modified gene sequence or a target gene 4 or a gene sequence shown as SEQ ID NO: 4 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fifth recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 and ketopantoate lactone reductase modified gene sequence or target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2 into a fifth vector to obtain a fifth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fifth recombinant vector comprises the following steps: firstly, modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 into a fifth vector, and then transforming the ketopantoate lactone reductase gene sequence or the target gene 2 or the nucleotide sequence shown as SEQ ID NO: 2 into a fifth vector to obtain a fifth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fifth recombinant vector comprises the following steps: firstly, ketopantoate lactone reductase is modified to a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 to a fifth vector, and then modifying the gene sequence of the L-pantoate lactone dehydrogenase or the target gene 1 or the nucleotide sequence shown as SEQ ID NO: 1 into a fifth vector to obtain a fifth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the sixth recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1, ketopantoate lactone reductase modified gene sequence or target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2, a formate dehydrogenase modified gene sequence or a target gene 3 or a nucleotide sequence shown as SEQ ID NO: 3, D-pantolactone hydrolase modification gene sequence or target gene 4 or a nucleotide sequence shown as SEQ ID NO: 4 into a sixth vector to obtain a sixth recombinant vector, wherein the cloning sequence of the 4 genes or the nucleotide sequences is not shown in sequence.
Another object of the present invention is to provide a recombinant engineered bacterium comprising any one or combination of a first recombinant vector capable of expressing L-pantolactone dehydrogenase, a second recombinant vector expressing ketopantolactone reductase, a third recombinant vector expressing formate dehydrogenase and a fourth recombinant vector expressing D-pantolactone hydrolase;
and/or the recombinant engineering bacteria comprise a fifth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase and ketopantoate lactone reductase;
and/or the recombinant engineering bacteria comprise a sixth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase, ketopantoate lactone reductase, formate dehydrogenase encoding gene and D-pantoate lactone hydrolase.
In a preferred technical scheme of the invention, the recombinant engineering bacteria comprise a fifth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase and ketopantoate lactone reductase and a third recombinant vector for expressing formate dehydrogenase.
In a preferred technical scheme of the invention, the recombinant engineering bacteria also comprise a fourth recombinant vector for expressing D-pantolactone hydrolase, or are combined with second recombinant engineering bacteria for expressing the fourth recombinant vector of the D-pantolactone hydrolase.
In the preferable technical scheme of the invention, the recombinant engineering bacteria are used for preparing D-pantolactone from DL-pantolactone.
In a preferred technical scheme of the invention, the first recombinant vector comprises an L-pantoate lactone dehydrogenase modified gene sequence.
In the preferable technical scheme of the invention, the L-pantoate lactone dehydrogenase encoding gene is subjected to codon optimization to obtain an L-pantoate lactone dehydrogenase modified gene sequence.
In a preferred technical scheme of the invention, the coding gene of the L-pantoate lactone dehydrogenase is derived from any one of rhodococcus erythropolis, mycobacterium, nocardia, streptomycete and actinomycetes.
In the preferable technical scheme of the invention, the L-pantoate lactone dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 1, and the nucleotide sequence of the target gene 1 is shown as SEQ ID NO. 1.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In a preferred embodiment of the present invention, the second recombinant vector comprises a ketopantoate lactone reductase-modified gene sequence.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase encoding gene is subjected to codon optimization to obtain a ketopantoate lactone reductase modified gene sequence.
In a preferred technical scheme of the invention, the ketopantoate lactone reductase coding gene is derived from any one of candida magnoliae, micromonospora and streptomyces.
In the preferred technical scheme of the invention, the ketopantoate lactone reductase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 2, and the nucleotide sequence of the target gene 2 is shown as SEQ ID NO. 2.
In a preferred embodiment of the present invention, the cleavage site is selected from SacI and NotI, or a combination thereof.
In a preferred embodiment of the present invention, the third recombinant vector comprises a formate dehydrogenase-modified gene sequence.
In the preferable technical scheme of the invention, the formate dehydrogenase encoding gene is subjected to codon optimization to obtain a formate dehydrogenase modified gene sequence.
In a preferred embodiment of the present invention, the formate dehydrogenase-encoding gene is derived from any one of Burkholderia, Escherichia coli and Aeromonas.
In the preferred technical scheme of the invention, the formate dehydrogenase modified gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 3, and the nucleotide sequence of the target gene 3 is shown as SEQ ID NO. 3.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In a preferred embodiment of the present invention, the fourth recombinant vector comprises a gene encoding D-pantolactone reductase.
In the preferred technical scheme of the invention, the D-pantolactone hydrolase coding gene is subjected to codon optimization to obtain a D-pantolactone modified gene sequence.
In a preferred technical scheme of the invention, the D-pantolactone hydrolase coding gene is derived from any one of fusarium moniliforme, fusarium solani and fusarium
In the preferred technical scheme of the invention, the D-pantoic acid lactone hydrolase modification gene sequence is artificially synthesized and added with enzyme cutting sites to prepare a target gene 4, and the nucleotide sequence of the target gene 4 is shown as SEQ ID NO. 4.
In a preferred embodiment of the present invention, the cleavage site is selected from any one of XhoI and NdeI or a combination thereof.
In a preferred embodiment of the present invention, the codon optimization is performed according to the codon preference of E.coli.
In a preferred embodiment of the present invention, the vector for recombination is selected from any one of pET-21a plasmid, pET-28a plasmid, pRSFDuet-I plasmid, or a combination thereof.
In a preferred embodiment of the present invention, the method for obtaining the first recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 into a first vector to obtain a first recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the second recombinant vector comprises the following steps: the ketopantoate lactone reductase is modified into a gene sequence or a target gene 2 or a gene sequence shown as SEQ ID NO: 2 into a second vector to obtain a second recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the third recombinant vector comprises the following steps: modifying a gene sequence of formate dehydrogenase or a target gene 3 or a gene sequence shown as SEQ ID NO: 3 into a third vector to obtain a third recombinant vector. In a preferred technical scheme of the present invention, the method for obtaining the fourth recombinant vector comprises: modifying a D-pantoic acid lactone hydrolase modified gene sequence or a target gene 4 or a gene sequence shown as SEQ ID NO: 4 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fifth recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 and ketopantoate lactone reductase modified gene sequence or target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2 into a fifth vector to obtain a fifth recombinant vector, wherein the cloning sequence of the 2 genes or the nucleic acid sequences is not shown in sequence.
In a preferred embodiment of the present invention, the method for obtaining the sixth recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1, ketopantoate lactone reductase modified gene sequence or target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2, a formate dehydrogenase modified gene sequence or a target gene 3 or a nucleotide sequence shown as SEQ ID NO: 3, D-pantolactone hydrolase modification gene sequence or target gene 4 or a nucleotide sequence shown as SEQ ID NO: 4 into a sixth vector to obtain a sixth recombinant vector, wherein the cloning sequence of the 4 genes or the nucleic acid sequences is not shown in sequence.
In a preferred embodiment of the present invention, any one or a combination of the first recombinant vector, the second recombinant vector, the third recombinant vector, the fourth recombinant vector, the fifth recombinant vector or the sixth recombinant vector is sequentially or simultaneously introduced into a host cell to obtain a recombinant engineered bacterium.
In a preferred embodiment of the present invention, the host cell is any one selected from the group consisting of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, and pantoea ananatis.
In a preferred technical scheme of the invention, the induction method of the recombinant engineering bacteria comprises the following steps:
s-1, inoculating the recombinant engineering bacteria into an LB culture medium according to the inoculation amount of 1-5%, and culturing for 6-16h at the temperature of 30-40 ℃ and at the speed of 50-500rpm to obtain a first-stage seed solution;
s-2, inoculating the primary seed solution into a TB culture medium according to the inoculation amount of 1-5%, culturing for 6-20h at the temperature of 25-40 ℃ and at the speed of 50-500rpm, centrifuging, washing with a phosphate buffer solution, and collecting thalli.
In the preferred technical scheme of the invention, the fermentation temperature in the S-1 step is 35-38 ℃, and the rotation speed is 100-300 rpm.
In the preferred technical scheme of the invention, in the S-2 step, the culture is firstly carried out for 0.5-3h under the conditions of 35-38 ℃ and 100-300rpm, and then the culture is carried out for 10-15h under the conditions of 28-30 ℃ and 100-300 rpm.
In the preferable technical scheme of the invention, the LB culture medium comprises antibiotics, yeast powder, tryptone and sodium chloride.
In the preferable technical scheme of the invention, the components of the LB culture medium comprise 50-200mg/L of antibiotics, 4-6g/L of yeast powder, 8-12g/L of tryptone and 8-12g/L of sodium chloride.
In a preferred technical scheme of the invention, the antibiotic is selected from any one of penicillin, amphotericin B, nystatin, polymyxin B, streptomycin, gentamicin, tetracycline, neomycin, ampicillin and kanamycin or a combination thereof.
In the preferable technical scheme of the invention, the composition of the LB culture medium comprises 100mg/L kanamycin, 5g/L yeast powder, 10g/L tryptone and 10g/L sodium chloride.
In the preferable technical scheme of the invention, the composition of the LB culture medium comprises 50mg/L of ampicillin, 100mg/L of kanamycin, 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
In the preferable technical scheme of the invention, the TB culture medium comprises antibiotics, tryptone, yeast powder, sodium chloride, glucose and lactose.
In the preferable technical scheme of the invention, the TB culture medium comprises 50-200mg/L of antibiotic, 18-22g/L of tryptone, 4-6g/L of yeast powder, 4-6g/L of sodium chloride, 1-3g/L of glucose and 0.1-3.0g/L of lactose.
In a preferred technical scheme of the invention, the antibiotic is selected from any one of penicillin, amphotericin B, nystatin, polymyxin B, streptomycin, gentamicin, tetracycline, neomycin, ampicillin and kanamycin or a combination thereof.
In the preferable technical scheme of the invention, the composition of the TB culture medium comprises 100mg/L kanamycin, 20g/L tryptone, 5g/L yeast powder, 5g/L sodium chloride, 2g/L glucose and 0.1-2.0g/L lactose.
In the preferable technical scheme of the invention, the composition of the TB culture medium comprises 50mg/L of ampicillin, 100mg/L of kanamycin, 20g/L of tryptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2g/L of glucose and 0.1-2.0g/L of lactose.
The invention also aims to provide a construction method of the recombinant engineering bacteria, which comprises the following steps:
(1) introducing any one or combination sequence of an L-pantolactone dehydrogenase modified gene sequence, a ketopantolactone reductase modified gene sequence, a formate dehydrogenase modified gene sequence and a D-pantolactone hydrolase modified gene sequence into a vector to obtain a recombinant vector;
(2) and (3) introducing the obtained recombinant vector into a host cell to obtain the recombinant engineering bacterium.
In a preferred technical scheme of the invention, the recombinant vector is selected from any one of a fifth recombinant vector which comprises a nucleotide sequence of an L-pantoate lactone dehydrogenase-modified gene sequence shown as SEQ ID No.1 and a nucleotide sequence of a ketopantoate lactone reductase-modified gene sequence shown as SEQ ID No.2, a third recombinant vector which comprises a nucleotide sequence of a formate dehydrogenase-modified gene sequence shown as SEQ ID No.3, and a fourth recombinant vector which comprises a nucleotide sequence of a D-pantoate lactone hydrolase-modified gene sequence shown as SEQ ID No. 4.
In a preferred embodiment of the present invention, the method for obtaining the third recombinant vector comprises the following steps: modifying a gene sequence of formate dehydrogenase or a target gene 3 or a gene sequence shown as SEQ ID NO: 3 into a third vector to obtain a third recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fourth recombinant vector comprises the following steps: modifying a D-pantoic acid lactone hydrolase modified gene sequence or a target gene 4 or a gene sequence shown as SEQ ID NO: 4 into a fourth vector to obtain a fourth recombinant vector.
In a preferred embodiment of the present invention, the method for obtaining the fifth recombinant vector comprises the following steps: modifying a gene sequence or a target gene 1 of L-pantoate lactone dehydrogenase or a gene sequence shown as SEQ ID NO: 1 and ketopantoate lactone reductase modified gene sequence or target gene 2 or a nucleotide sequence shown as SEQ ID NO: 2 into a fifth vector to obtain a fifth recombinant vector, wherein the cloning sequence of the two genes or nucleic acid sequences is not shown in sequence.
In a preferred embodiment of the present invention, the vector for recombination is selected from any one of pET-21a plasmid, pET-28a plasmid, pRSFDuet-I plasmid, or a combination thereof.
In a preferred technical scheme of the invention, any one or a combination of a third recombinant vector, a fourth recombinant vector or a fifth recombinant vector is sequentially or synchronously introduced into a host cell to obtain the recombinant engineering bacteria.
In a preferred technical scheme of the invention, the recombinant engineering bacteria comprise a fifth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase and ketopantoate lactone reductase and a third recombinant vector for expressing formate dehydrogenase.
In a preferred technical scheme of the invention, the recombinant engineering bacteria also comprise a fourth recombinant vector for expressing D-pantolactone hydrolase, or are combined with second recombinant engineering bacteria for expressing the fourth recombinant vector of the D-pantolactone hydrolase.
In a preferred embodiment of the present invention, the host cell is any one selected from the group consisting of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, and pantoea ananatis.
The invention also aims to provide an induced expression method of the recombinant engineering bacteria, which comprises the following steps:
s-1, inoculating the recombinant engineering bacteria into an LB culture medium according to the inoculation amount of 1-5%, and culturing for 6-16h at the temperature of 30-40 ℃ and at the speed of 50-500rpm to obtain a first-stage seed solution;
s-2, inoculating the primary seed solution into a TB culture medium according to the inoculation amount of 1-5%, culturing for 6-20h at the temperature of 25-40 ℃ and at the speed of 50-500rpm, centrifuging, washing with a phosphate buffer solution, and collecting thalli.
In the preferred technical scheme of the invention, in the step S-1, the fermentation temperature is 35-38 ℃, and the rotation speed is 100-300 rpm.
In the preferred technical scheme of the invention, the S-2 step is firstly cultured for 0.5-3h under the conditions of 35-38 ℃ and 100-300rpm and then cultured for 10-15h under the conditions of 28-30 ℃ and 100-300 rpm.
In the preferable technical scheme of the invention, the LB culture medium comprises antibiotics, yeast powder, tryptone and sodium chloride.
In the preferable technical scheme of the invention, the composition of the LB culture medium comprises 50-200mg/L of antibiotic, 4-6g/L of yeast powder, 8-12g/L of tryptone and 8-12g/L of sodium chloride.
In a preferred technical scheme of the invention, the antibiotic is selected from any one of penicillin, amphotericin B, nystatin, polymyxin B, streptomycin, gentamicin, tetracycline, neomycin, ampicillin and kanamycin or a combination thereof.
In the preferable technical scheme of the invention, the composition of the LB culture medium comprises 100mg/L kanamycin, 5g/L yeast powder, 10g/L tryptone and 10g/L sodium chloride.
In the preferable technical scheme of the invention, the composition of the LB culture medium comprises 50mg/L of ampicillin, 100mg/L of kanamycin, 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
In the preferable technical scheme of the invention, the TB culture medium comprises antibiotics, tryptone, yeast powder, sodium chloride, glucose and lactose.
In the preferable technical scheme of the invention, the components of the TB culture medium comprise 50-200mg/L of antibiotics, 18-22g/L of tryptone, 4-6g/L of yeast powder, 4-6g/L of sodium chloride, 1-3g/L of glucose and 0.1-3.0g/L of lactose.
In a preferred technical scheme of the invention, the antibiotic is selected from any one of penicillin, amphotericin B, nystatin, polymyxin B, streptomycin, gentamicin, tetracycline, neomycin, ampicillin and kanamycin or a combination thereof.
In the preferable technical scheme of the invention, the composition of the TB culture medium comprises 100mg/L kanamycin, 20g/L tryptone, 5g/L yeast powder, 5g/L sodium chloride, 2g/L glucose and 0.1-2.0g/L lactose.
In the preferable technical scheme of the invention, the composition of the TB culture medium comprises 50mg/L of ampicillin, 100mg/L of kanamycin, 20g/L of tryptone, 5g/L of yeast powder, 5g/L of sodium chloride, 2g/L of glucose and 0.1-2.0g/L of lactose.
Another object of the present invention is to provide a process for the preparation of D-pantolactone, comprising the steps of: putting DL-pantoic acid lactone with the concentration of 10-300g/L into a reaction system with formic acid and recombinant engineering bacteria OD value of 1-5, and reacting for 20-40h at 30-40 ℃ under the condition of pH5-7 to prepare the D-pantoic acid lactone.
In the preferable technical scheme of the invention, the OD value of the recombinant engineering bacteria is 2-3.
In the preferable technical scheme of the invention, the recombinant engineering bacteria can express L-pantolactone dehydrogenase, ketopantolactone reductase and formate dehydrogenase.
In the preferred technical scheme of the invention, recombinant engineering bacteria for expressing D-pantolactone hydrolase can be added.
In the preferred technical scheme of the invention, the reaction solution is filtered, the filtrate is collected, the pH value is adjusted to 1-3, and the filtrate is placed.
In a preferred embodiment of the present invention, DL-pantoic acid lactone: the molar ratio of formic acid is 1 (0.8-1.2), preferably 1 (0.9-1.1).
In a preferred embodiment of the present invention, the concentration of DL-pantoic acid lactone is 50-250g/L, preferably 100-200 g/L.
In the preferred technical scheme of the invention, the temperature of the reaction system is 35-37 ℃.
In the preferred technical scheme of the invention, the reaction time is 25-35 h.
In the preferred technical scheme of the invention, the pH of the reaction system is 5.5-6.5, and the preferred pH is 5.8-6.2.
In a preferred embodiment of the present invention, the pH adjuster is selected from any one of ammonia water, sodium hydroxide, sodium bicarbonate, triethylamine, potassium hydroxide, sodium phosphate, sodium citrate, sodium malate, phosphate buffer, Tris buffer, and sulfuric acid.
In a preferred embodiment of the present invention, NADPH is optionally added, and preferably, the concentration of NADPH in the reaction system is 10 to 100mmol/L, preferably 30 to 90mmol/L, and more preferably 50 to 70 mmol/L.
In the preferred technical scheme of the invention, the ee value of the obtained D-pantolactone is more than or equal to 95 percent, preferably more than or equal to 96 percent, more preferably more than or equal to 97 percent, still more preferably more than or equal to 98 percent, and still more preferably more than or equal to 99 percent.
The invention also aims to provide application of the recombinant engineering bacteria in preparing D-pantolactone from DL-pantolactone.
In a preferred embodiment of the present invention, the use of said D-pantolactone for the preparation of a panto-compound, preferably said panto-compound is selected from any one of D-pantolactone, D-pantothenic acid, D-calcium pantothenate, D-panthenol, pantethine.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
1. Conversion rate
Instruments and working conditions: shimadzu LC-16 liquid chromatograph and chromatographic column
IE5 μm, 4.6 × 250mm, column temperature 30 deg.C, collection time 30min, wavelength 210nm, flow rate 1.0ml/min, mobile phase 0.05mol/L sodium dihydrogen phosphate water solution: methanol 60: 40.
The experimental steps are as follows: respectively diluting the reaction solution by 100 times when the conversion time T is 0 and T is M (M is any value more than 0), filtering, injecting sample with the sample amount of 10ul, and respectively recording the peak area S of L-pantolactone0And SM。
Conversion rate(M time)＝(S0-SM)/S0。
2. ee value calculation formula
ee value ═ L-pantolactone content (D-pantolactone content)/(D-pantolactone content + L-pantolactone content)
Compared with the prior art, the invention has the following beneficial technical effects:
1. the invention provides a recombinant engineering bacterium and a construction method thereof, the recombinant engineering bacterium can co-express an L-pantolactone dehydrogenase encoding gene, a ketopantolactone reductase encoding gene, a formate dehydrogenase encoding gene and a D-pantolactone hydrolase encoding gene through induction culture, is used for efficiently preparing D-pantolactone with high optical purity, and has the advantages of simple operation, environmental protection, suitability for industrial production and the like.
2. The invention also provides a method for synthesizing D-pantolactone by efficiently catalyzing DL-pantolactone or L-pantolactone with enzyme generated by the induction of the recombinant engineering bacteria, which obviously improves the selectivity and reaction efficiency of L-pantolactone dehydrogenase and the product conversion rate.
3. The method does not adopt a chemical resolution reagent, avoids the cyclic racemization of the L-pantoic acid lactone, shortens the production period, and has the advantages of simple and convenient operation, environmental protection, better cost, suitability for large-scale industrial production and the like.
Detailed Description
The present invention is further illustrated in detail by the following examples. These examples and experimental examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 construction of a multienzyme Co-expression recombinant engineered bacterium
1. Design and Synthesis of Gene of interest
Step one, carrying out codon optimization on a nucleotide sequence of an L-pantoate lactone dehydrogenase encoding gene derived from Humibacter sp.BT305 (actinomycetes) according to codon preference of escherichia coli (E.coli) to obtain an L-pantoate lactone dehydrogenase modified gene sequence, wherein the nucleotide sequence is shown as SEQ ID No. 1;
and step two, carrying out codon optimization on the nucleotide sequence of the ketopantoate lactone reductase coding gene derived from the candida magnolifolia according to the codon preference of escherichia coli (E.coli) to obtain a D-ketopantoate lactone modified gene sequence, wherein the nucleotide sequence is shown as SEQ ID No. 2.
And step three, carrying out codon optimization on a nucleotide sequence coded by the formate dehydrogenase derived from Burkholderia stabilis according to the codon preference of escherichia coli (E.coli) to obtain a modified gene sequence of the formate dehydrogenase, wherein the nucleotide sequence is shown as SEQ ID No. 3.
And step four, carrying out codon optimization on the nucleotide sequence of the D-pantoate lactonohydrolase encoding gene derived from Fusarium moniliforme CGMCC0536 (Fusarium moniliforme) according to the codon preference of escherichia coli (E.coli) to obtain a modified gene sequence of the D-pantoate lactonohydrolase, wherein the nucleotide sequence is shown as SEQ ID NO. 4.
Step five, synthesizing nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, and respectively adding XhoI and NdeI enzyme cutting sites, SacI and NotI enzyme cutting sites, XhoI and NdeI enzyme cutting sites and XhoI and NdeI enzyme cutting sites to prepare the target gene 1, the target gene 2, the target gene 3 and the target gene 4.
2. Construction of recombinant expression vectors
Step one, taking a DNA molecule of the target genome 1 as a template, carrying out PCR amplification on lpldh-for and lpldh-rev by using primers, carrying out electrophoresis on 1% agarose gel to separate PCR products, and recovering a gene fragment of the target genome 1 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites underlined)
lpldh-for：GGAATTCCATATGATGAACCCGTGGTTTGAAAC
lpldh-rev：CCGCTCGAGTGCGCTTTCTGCTTCTGC
The PCR system was as follows:
the PCR process was as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 57 deg.C for 30s, extension at 72 deg.C for 1min for 30s, circulating for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
And step two, taking the DNA molecule of the target genome 2 as a template, carrying out PCR amplification on KPR-for and KPR-rev by adopting a primer pair, carrying out electrophoresis separation on a PCR product by using 1% agarose gel, and recovering the gene fragment of the target genome 2 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites underlined)
KPR-for：
GGAATTCCATATGATGGCTAAAAACTTCTCTAACGTTGAATACC
KPR-rev：CCGCTCGAGCGGCAGGGTGTAACCACCGTCAAC
The PCR system was as follows:
the PCR process was as follows:
pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min, circulation for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
And step three, double-digesting the pRSFDuet-I plasmid and the gene fragment of the genome 1 by using restriction enzymes XhoI and NdeI, recovering a vector framework and a digested PCR product, connecting the vector framework and the digested PCR product by using T4 DNA ligase, transforming the connected product (named as pRSF-lpldhh) into E.coli BL21(DE3) competent cells, screening positive clones, extracting plasmids, sequencing and identifying, and naming the correct clone as E-pRSF-lpldh.
And step four, carrying out double enzyme digestion on pRSF-lpldh plasmid and the gene fragment of the genome 2 by using restriction enzymes SacI and NotI, recovering a vector skeleton and an enzyme digestion PCR product, connecting the vector skeleton and the enzyme digestion PCR product by using T4 DNA ligase, transforming the connection product (named as pRSFDuet-lpldh-kpr) into E.coli BL21(DE3) competent cells, screening positive clones, extracting plasmids for sequencing identification, and naming the correct clone as E-lpldh-kpr to prepare the single-particle double-enzyme recombinant engineering bacteria for co-expressing L-pantoate dehydrogenase and ketopantoate lactone reductase.
And step five, taking the DNA molecule of the target genome 3 as a template, performing PCR amplification on FDH-for and FDH-rev by adopting a primer pair, separating a PCR product by 1% agarose gel electrophoresis, and recovering the gene fragment of the target genome 3 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites underlined)
FDH-for：CGGAATTCATGGCTACCGTTCTGTGCG
FDH-rev：
ATAAGAATGCGGCCGCGGTCAGACGGTAAGACTGAGC
The PCR system was as follows:
the PCR process was as follows:
pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min, circulation for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
And sixthly, double enzyme digestion is carried out on the pET-21a plasmid and the gene fragment of the genome 3 by using restriction endonuclease XhoI and NdeI, a vector framework and a PCR product are recovered, T4 DNA ligase is used for connecting the plasmid and the PCR product, the ligation product (named as pET-FDH) is transformed into E-lpldh-kpr competent cells, positive clones are screened, the plasmid is extracted for sequencing identification, the correct clone is named as E-lpldh-kpr-FDH, and the double plasmid three-enzyme recombinant engineering bacteria for co-expressing L-pantoate lactone dehydrogenase, ketopantoate lactone reductase and formate dehydrogenase is prepared.
Example 2 construction of recombinant engineering bacteria expressing D-pantoate hydrolase
1. Design and Synthesis of Gene of interest
Carrying out codon optimization on a nucleotide sequence of a D-pantolactone hydrolase coding gene derived from Fusarium moniliforme CGMCC0536 according to the codon preference of escherichia coli (E.coli), and adding XhoI and NdeI enzyme cutting sites to obtain a D-pantolactone hydrolase modification gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 4;
2. construction of recombinant engineering bacteria
Step one, taking a DNA molecule of a target genome 4 as a template, carrying out PCR amplification on HYD-for and HYD-rev by adopting a primer pair, carrying out electrophoresis separation on a PCR product by using 1% agarose gel, and recovering a gene fragment of the target genome 4 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites underlined)
HYD-for：GGAATTCCATATGATGGCTAAGCTTCCTTCTAC
HYD-rev：CCGCTCGAGCTAATCATAGAGCTTGGGACCC
The PCR system was as follows:
the PCR process was as follows:
pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 53 deg.C for 30s, extension at 72 deg.C for 1min for 30s, circulating for 28 times, keeping at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
And step two, double-enzyme digestion is carried out on the PCR products of the pET-28a plasmid and the genome 4 by using restriction enzyme XhoI and NdeI, the carrier skeleton and the PCR products are recovered, T4 DNA ligase is used for connecting the plasmid and the PCR products, the connection products (named as pET-hyd) are transformed into E.coli BL21(DE3) competent cells, positive clones are screened, the plasmid is extracted for sequencing identification, and the correct clone is named as E-hyd.
Example 3 induced expression of recombinant engineering bacterium E-lpldh-kpr-FDH
The recombinant engineering bacterium E-lpldh-kpr-FDH prepared in the example 1 is inoculated in 5mL LB culture medium and is cultured overnight at the temperature of 37 ℃ and the speed of 200 rpm;
inoculating the seeds into 100mL of TB culture medium according to the inoculation amount of 2%, culturing for 2h under the conditions of 37 ℃ and 200rpm, adjusting the culture temperature to 30 ℃, and continuing culturing for 15 h;
6600rpm for 3min, discarding supernatant, washing with 0.2moL/L phosphoric acid buffer (pH6.5) for 2 times, and collecting thallus.
Wherein, the LB culture medium comprises the following components: 50ug/mL of ampicillin, 100mg/L of kanamycin, 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
The composition of TB medium is as follows: tryptone 20g/L, yeast powder 5g/L, sodium chloride 5g/L, glucose 2g/L, lactose 0.1-2.0g/L, ampicillin 50mg/L, kanamycin 100 mg/L.
Example 4 inducible expression of recombinant engineered bacteria E-hyd
Inoculating the recombinant engineering bacterium E-hyd prepared in the embodiment 2 into 5mL LB culture medium, and culturing at 37 ℃ and 200rpm overnight to obtain seed liquid;
inoculating the seed solution into 100mL TB culture medium according to the inoculation amount of 2%, culturing for 2h under the conditions of 37 ℃ and 200rpm, adjusting the culture temperature to 30 ℃, and continuing to culture for 15 h; 6600rpm for 3min, discarding supernatant, washing with 0.2moL/L phosphoric acid buffer (pH6.5) for 2 times, and collecting thallus.
Wherein, the LB culture medium comprises the following components: 100mg/L kanamycin, 5g/L yeast powder, 10g/L tryptone and 10g/L sodium chloride.
The composition of TB medium is as follows: tryptone 20g/L, yeast powder 5g/L, sodium chloride 5g/L, glucose 2g/L, lactose 0.1-2.0g/L, kanamycin 100 mg/L.
Example 5 conversion of DL-pantoic acid lactone
Adding 130g of DL-pantolactone and 46g of formic acid into a reaction vessel, adding water until the total volume is 1L, and stirring and dissolving; the solution pH was adjusted to 6.2 with 20-25% ammonia, the temperature was raised to 37 ℃, the recombinant engineered bacterial cells (OD ═ 2) collected in example 1 were added, and the mixture was left at 37 ℃ and stirred to react for 30 hours, to obtain a reaction solution. The reaction solution is subjected to high performance liquid chromatography detection, the final concentration of D-pantolactone is 125.45g/L, the conversion rate of DL-pantolactone is 96.5%, and the ee value is 93%.
Example 6 conversion of DL-pantoic acid lactone
Adding 130g of DL-pantolactone and 46g of formic acid into a reaction vessel, adding water until the total volume is 1L, and stirring and dissolving; after adjusting the pH of the solution to 6.2 with 20 to 25% ammonia water and raising the temperature to 37 ℃ and adding the recombinant engineered bacterial cells (OD 2) collected in example 1 and 50mM NADPH (reaction system concentration), the mixed solution was left at 37 ℃ and stirred for 30 hours to obtain a reaction solution. The reaction solution is subjected to high performance liquid chromatography detection, the final concentration of D-pantolactone is 127.54g/L, the conversion rate of DL-pantolactone is 98.1%, and the ee value is 96.2%.
Example 7 conversion of DL-pantoic acid lactone
Adding 130g of DL-pantolactone and 46g of formic acid into a reaction vessel, adding water until the total volume is 1L, and stirring and dissolving; the solution pH was adjusted to 6.2 with 20-25% ammonia water, the temperature was raised to 37 ℃, then the recombinant engineered bacterial cells collected in example 1 (OD ═ 2) and the recombinant engineered bacterial cells collected in example 2 (OD ═ 3) were added, and the mixed solution was left at 37 ℃ and stirred to react for 30 hours, thereby obtaining a reaction solution. The reaction solution was subjected to chromatographic detection, and the final concentration of D-pantoic acid was 147.97g/L, and the conversion of DL-pantoic lactone was 99.98%.
And (3) passing the reaction solution through a ceramic membrane, collecting clear solution of the ceramic membrane, passing the clear solution through a nanofiltration membrane, collecting clear solution of the nanofiltration membrane, adjusting the pH to 1.5 by using concentrated sulfuric acid, and standing for 30min to obtain a D-pantolactone solution, wherein the concentration of the D-pantolactone is 129.97g/L, and the ee value is 99.95%.
Example 8 conversion of DL-pantoic acid lactone
Adding 130g of DL-pantolactone and 46g of formic acid into a reaction vessel, adding water until the total volume is 1L, and stirring and dissolving; the pH of the solution was adjusted to 6.2 with 20-25% aqueous ammonia, the temperature was raised to 37 ℃ and the recombinant engineered bacterial cells (OD 2) collected in example 1 and the recombinant engineered bacterial cells (OD 2) collected in example 2 were added thereto, and the mixture was stirred at 37 ℃ for 30 hours to obtain a reaction solution. The reaction solution was subjected to chromatographic detection, and the final concentration of D-pantoic acid was 146.82g/L, and the conversion of DL-pantoic lactone was 99.2%.
And (3) passing the reaction solution through a ceramic membrane, collecting a ceramic membrane clear solution and a nanofiltration membrane, collecting a nanofiltration membrane clear solution, adjusting the pH to be about 1.5, and standing for 30min to obtain a D-pantolactone solution, wherein the concentration of D-pantolactone is 129.41g/L, and the ee value is 99.09%.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.
Sequence listing
<110> Anhui Hua constant Biotech, Inc
Bayannur Huaheng Biotechnology Co.,Ltd.
QINHUANGDAO HUAHENG BIOENGINEERING Co.,Ltd.
HEFEI HUAHENG BIOLOGICAL ENGINEERING Co.,Ltd.
<120> recombinant engineering bacterium, construction method and application thereof
<150> 2020110463081
<151> 2020-09-29
<150> 2021109419627
<151> 2021-08-17
<150> 2021110873038
<151> 2021-09-16
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cgtttcgaaa aagaaggtaa aaaaggtgct ctggttttca ccgcttctat gtctggtcac 540
atcgttaacg ttccgcagtt ccaggctacc tacaacgctg ctaaagctgg tgttcgtcac 600
ttcgctaaat ctctggctgt tgaattcgct ccgttcgctc gtgttaactc tgtttctccg 660
ggttacatca acaccgaaat ctctgacttc gttccgcagg aaacccagaa caaatggtgg 720
tctctggttc cgctgggtcg tggtggtgaa accgctgaac tggttggtgc ttacctgttc 780
ctggcttctg acgctggttc ttacgctacc ggtaccgaca tcatcgttga cggtggttac 840
accctgccg 849
<210> 3
<211> 1152
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggctaccg ttctgtgcgt tctgtacccg gacccggttg acggttaccc gccgcactac 60
gttcgtgaca ccatcccggt tatcacccgt tacgctgacg gtcagaccgc tccgaccccg 120
gctggtccgc cgggtttccg tccgggtgaa ctggttggtt ctgtttctgg tctgggtctg 180
cgtggttacc tggaagctca cggtcacacc ctgatcgtta cctctgacaa agacggtccg 240
gactctgaat tcgaacgtcg tctgccggac gctgacgttg ttatctctca gccgttctgg 300
ccggcttacc tgaccgctga acgtatcgct cgtgctccga aactgcgtct ggctctgacc 360
gctggtatcg gttctgacca cgttgacctg gacgctgctg ctcgtgctca catcaccgtt 420
gctgaagtta ccggttctaa ctctatctct gttgctgaac acgttgttat gaccaccctg 480
gctctggttc gtaactacct gccgtctcac gctatcgctc agcagggtgg tatgaacatc 540
gctgactgcg tttctcgttc ttacgacgtt gaaggtatgc acttcggtac cgttggtgct 600
ggtcgtatcg gtctggctgt tctgcgtcgt ctgccgttcg gtctgcacct gcactacacc 660
cagcgtcacc gtctggacgc tgctatcgaa caggaactgg gtctgaccta ccacgctgac 720
ccggcttctc tggctgctgc tgttgacatc gttaacctgc agatcccgct gtacccgtct 780
accgaacacc tgttcgacgc tgctatgatc gctcgtatga aacgtggtgc ttacctgatc 840
aacaccgctc gtgctaaact ggttgaccgt gacgctgttg ttcgtgctgt tacctctggt 900
cacctggctg gttacggtgg tgacgtttgg ttcccgcagc cggctccggc tgaccacccg 960
tggcgtgcta tgccgttcaa cggtatgacc ccgcacatct ctggtacctc tctgtctgct 1020
caggctcgtt acgctgctgg taccctggaa atcctgcagt gctggttcga cggtcgtccg 1080
atccgtaacg aatacctgat cgttgacggt ggtaccctgg ctggtaccgg tgctcagtct 1140
taccgtctga cc 1152
<210> 4
<211> 1146
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggcaaaac taccctcaac agctcaaata attgatcaga aatccttcaa cgtgctgaag 60
gacgtgccac cgccagcggt ggctaatgat tccttggttt ttacctggcc tggtgttacg 120
gaggaaagct tggttgaaaa accgtttcac gtgtacgatg aagagttcta cgacgtgatc 180
ggcaaggacc cgtccttgac cctgatcgct accagtgata cggacccgat tttccatgaa 240
gccgtcgtgt ggtatccgcc gaccgaggag gtgttctttg ttcagaacgc aggcgctccg 300
gctgcgggca ctggtctgaa caaaagcagc attatccaga aaatttcgct gaaagaagcg 360
gatgaggttc gtaagggcaa gaaagatgag gttaaagttg cagttgttga ttctaatccg 420
caggtcatca acccgaacgg cggtacctat tacaaaggca acattatctt cgcgggcgaa 480
ggtcaaggtg acgatgtgcc gagcgcactg tacctgatga atccgctccc gccgtacaac 540
accactacgc tgctgaataa ctattttggt cgccagttca acagcttgaa cgatgttggt 600
atcaacccgc gtaatggcga cctgtatttc accgacaccc tttatggcta tctgcaggat 660
tttcgtccgg ttccaggtct gcgtaaccaa gtctaccgct acaacttcga tacgggtgcg 720
gtgacggtgg tcgccgacga cttcaccttg ccgaacggta ttggcttcgg tccggatggt 780
aaaaaggtgt atgttactga cacaggcatc gccctgggtt tttacggccg caacctgagc 840
tccccggcgt ctgtgtacag ctttgacgta aatcaagatg gcaccttaca aaacagaaag 900
acctttgcgt atgtcgcgag ctttatcccg gacggggttc acaccgacag caaaggtcgt 960
gtttacgcag gatgcggtga cggcgttcat gtgtggaatc cgtctggcaa gctgatcggt 1020
aagatctata ccggcaccgt tgcggcaaat ttccagttcg ctggtaaggg ccgtatgatt 1080
attaccggtc aaaccaagct attttacgtg accttgggtg cgtctggtcc gaaactgtac 1140
gactaa 1146
Claims (10)
1. A recombinant vector contains a recombinant vector, wherein the nucleotide sequence of the L-pantolactone dehydrogenase modification gene sequence is shown as SEQ ID No.1, the nucleotide sequence of the ketopantolactone reductase modification gene sequence is shown as SEQ ID No.2, the nucleotide sequence of the formate dehydrogenase modification gene sequence is shown as SEQ ID No.3, and the nucleotide sequence of the D-pantolactone hydrolase modification gene sequence is shown as SEQ ID No. 4.
2. The recombinant vector according to claim 1, wherein the recombinant vector optionally comprises either a fifth recombinant vector or a sixth recombinant vector, wherein the fifth recombinant vector comprises the nucleotide sequence of the L-pantoate lactone dehydrogenase-modified gene sequence shown in SEQ ID No.1 and the nucleotide sequence of the ketopantoate lactone reductase-modified gene sequence shown in SEQ ID No.2, the sixth recombinant vector comprises the nucleotide sequence of the L-pantoate lactone dehydrogenase-modified gene sequence shown in SEQ ID No.1, the nucleotide sequence of the ketopantoate lactone reductase-modified gene sequence shown in SEQ ID No.2, the nucleotide sequence of the formate dehydrogenase-modified gene sequence shown in SEQ ID No.3 and the nucleotide sequence of the D-pantoate lactone hydrolase-modified gene sequence shown in SEQ ID No. 4.
3. A recombinant engineered bacterium comprising any one or combination of a first recombinant vector capable of expressing L-pantolactone dehydrogenase, a second recombinant vector expressing ketopantolactone reductase, a third recombinant vector expressing formate dehydrogenase and a fourth recombinant vector expressing D-pantolactone hydrolase;
and/or the recombinant engineering bacteria comprise a fifth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase and ketopantoate lactone reductase;
and/or the recombinant engineering bacteria comprise a sixth recombinant vector capable of co-expressing L-pantoate lactone dehydrogenase, ketopantoate lactone reductase, formate dehydrogenase and D-pantoate lactone hydrolase.
4. The recombinant engineered bacterium of claim 3, wherein the recombinant engineered bacterium comprises a fifth recombinant vector capable of co-expressing L-pantolactone dehydrogenase, ketopantolactone reductase and a third recombinant vector expressing formate dehydrogenase; preferably, the recombinant engineering bacteria also comprise a fourth recombinant vector for expressing the D-pantolactone hydrolase, or are combined with a second recombinant engineering bacteria for expressing the fourth recombinant vector for expressing the D-pantolactone hydrolase.
5. A construction method of recombinant engineering bacteria comprises the following steps:
(1) respectively introducing any one or combination of an L-pantolactone dehydrogenase modified gene sequence, a ketopantolactone reductase modified gene sequence, a formate dehydrogenase modified gene sequence and a D-pantolactone hydrolase modified gene sequence into a vector to obtain a recombinant vector;
(2) and (3) introducing the obtained recombinant vector into a host cell to obtain the recombinant engineering bacterium.
6. An inducible expression method of recombinant engineering bacteria comprises the following steps:
s-1, inoculating the recombinant engineering bacteria into an LB culture medium according to the inoculation amount of 1-5%, and culturing for 6-16h at the temperature of 30-40 ℃ and at the speed of 50-500rpm to obtain a first-stage seed solution;
s-2, inoculating the primary seed solution into a TB culture medium according to the inoculation amount of 1-5%, culturing for 6-20h at the temperature of 25-40 ℃ and at the speed of 50-500rpm, centrifuging, washing with a phosphate buffer solution, and collecting thalli.
7. A preparation method of D-pantolactone comprises the following steps: placing DL-pantoic acid lactone with the concentration of 10-300g/L into a reaction system with formic acid and the OD value of the recombinant engineering bacteria of 1-5, and placing the reaction system at the temperature of 30-40 ℃ and under the condition of pH5-7 for 20-40h to prepare the D-pantoic acid lactone.
8. The preparation method according to claim 7, wherein the recombinant engineered bacteria can express L-pantolactone dehydrogenase, ketopantolactone reductase and formate dehydrogenase; preferably, recombinant engineering bacteria for expressing D-pantolactone hydrolase can be added.
9. The production method according to any one of claims 7 to 8, wherein the ratio of DL-pantoic acid lactone: the molar ratio of formic acid is 1 (0.8-1.2), preferably 1 (0.9-1.1).
10. The use of a recombinant engineered bacterium according to any one of claims 3-4 for the preparation of D-pantolactone.
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| CN2020110463081 | 2020-09-29 | ||
| CN202011046308.1A CN113106129A (en) | 2020-09-29 | 2020-09-29 | Preparation method of D pantolactone with high conversion rate |
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| CN202011046308.1A Withdrawn CN113106129A (en) | 2020-09-29 | 2020-09-29 | Preparation method of D pantolactone with high conversion rate |
| CN202110941962.7A Pending CN114426978A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110940857.1A Pending CN114426977A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942787.3A Pending CN114426979A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942171.6A Pending CN115044594A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111085127.4A Pending CN115011624A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111087987.1A Pending CN115029364A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111084984.2A Pending CN115011623A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111087303.8A Pending CN114774446A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111133610.5A Withdrawn CN114457128A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111135083.1A Withdrawn CN114457129A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111135111.XA Withdrawn CN114457130A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
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| CN202110941962.7A Pending CN114426978A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110940857.1A Pending CN114426977A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942787.3A Pending CN114426979A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and construction method and application thereof |
| CN202110942171.6A Pending CN115044594A (en) | 2020-09-29 | 2021-08-17 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111085127.4A Pending CN115011624A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and application thereof in efficient conversion of L-pantolactone |
| CN202111087987.1A Pending CN115029364A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
| CN202111084984.2A Pending CN115011623A (en) | 2020-09-29 | 2021-09-16 | Recombinant engineering bacterium and construction method and application thereof |
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| CN202111135111.XA Withdrawn CN114457130A (en) | 2020-09-29 | 2021-09-27 | Recombinant engineering bacterium and construction method and application thereof |
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| CN115044638A (en) * | 2022-07-21 | 2022-09-13 | 上海奥萝拉医药科技有限公司 | Preparation method of D-pantolactone with high optical selectivity |
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- 2021-09-27 CN CN202111135111.XA patent/CN114457130A/en not_active Withdrawn
- 2021-09-27 CN CN202111132818.5A patent/CN114457127A/en not_active Withdrawn
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| CN114457129A (en) | 2022-05-10 |
| CN114426977A (en) | 2022-05-03 |
| CN115011624A (en) | 2022-09-06 |
| CN113106129A (en) | 2021-07-13 |
| CN114426978A (en) | 2022-05-03 |
| CN114457130A (en) | 2022-05-10 |
| CN114774446A (en) | 2022-07-22 |
| CN115044594A (en) | 2022-09-13 |
| CN115029364A (en) | 2022-09-09 |
| CN114426979A (en) | 2022-05-03 |
| CN114457127A (en) | 2022-05-10 |
| CN115011623A (en) | 2022-09-06 |
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