CN114657198A - Recombinant engineering bacterium and application thereof in preparing pan-compound - Google Patents

Recombinant engineering bacterium and application thereof in preparing pan-compound Download PDF

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CN114657198A
CN114657198A CN202111156122.6A CN202111156122A CN114657198A CN 114657198 A CN114657198 A CN 114657198A CN 202111156122 A CN202111156122 A CN 202111156122A CN 114657198 A CN114657198 A CN 114657198A
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周芳芳
刘树蓬
刘磊
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Hefei Huaheng Biological Engineering Co ltd
Anhui Huaheng Biotechnology Co Ltd
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Hefei Huaheng Biological Engineering Co ltd
Anhui Huaheng Biotechnology Co Ltd
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Abstract

The invention discloses recombinant engineering bacteria and application thereof in preparing panto-compounds, and relates to recombinant engineering bacteria and application thereof in preparing D-pantoic acid. The invention successfully constructs the gene engineering bacteria for biosynthesis of D-pantoic acid for the first time, and the D-pantoic acid is generated by fermentation and conversion with valine as a substrate, and the method has the advantages of cheap and easily obtained raw materials and low reaction cost.

Description

Recombinant engineering bacterium and application thereof in preparing pan-compound
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium and application thereof in preparing pan-compound.
Background
Pantothenic acid also called vitamin B5Are essential nutrients for mammals including humans and domestic animals, and are used in the biosynthesis of coenzyme a (coa) and Acyl Carrier Protein (ACP) in cells of the body, thereby participating in over a hundred cellular metabolic reactions.
D-pantolactone is an important precursor for the synthesis of D-pantothenic acid. CN110423717A discloses a preparation method of D-pantolactone, which comprises the step of carrying out enzymatic selective hydrolysis separation or enzymatic catalytic synthesis on DL-pantolactone to prepare the D-pantolactone. However, the synthesis of DL-pantoic acid lactone needs to use a large amount of isobutyraldehyde and formaldehyde, the formaldehyde has irritation harm to human body, and the synthesis method adopts an organic reagent for extraction and separation, so that the reaction substrate is expensive, and the limitation of resolving racemic intermediate (such as resolving DL-pantoic acid lactone to obtain D-pantoic acid lactone for polymerization with beta-alanine) is required, and the economic benefit and the environmental benefit are not satisfactory.
CN108456701A discloses a preparation method of D-pantolactone. The method takes valine as a substrate and prepares D-pantolactone through multi-enzyme combined catalysis. But has the defects of complicated reaction steps, long reaction time, high cost caused by the consumption of coenzyme NADPH in the process of converting ketopantoate into pantoate, and the like, and limits the industrialized application prospect of the ketopantoate.
Disclosure of Invention
The object of the present invention is to provide a recombinant plasmid comprising:
a first recombinant plasmid containing an L-amino acid deaminase-encoding gene;
and/or, a second recombinant plasmid comprising a methanol dehydrogenase-encoding gene and an aldolase-encoding gene;
and/or a third recombinant plasmid comprising a formate dehydrogenase-encoding gene and a ketopantoate reductase-encoding gene.
According to the preferable technical scheme, the coding gene of the L-amino acid deaminase is derived from proteus mirabilis, and the coded amino acid sequence is shown as SEQ ID NO. 6.
According to the preferable technical scheme, the L-amino acid deaminase coding gene is subjected to codon optimization to obtain an L-amino acid deaminase optimized gene sequence.
In the preferred technical scheme of the invention, the nucleotide sequence of the L-amino acid deaminase optimized gene sequence is shown as SEQ ID NO. 1.
According to the preferable technical scheme, the L-amino acid deaminase optimized gene sequence is artificially synthesized and added with enzyme cutting sites to obtain the target gene 1.
According to the preferable technical scheme, the plasmid adopted by the first recombinant plasmid is pET-28a plasmid.
According to the preferable technical scheme, the methanol dehydrogenase encoding gene is derived from bacillus, and the encoded amino acid sequence is shown as SEQ ID NO. 7.
According to the preferable technical scheme, the methanol dehydrogenase encoding gene is subjected to codon optimization to obtain a methanol dehydrogenase optimized gene sequence.
In the preferred technical scheme of the invention, the nucleotide sequence of the methanol dehydrogenase optimized gene sequence is shown as SEQ ID NO. 2.
According to the preferable technical scheme, the methanol dehydrogenase optimized gene sequence is artificially synthesized, and a restriction enzyme site is added to obtain the target gene 2.
According to the preferred technical scheme, the aldolase encoding gene is derived from escherichia coli, and the encoded amino acid sequence is shown as SEQ ID NO. 8.
According to the preferable technical scheme, the aldolase encoding gene is subjected to codon optimization to obtain an aldolase optimized gene sequence.
In the preferred technical scheme of the invention, the nucleotide sequence of the aldolase optimized gene sequence is shown as SEQ ID NO. 3.
According to the preferable technical scheme, the aldolase optimized gene sequence is artificially synthesized, and enzyme cutting sites are added to obtain the target gene 3.
In a preferred technical scheme of the invention, the plasmid adopted by the second recombinant plasmid is pRSFDuet-I plasmid.
According to the preferable technical scheme, the formate dehydrogenase encoding gene is derived from Burkholderia, and the encoded amino acid sequence is shown as SEQ ID NO. 9.
According to the preferable technical scheme, the formate dehydrogenase encoding gene is subjected to codon optimization to obtain a formate dehydrogenase optimized gene sequence.
In the preferred technical scheme of the invention, the nucleotide sequence of the formate dehydrogenase optimized gene sequence is shown as SEQ ID NO. 4.
According to the preferable technical scheme, the formate dehydrogenase optimized gene sequence is artificially synthesized, and enzyme cutting sites are added to obtain the target gene 4.
According to the preferable technical scheme, the ketopantoate reductase coding gene is derived from stenotrophomonas maltophilia, and the coded amino acid sequence is shown as SEQ ID NO. 10.
According to the preferable technical scheme, the ketopantoate reductase encoding gene is subjected to codon optimization to obtain a ketopantoate reductase optimized gene sequence.
In the preferred technical scheme of the invention, the nucleotide sequence of the ketopantoate reductase optimized gene sequence is shown as SEQ ID NO. 5.
According to the preferable technical scheme, the ketopantoate reductase optimized gene sequence is artificially synthesized, and enzyme cutting sites are added to obtain the target gene 5.
In a preferred technical scheme of the invention, the plasmid adopted by the third recombinant plasmid is pRSFDUet-I plasmid.
Another object of the present invention is to provide a recombinant engineered bacterium, comprising:
a first recombinant engineered bacterium capable of converting valine to alpha-ketoisovalerate;
and/or, a second recombinant engineered bacterium capable of converting alpha-ketoisovalerate to ketopantoate;
and/or a third recombinant engineered bacterium capable of converting ketopantoate to D-pantoate.
In a preferred embodiment of the present invention, the valine is selected from any one of L-valine, a mixture of L-valine and D-valine, and L-valine.
According to the preferable technical scheme, the first recombinant engineering bacterium comprises an L-amino acid deaminase coding gene or a target gene 1 or a gene sequence shown as SEQ ID NO: 1.
According to a preferable technical scheme of the invention, the second recombinant engineering bacterium comprises a methanol dehydrogenase encoding gene and an aldolase encoding gene, or the second recombinant engineering bacterium comprises a target gene 2 and a target gene 3, or the second recombinant engineering bacterium comprises a nucleotide sequence shown as SEQ ID NO:2 and SEQ ID NO: 3.
According to a preferable technical scheme of the invention, the third recombinant engineering bacterium comprises a formate dehydrogenase encoding gene and a ketopantoate reductase encoding gene, or the third recombinant engineering bacterium comprises a target gene 4 and a target gene 5, or the third recombinant engineering bacterium comprises a nucleotide sequence shown as SEQ ID NO:4 and SEQ ID NO: 5.
According to the preferable technical scheme, the method for obtaining the first recombinant engineering bacterium comprises the following steps: the coding gene or the target gene 1 of the L-amino acid deaminase or the gene shown as SEQ ID NO:1 into host cell to obtain the first recombinant engineering bacterium.
In a preferred embodiment of the present invention, the host cell is selected from any one of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, pantoea ananatis, or a combination thereof.
According to the preferable technical scheme, the method for obtaining the first recombinant engineering bacterium comprises the following steps: the coding gene or the target gene 1 of the L-amino acid deaminase or the gene shown as SEQ ID NO:1 to a vector pET-28a, and introducing the obtained recombinant plasmid into E.coli BL21(DE3) competent cells to obtain a first recombinant engineering bacterium.
According to the preferable technical scheme, the method for obtaining the second recombinant engineering bacteria comprises the following steps: and (2) carrying out gene recombination on a methanol dehydrogenase encoding gene and an aldolase encoding gene, or a target gene 2 and a target gene 3, or a gene sequence shown as SEQ ID NO:2 and SEQ ID NO:3 into host cell to obtain the second recombinant engineering bacterium.
In a preferred embodiment of the present invention, the host cell is selected from any one of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, pantoea ananatis, or a combination thereof.
According to the preferable technical scheme, the method for obtaining the second recombinant engineering bacteria comprises the following steps:
(a) and (2) a methanol dehydrogenase encoding gene or a target gene 2 or a gene shown as SEQ ID NO:2 to the vector pRSFDuet-I to obtain a recombinant plasmid pRSF-mdh;
(b) aldolase encoding gene or gene of interest 3 or a gene as set forth in SEQ ID NO:3 is cloned on the recombinant plasmid pRSF-mdh to obtain the recombinant plasmid pRSF-mdh-ald;
(c) and (3) introducing the vector pRSF-mdh-ald into E.coli BL21(DE3) competent cells to obtain a second recombinant engineering bacterium.
According to the preferable technical scheme of the invention, the method for obtaining the third recombinant engineering bacterium comprises the following steps: the formate dehydrogenase encoding gene and the ketopantoate reductase encoding gene, or the target gene 4 and the target gene 5, or the nucleotide sequence shown as SEQ ID NO:4 and SEQ ID NO:5 into host cell to obtain the third recombinant engineering bacterium.
In a preferred embodiment of the present invention, the host cell is selected from any one of bacillus, yeast, escherichia, pantoea, salmonella, corynebacterium glutamicum, escherichia coli, pantoea ananatis, or a combination thereof.
According to the preferable technical scheme of the invention, the third recombinant engineering bacterium is obtained by the following steps:
(a) and (2) a formate dehydrogenase encoding gene or a target gene 4 or a gene shown as SEQ ID NO:4 is cloned on a vector pRSFDUet-I to obtain a recombinant plasmid pRSF-fdh;
(b) and (2) carrying out gene encoding ketopantoate reductase or target gene 5 or the gene shown as SEQ ID NO:5 to obtain a recombinant plasmid pRSF-fdh-kur;
(c) and (3) introducing the recombinant plasmid pRSF-fdh-kur into E.coli BL21(DE3) competent cells to obtain a third recombinant engineering bacterium. According to the preferable technical scheme, the coding gene of the L-amino acid deaminase is derived from proteus mirabilis, and the coded amino acid sequence is shown as SEQ ID NO. 6.
According to the preferable technical scheme, the methanol dehydrogenase encoding gene is derived from bacillus, and the encoded amino acid sequence is shown as SEQ ID NO. 7.
According to the preferred technical scheme, the aldolase encoding gene is derived from escherichia coli, and the encoded amino acid sequence is shown as SEQ ID NO. 8.
According to the preferable technical scheme, the formate dehydrogenase encoding gene is derived from Burkholderia, and the encoded amino acid sequence is shown as SEQ ID NO. 9.
According to the preferable technical scheme, the ketopantoate reductase coding gene is derived from stenotrophomonas maltophilia, and the coded amino acid sequence is shown as SEQ ID NO. 10.
The invention also aims to provide an application of the recombinant engineering bacteria in the preparation of D-pantoic acid, which specifically comprises the following steps: the first recombinant engineering bacterium is induced and expressed to obtain a first thallus, the second recombinant engineering bacterium is induced and expressed to obtain a second thallus, the third recombinant engineering bacterium is induced and expressed to obtain a third thallus, and the first thallus, the second thallus and the third thallus are used for preparing D-pantoic acid.
In a preferred technical scheme of the invention, the induced expression method comprises the following steps:
(1) inoculating the recombinant engineering bacteria into an LB culture medium according to the inoculation amount of 1-5%, and culturing for 6-12h at 30-40 ℃ and 50-500rpm to obtain a seed solution;
(2) inoculating the seed solution into fermentation culture medium according to 1-10% of inoculation amount, culturing at 30-40 deg.C and pH of 6.0-8.0 until OD value of fermentation broth is 0.5-2, adding isopropyl thiogalactoside to final concentration of 0.5-1mM/L, fermenting at 30 deg.C for 12-24 hr, collecting wet thallus, and storing at-20 deg.C.
According to the preferable technical scheme, the LB culture medium comprises the following components: 50mg/L kanamycin, 10g tryptone, 10g/L sodium chloride and 5g/L yeast powder.
According to the preferable technical scheme, the fermentation medium comprises the following components: magnesium sulfate heptahydrate 2g/L, potassium dihydrogen phosphate 7g/L, citric acid monohydrate 2g/L, ammonium sulfate 3g/L, yeast powder 1g/L, and glucose 6 g/L.
According to the preferable technical scheme, the culture temperature is 30-37 ℃.
The rotation speed is 100-400rpm, preferably 200-300 rpm.
The preferable technical proposal of the invention is that the OD value of the fermentation liquor is 0.6-1 after the culture is carried out at 37 ℃ and pH7.0.
In the preferred technical scheme of the invention, isopropyl thiogalactoside is added to the final concentration of 0.6-0.8 mM/L.
Another object of the present invention is to provide a process for preparing D-pantoic acid, which comprises the steps of: mixing the first thallus, the second thallus and the third thallus, valine and methanol with water, adding ammonium formate, and reacting for 10-60 hours at the temperature of 30-40 ℃ and the pH value of 4-7 to obtain D-pantoic acid.
According to the preferable technical scheme, the first thallus is a thallus containing a sequence shown as SEQ ID NO:1, or a cell thereof.
According to the preferable technical scheme, the first thallus is thallus containing an L-amino acid deaminase coding gene or a target gene 1, wherein an amino acid sequence coded by the L-amino acid deaminase coding gene is shown as SEQ ID NO: and 6.
In a preferred embodiment of the present invention, the OD of the first cell in the reaction system is 1 to 15, preferably 5 to 15, and more preferably 8 to 12.
According to the preferable technical scheme, the second thallus is a thallus containing a polypeptide shown as SEQ ID NO:2 and SEQ ID NO:3 in a microorganism having the nucleotide sequence shown in (3).
According to a preferable technical scheme of the invention, the second thallus contains a methanol dehydrogenase coding gene and an aldolase coding gene, or a target gene 2 and a target gene 3, wherein an amino acid sequence coded by the methanol dehydrogenase coding gene is shown as SEQ ID NO:7, the amino acid sequence coded by the aldolase coding gene is shown as SEQ ID NO: shown in fig. 8.
In a preferred embodiment of the present invention, the OD value of the second cell in the reaction system is 1 to 15, preferably 8 to 15, and more preferably 10 to 12.
According to the preferable technical scheme, the third thallus is a thallus containing a microorganism shown as SEQ ID NO:4 and the nucleotide sequence shown as SEQ ID NO:5 in a microorganism having the nucleotide sequence shown in (a).
According to a preferable technical scheme of the invention, the third thallus contains a formate dehydrogenase coding gene and a ketopantoate reductase coding gene, or a target gene 4 and a target gene 5, wherein an amino acid sequence coded by the formate dehydrogenase coding gene is shown as SEQ ID NO:9, the amino acid sequence coded by the ketopantoate reductase coding gene is shown as SEQ ID NO: shown at 10.
In a preferred embodiment of the present invention, the OD of the third bacterial cell in the reaction system is 1 to 15, preferably 8 to 15, and more preferably 10 to 12.
According to a preferred embodiment of the present invention, the valine: the molar ratio of methanol is (0.9-1.1) to (0.9-1.1), preferably (0.95-1.05) to (0.95-1.05)
According to the preferable technical scheme, the ammonium formate is added in a fed-batch mode.
In the preferred technical scheme of the invention, the concentration of the ammonium formate is 1.5g/L-2.5g/L, preferably 1.8g/L-2.2 g/L.
In the preferred technical scheme of the invention, metal salt or phosphate solution can be added into the reaction system.
In a preferred embodiment of the present invention, the metal salt or phosphate is selected from any one of zinc salt, calcium salt, copper salt, magnesium salt, sodium salt, and potassium salt, or a combination thereof, and is preferably selected from any one of magnesium chloride, zinc chloride, calcium chloride, copper chloride, sodium chloride, potassium chloride, sodium sulfate, sodium bisulfate, potassium sulfate, magnesium sulfate, zinc sulfate, calcium sulfate, copper sulfate, magnesium phosphate, zinc phosphate, calcium phosphate, copper phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, calcium hydrogen phosphate, calcium pyrophosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acid pyrophosphate, sodium phosphate, and sodium pyrophosphate, or a combination thereof. According to the preferable technical scheme of the invention, the concentration of the metal salt or phosphate solution is 0-50mmol/L, preferably 1-40mol/L, and more preferably 5-30 mol/L.
Preferred technical solution of the present invention, Zn in metal salt solution2+The concentration is 0-15mM, preferably 5-10 mM.
Preferred technical solution of the present invention, Cu in metal salt solution2+The concentration is 0-15mM, preferably 5-10 mM.
Preferred technical solution of the present invention, Ca in the metal salt solution2+The concentration is 0-9mM, preferably 2-7 mM.
Preferred technical solution of the present invention, Mg in a metal salt solution2+The concentration is 0-9mM, preferably 2-7 mM.
In the preferred embodiment of the present invention, Na is contained in the metal salt solution+The concentration is 0-9mM, preferably 2-7 mM.
Preferred embodiment of the invention, PO of phosphate solution4 3-The concentration is 0-21mM, preferably 0-15mM, preferably 2-10 mM.
Preferred embodiment of the invention, HPO in phosphate solution4 2-The concentration is 0 to 15mM, preferably 2 to 10 mM.
Preferred embodiment of the invention, H in phosphate solution2PO4 -The concentration is 0 to 15mM, preferably 2 to 10 mM.
According to the preferable technical scheme, the reaction temperature is 35-38 ℃.
According to the preferable technical scheme, the reaction time is 20-40 h.
According to the preferable technical scheme of the invention, the pH value of the reaction system is 5-7.
In a preferred embodiment of the present invention, the pH adjusting agent for adjusting the pH of the reaction system 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.
It is another object of the present invention to provide a use of D-pantoic acid produced by the process as described above for the preparation of panto-compounds selected from any one of D-pantolactone, calcium D-pantothenate, D-panthenol, pantethine.
The invention also aims to provide a preparation method of D-pantoic acid lactone, which is characterized in that D-pantoic acid is subjected to lactonization reaction to obtain the D-pantoic acid lactone.
According to the preferable technical scheme, D-pantoic acid reacts under an acidic condition at the temperature of 30-80 ℃ to obtain D-pantoic acid lactone.
In a preferred embodiment of the present invention, the acidic condition is pH3.0 or less, preferably pH2.0 or less.
According to the preferable technical scheme of the invention, the reaction temperature is 40-70 ℃, and preferably 50-60 ℃.
In the preferred technical scheme of the invention, the D-pantoic acid can adopt clear liquid obtained by filtering fermentation liquor.
According to the preferable technical scheme of the invention, the D-pantoic acid can be clear liquid obtained by filtering the recombinant engineering bacterium fermentation liquid. According to the preferable technical scheme, the lactonization reaction comprises the steps of taking a reaction solution of D-pantoic acid, passing through a ceramic membrane, collecting a clear solution, passing through a nanofiltration membrane, collecting a clear solution, adjusting the pH value of the clear solution to be below 2.0 by using sulfuric acid, and stirring the clear solution at the temperature of 40-60 ℃ for reaction; adding 0.1mol/LNaOH solution, adjusting the pH value of the solution to 4-7, and obtaining the D-pantoic acid lactone.
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.
Unless otherwise stated, the present invention measures conversion and yield as follows.
1. Enzyme activity
The enzyme activity is a unit for measuring the enzyme activity, and the unit is U. In the present application, 1 enzyme activity unit is defined to mean the amount of enzyme converted to 1 umOLD-pantoic acid in1 minute per 1ml of substrate solution.
2. Conversion rate
Instruments and working conditions: and (3) chromatographic column: InertsilNH25um 4.6*250mm；
Mobile phase: acetonitrile: 0.04moL/L potassium dihydrogen phosphate in water (pH 3.0 adjusted with phosphoric acid) 75: 25; column temperature: 30 ℃; wavelength: 205 nm; flow rate: 1.0 mL/min; sample introduction amount: 5 uL.
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, introducing sample amount of 10ul, and recording valine peak area S0And SM。
Conversion (time M) 0.79 Nm/(S)0-SM)。
Compared with the prior art, the invention has the beneficial effects that:
1. successfully constructs recombinant engineering bacteria for biosynthesis of D-pantoic acid for the first time, and generates the D-pantoic acid by fermentation and conversion with valine as a substrate, and the method has the advantages of cheap and easily obtained raw materials and low reaction cost.
2. The invention takes the methanol as the substrate, effectively avoids directly adding the formaldehyde, simultaneously does not cause extra formaldehyde to overflow out of the system, is favorable for saving the cost and is good for the environment.
3. The method converts the formic acid into volatile carbon dioxide, is beneficial to improving the reaction efficiency, avoids complicated steps caused by separating and removing the formic acid, shortens the production period, reduces the generation of three wastes and the recovery and treatment cost thereof, and is suitable for industrialized generation.
Drawings
FIG. 1 is a schematic diagram of a technical principle of an embodiment of the present invention;
FIG. 2 comparison of D-pantoic acid conversion of test examples 1-6 with comparative example 1;
FIG. 3 the yield of D-pantolactone of test examples 1 to 6 is compared with that of comparative example 1.
Detailed Description
The present invention will be further described with reference to the following examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1Construction of first recombinant engineering bacterium for expressing L-amino acid deaminase
Step one, carrying out codon optimization on a nucleotide sequence of an L-amino acid deaminase coding gene (the coded amino acid sequence of the coding gene is shown as SEQ ID NO: 6) derived from Proteus mirabilis according to the codon preference of escherichia coli (E.coli) to obtain an L-amino acid deaminase optimized gene sequence, wherein the nucleotide sequence of the L-amino acid deaminase optimized gene sequence is shown as SEQ ID NO: 1;
the nucleotide sequence shown in SEQ ID NO. 1 is artificially synthesized, and XhoI and NdeI restriction sites are added to obtain the target gene 1.
And step two, taking the DNA molecule of the target gene 1 as a template, performing PCR amplification on LA-for and LA-rev by using primers, separating PCR products by electrophoresis of 1% agarose gel, and recovering the gene fragment of the target gene 1 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites are underlined)
LA-for：GGAATTCCATATGATGGCTATAAGTAGGAGAAAATTTATTC；
LA-rev：CCGCTCGAGAAAGCGGTACAAGCTGAACGG。
The PCR system was as follows:
Figure BDA0003288668740000141
Figure BDA0003288668740000151
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 for 20s, 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 three, double enzyme digestion of pET-28a plasmid and the gene fragment of the target gene 1 by using restriction enzyme XhoI and NdeI, recovery of a vector framework and an enzyme digestion product, connection of the vector framework and the enzyme digestion product by using T4 DNA ligase, transformation of the connection product (named as pET-28a-LA) into E.coli BL21(DE3) competent cells, screening of positive clones, extraction of plasmids, sequencing and identification, and naming of correct clones as E-28 a-LA.
Example 2Construction of second recombinant engineering bacterium for co-expression of methanol dehydrogenase and aldolase
Step one, carrying out codon optimization on a nucleotide sequence of a methanol dehydrogenase encoding gene sequence (the encoded amino acid sequence is shown as SEQ ID NO: 7) from Bacillus (Bacillus methanolicus) according to the codon preference of escherichia coli (E.coli) to obtain a methanol dehydrogenase optimized gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO: 2;
carrying out codon optimization on a nucleotide sequence of an aldolase encoding gene sequence (the encoded amino acid sequence is shown as SEQ ID NO: 8) derived from Escherichia coli (Escherichia coli) according to the codon preference of the Escherichia coli (E.coli) to obtain an aldolase optimized gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO: 3;
the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3 are artificially synthesized, and BamHI and NotI enzyme cutting sites and XhoI and NdeI enzyme cutting sites are respectively added to obtain a target gene 2 and a target gene 3.
And step two, using the DNA molecule of the target gene 2 as a template, adopting primers for mdh-for and mdh-rev to carry out PCR amplification, carrying out electrophoresis separation on a PCR product by 1% agarose gel, and then recovering the gene fragment of the target gene 2 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites are underlined)
mdh-for：CGGGATCCATGAAAAACACCCAGTCTGCTTTC；
mdh-rev：
ATAAGAATGCGGCCGCCATAGCGTTTTTGATGATCTGGATAAC。
The PCR system was as follows:
Figure BDA0003288668740000161
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 1min30s, 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.
Using DNA molecule of target gene 3 as template, adopting primer pair ald-for and ald-rev to make PCR amplification, 1% agarose gel electrophoresis separating PCR product, then using gel recovery kit to recover gene fragment of target gene 3.
The primer sequences are as follows: (restriction sites underlined)
ald-for：
GGAATTCCATATGATGAAAAACTGGAAAACAAGTGCAGAATCAATC；
ald-rev：CCGCTCGAGCAGCTTAGCGCCTTCTACAGCTTCAC。
The PCR system was as follows:
Figure BDA0003288668740000171
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 50s, circulating for 28 times, maintaining at 72 deg.C for 10min, cooling to 4 deg.C, and storing in refrigerator at 4 deg.C for use.
Step three, carrying out double restriction enzyme digestion on pRSFDuet-I plasmid and a gene fragment of a target gene 2 by using restriction enzymes, recovering a vector framework and a restriction enzyme digestion product, connecting the vector framework and the restriction enzyme digestion product by using T4 DNA ligase, transforming the connection product pRSF-mdh into E.coli BL21(DE3) competent cells, screening positive clones, extracting the plasmid, carrying out sequencing identification, and naming the correct clone as E-pRSF-mdh;
the recombinant plasmid pRSF-mdh and the gene fragment of the target gene 3 are subjected to double enzyme digestion by restriction enzymes, a vector framework and an enzyme digestion product are recovered, the recombinant plasmid pRSF-mdh and the gene fragment are connected by T4 DNA ligase, the connection product pRSF-mdh-ald is transformed into E.coli BL21(DE3) competent cells, positive clones are screened, plasmids are extracted, sequencing identification is carried out, and the correct clone is named as E-pRSF-mdh-ald.
Example 4Construction of a third recombinant engineered bacterium co-expressing formate dehydrogenase and ketopantoate reductase
Firstly, carrying out codon optimization on a nucleotide sequence of a formate dehydrogenase encoding gene sequence (an encoded amino acid sequence of which is shown as SEQ ID NO: 9) derived from Burkholderia (burkholderia stabilis) according to the codon preference of escherichia coli (E.coli), and adding BamHI and NotI enzyme cutting sites to obtain a formate dehydrogenase optimizing gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO: 4;
carrying out codon optimization on a nucleotide sequence of a ketopantoate reductase coding gene sequence (the coded amino acid sequence is shown as SEQ ID NO: 10) derived from Stenotrophomonas maltophilia according to the codon preference of escherichia coli (E.coli), and adding XhoI and NdeI enzyme cutting sites to obtain the ketopantoate reductase optimized gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO: 5;
the nucleotide sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5 are artificially synthesized to obtain the target gene 4 and the target gene 5.
And step two, using the DNA molecule of the target gene 4 as a template, adopting primers for performing PCR amplification on fdh-for and fdh-rev, carrying out electrophoresis separation on a 1% agarose gel to obtain a PCR product, and recovering the gene fragment of the target gene 4 by using a gel recovery kit.
The primer sequences are as follows: (restriction sites underlined)
fdh-for：CGGGATCCATGGCTACCGTTCTGTGCGTTC；
fdh-rev：ATAAGAATGCGGCCGCGGTCAGACGGTAAGACTG。
The PCR system was as follows:
Figure BDA0003288668740000191
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 10s, 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.
Using DNA molecule of target gene 5 as template, adopting primer pair kur-for and kur-rev to make PCR amplification, 1% agarose gel electrophoresis separating PCR product, then using gel recovery kit to recover gene fragment of target gene 5.
The primer sequences are as follows:
kur-for：GGAATTCCATATGATGACCCAGCAACGGTGGCGCC
kur-rev：CCGCTCGAGTCAGAACCCGTAGCGCAGG
the PCR system was as follows:
Figure BDA0003288668740000192
Figure BDA0003288668740000201
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 50s, circulating for 28 times, maintaining at 72 deg.C for 10min, cooling to 4 deg.C, and storing in 4 deg.C refrigerator for use.
And step three, carrying out double digestion on pRSFDUet-I plasmid and the gene fragment of the target gene 4 by using restriction enzymes, recovering a vector framework and a digestion product, connecting the vector framework and the digestion product by using T4 DNA ligase, transforming the ligation product pRSF-fdh into E.coli BL21(DE3) competent cells, screening positive clones, extracting the plasmid, carrying out sequencing identification, and naming the correct clone as E-pRSF-fdh.
The recombinant plasmid pRSF-fdh and the gene fragment of the target gene 5 are subjected to double enzyme digestion by using restriction enzymes, a vector framework and an enzyme digestion product are recovered, the recombinant plasmid pRSF-fdh and the gene fragment are connected by using T4 DNA ligase, the connection product pRSF-fdh-kur is transformed into E.coli BL21(DE3) competent cells, positive clones are screened, plasmids are extracted, sequencing identification is carried out, and the correct clone is named as E-pRSF-fdh-kur.
Example 4Induced expression of first recombinant engineering bacterium E-28a-LA
Inoculating the first recombinant engineering bacterium E-28a-LA prepared in the example 1 into 5ml of LB culture medium according to the inoculation amount of 2%, and performing constant temperature shaking culture at 37 ℃ and 200rpm for 8h to obtain a seed solution; the composition of LB medium was as follows: 50mg/L kanamycin, 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast powder.
Inoculating the seed solution into 10L fermentation tank containing 6L fermentation medium at 2%, culturing at 37 deg.C and pH of 7.0 to OD of fermentation liquid600Adding isopropyl thiogalactoside (IPTG) to a final concentration of 0.5mM/L at a value of 0.6, and culturing at 30 deg.C for 18 h; collecting thallus I, and storing at-20 deg.C. The fermentation medium comprises 2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 2g/L citric acid monohydrate, 3g/L ammonium sulfate, 1g/L yeast powder and 6g/L glucose.
Example 5Induced expression of second recombinant engineering bacterium E-pRSF-mdh-ald
Inoculating the second recombinant engineering bacterium E-pRSF-mdh-ald in example 2 into 5ml of LB culture medium according to the inoculation amount of 2%, and carrying out constant temperature shaking culture at 37 ℃ and 200rpm for 8h to obtain a seed solution; the composition of LB medium was as follows: 50mg/L kanamycin, 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast powder.
Inoculating the seed solution into a 10L fermentation tank containing 6L fermentation medium according to the inoculation amount of 2%, culturing at 37 deg.C and pH7.0 until the OD value of the fermentation liquid is 0.6, adding IPTG until the final concentration is 0.5mM/L, and culturing at 30 deg.C for 18 h; collecting thallus II, and storing at-20 deg.C. The culture medium in the fermentation tank comprises 2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 2g/L citric acid monohydrate, 3g/L ammonium sulfate, 1g/L yeast powder and 6g/L glucose.
Example 6Induced expression of the third recombinant engineering bacterium E-pRSF-fdh-kur
Inoculating the third recombinant engineering bacterium E-pRSF-fdh-kur in example 3 into 5ml of LB culture medium according to the inoculation amount of 2%, and carrying out constant temperature shaking culture at 37 ℃ and 200rpm for 8h to obtain a seed solution; the composition of the LB medium was as follows: 50mg/L kanamycin, 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast powder.
Inoculating the seed solution into a 10L fermentation tank containing 6L fermentation medium according to the inoculation amount of 2%, culturing at 37 deg.C and pH7.0 until the OD value of the fermentation liquid is 0.6, adding IPTG until the final concentration is 0.5mM/L, and culturing at 30 deg.C for 18 h; collecting thallus III, and storing at-20 deg.C. The fermentation medium comprises 2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 2g/L citric acid monohydrate, 3g/L ammonium sulfate, 1g/L yeast powder and 6g/L glucose.
The bacterial cell one obtained in example 4, the bacterial cell two obtained in example 5, and the bacterial cell three obtained in example 6 were used in the production of D-pantoic acid and D-pantolactone in test examples 1 to 6.
Test example 1Preparation of D-pantoic acid and D-pantoic acid lactone
249.5g of valine, 68.25g of methanol, a first cell, a second cell and a third cell were added to a reaction vessel, water was added to the reaction vessel until the total volume became 5L, the mixture was dissolved by stirring, the OD value of the first cell in the reaction system was 12, the OD value of the second cell was 10, the OD value of the third cell was 8, and ammonium formate was fed to the reaction vessel to maintain the ammonium formate concentration in the reaction solution at 2 g/L. Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, wherein the concentration of the D-pantoic acid is 55.3g/L through detection, and the conversion rate is shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring and reacting for 0.5 hour at 50 ℃; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, wherein the yield is shown in figure 3.
Test example 2Preparation of D-pantoic acid and D-pantoic acid lactone
And (3) crushing the first thallus, the second thallus and the third thallus to respectively obtain L-amino acid deaminase, aldolase, methanol dehydrogenase, formate dehydrogenase and ketopantoate reductase.
249.5g of valine and 68.25g of methanol are added into a reaction vessel, water is added until the total volume is 5L, and the L-amino acid deaminase, methanol dehydrogenase, aldolase, formate dehydrogenase and ketopantoate reductase are dissolved by stirring, wherein the L-amino acid deaminase, the methanol dehydrogenase and the aldolase are 10U/L, and the formate dehydrogenase and the ketopantoate reductase are 12U/L. Ammonium formate is fed in a flow manner, and the concentration of the ammonium formate in the reaction liquid is maintained to be 2 g/L.
Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, wherein the concentration of the D-pantoic acid is 56.7g/L through detection, and the conversion rate is shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring the solution at the temperature of 50 ℃ for reaction for 0.5 hour; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, and the yield is shown in figure 3.
Test example 3Preparation of D-pantoic acid and D-pantoic acid lactone
249.5g of valine, 68.25g of methanol, a first cell, a second cell and a third cell were added to a reaction vessel, water was added to the reaction vessel until the total volume became 5L, the mixture was dissolved by stirring, the OD value of the first cell, the OD value of the second cell and the OD value of the third cell were 12 and 8, and the concentration of ammonium formate in the reaction solution was maintained at 2 g/L. Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, wherein the concentration of the D-pantoic acid is 55.0g/L through detection, and the conversion rate is shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring the solution at the temperature of 50 ℃ for reaction for 0.5 hour; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, wherein the yield is shown in figure 3.
Test example 4Preparation of D-pantoic acid and D-pantoic acid lactone
Adding 249.5g of valine, 68.25g of methanol, the first thalli, the second thalli and the third thalli into a reaction vessel, adding water until the total volume is 5L, stirring and dissolving, wherein the OD value of the first thalli in the reaction system is 12, the OD value of the second thalli is 10, the OD value of the third thalli is 10, and adding ammonium formate in a flowing manner to maintain the concentration of the ammonium formate in the reaction liquid to be 2 g/L. Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, wherein the concentration of the D-pantoic acid is 56.9g/L through detection, and the conversion rate is shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring the solution at the temperature of 50 ℃ for reaction for 0.5 hour; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, wherein the yield is shown in figure 3.
Test example 5Preparation of D-pantoic acid and D-pantoic acid lactone
249.5g of valine, 68.25g of methanol, 3.4g of zinc chloride, the first thallus, the second thallus and the third thallus are added into a reaction vessel, water is added until the total volume is 5L, stirring and dissolving are carried out, the OD value of the first thallus in the reaction system is 12, the OD value of the second thallus is 10, the OD value of the third thallus is 10, ammonium formate is fed back, and the concentration of the ammonium formate in the reaction liquid is maintained to be 2 g/L. Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, wherein the concentration of the D-pantoic acid is 59.56g/L through detection, and the conversion rate is shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring the solution at the temperature of 50 ℃ for reaction for 0.5 hour; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, wherein the yield is shown in figure 3.
Test example 6Preparation of D-pantoic acid and D-pantoic lactone
249.5g of valine, 68.25g of methanol, 6g of sodium dihydrogen phosphate, the first thallus, the second thallus and the third thallus are added into a reaction vessel, water is added until the total volume is 5L, the mixture is stirred and dissolved, the OD value of the first thallus in the reaction system is 12, the OD value of the second thallus is 10, the OD value of the third thallus is 10, ammonium formate is fed, and the concentration of the ammonium formate in the reaction liquid is maintained to be 2 g/L. Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, wherein the concentration of the D-pantoic acid is 60.44g/L through detection, and the conversion rate is shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring and reacting for 0.5 hour at 50 ℃; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, and the yield is shown in figure 3.
Comparative example 1Preparation of D-pantoic acid and D-pantoic acid lactone
249.5g of valine, 68.25g of methanol, L-amino acid deaminase, methanol dehydrogenase, aldolase, ketopantoate reductase and formate dehydrogenase are added into a reaction vessel, water is added until the total volume is 5L, the mixture is stirred and dissolved, 10U/L of L-amino acid deaminase, 10U/L of methanol dehydrogenase, 12U/L of aldolase, 12U/L of ketopantoate reductase and 12U/L of formate dehydrogenase are fed in the reaction system, and ammonium formate is maintained at the concentration of 2g/L in the reaction liquid. Adjusting the temperature of the solution to 37 ℃, adjusting the pH value of the solution to 5-7 by using dilute sulfuric acid and ammonia water in the reaction process, continuously reacting for 20 hours under the stirring condition to obtain a D-pantoic acid solution, and detecting the conversion rate shown in figure 2.
Taking the D-pantoic acid reaction solution, and passing through a ceramic membrane; the obtained clear phase solution passes through a nanofiltration membrane; adjusting the pH value of the solution to be below 2.0 by using sulfuric acid, and stirring the solution at the temperature of 50 ℃ for reaction for 0.5 hour; adding 0.1mol/LNaOH, adjusting the pH value of the solution to 6.0, concentrating and crystallizing to obtain D-pantolactone, wherein the yield is shown in figure 3.
Sequence listing
<110> Anhui Hua constant Biotech, Inc
HEFEI HUAHENG BIOLOGICAL ENGINEERING Co.,Ltd.
<120> recombinant engineering bacteria and application thereof in preparation of pan-compound
<150> 202011524368X
<151> 2020-12-22
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1413
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggctatat caagaaggaa atttattcta ggtggcacgg tggtggccgt tgctgcgggc 60
gcgggcgttt tgaccccgat gctgacccgt gaaggtcgtt tcgtgccggg tactccgcgc 120
cacggctttg tggagggtac tggtggtcca ctgccaaaac aagatgatgt tgtggtcatt 180
ggcgcgggca tcctcgggat catgactgcg attaacctgg cggaacgtgg cctgagcgtt 240
acgattgttg aaaaaggtaa tattgcaggc gaacaatcca gccgcttcta tggtcaggcg 300
atcagctaca aaatgccgga cgaaacgttt ctgctgcatc acctgggtaa gcaccgttgg 360
cgtgagatga acgcgaaagt tggcatcgac accacgtacc gcacccaggg acgtgttgag 420
gttccgcttg acgaggagga tctggagaat gttcgtaaat ggattgacgc caaatccaaa 480
gatgtgggtt ctgacatccc gttccgcact aaaatgattg aaggtgctga gctgaagcaa 540
cgtctgagag gcgccaccac cgattggaaa attgcaggct tcgaagagga cagcggttcg 600
ttcgatccgg aggtggctac gttcgtgatg gcagaatacg ccaaaaagat gggcatcaag 660
atctttacca actgcgcagc gcgtggcctg gaaacccaag cgggggtgat cagcgacgtg 720
gtgaccgaaa agggtccgat taaaaccagc cgtgttgttg tcgcgggcgg tgtcggttct 780
cgcctgttta tgcagaattt gaatgtcgat gttccgaccc taccggcgta tcagtcgcaa 840
caactgatca gcgccgctcc gaatgcgcct ggtggcaacg tggcgttgcc gggcggtatc 900
ttttttcgtg atcaggcgga cggcacctat gcaacgagcc cgcgcgttat cgtcgctccg 960
gttgtaaagg agtctttcac ctacggctat aaatacctgc cgctcctggc attgccggac 1020
tttccggtcc acatttcctt gaatgaacag ctgatcaaca gcttcatgca gtccacccat 1080
tgggatttga acgaagaaag tccgttcgag aagtaccgtg atatgaccgc cctgccagat 1140
ctgccggaac tgaacgcgag cctggagaag ttgaagaagg agttcccggc atttaaagag 1200
tcaacgttaa ttgaccagtg gagcggtgct atggcgattg cgccagacga gaacccgatc 1260
atctccgacg ttaaggagta cccgggtctg gtgatcaaca ccgcgaccgg ttggggtatg 1320
accgaatctc cggtgagcgc agaaattacc gcggatttgc tgcttggtaa gaagccggta 1380
ctcgacgcaa agccgttttc gctgtatcgc ttc 1413
<210> 2
<211> 1155
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<213> Artificial Sequence (Artificial Sequence)
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atgaaaaata cacaatcagc tttttatatg ccatccgtga atttgtttgg tgctggctcc 60
gtcaacgaag ttggtactcg cctggcaggt ttgggtgtga aaaaagcgtt gctggtaacc 120
gacgccggcc tgcacagcct agggctctcg gagaagatcg cgggcattat tcgtgaagcg 180
ggcgtcgagg ttgccatctt tccgaaggct gagccgaatc cgaccgacaa gaacgttgcg 240
gaaggtcttg aagcctacaa cgcggagaac tgcgactcca tcgttacctt gggaggcggc 300
tctagccacg atgctggcaa ggcgattgca ttggttgcgg cgaatggtgg caccatccac 360
gactacgagg gtgtcgacgt gagcaagaag ccgatggtgc cgctgattgc aattaacacc 420
accgcgggca ctggttcaga actgacgaaa ttcaccatta tcaccgacac cgaacgcaaa 480
gttaaaatgg ctattgtgga taaacatgtg accccgacgc tgtctatcaa cgacccggag 540
ctgatggttg gtatgccgcc aagcctgacg gcagcgacgg gcctggatgc gctgacgcat 600
gcgattgaag cgtacgtgtc caccggcgcg accccgatta ccgacgcctt ggcaatccag 660
gcaatcaaga tcatcagcaa gtacctgccg cgcgcagttg cgaacggtaa agacatcgag 720
gcgagagaac agatggcgtt cgcccaaagc ttggcgggca tggcgtttaa caacgcaggt 780
ctgggctatg ttcacgcaat cgcgcatcag ctgggtggct tttataattt cccgcatggt 840
gtttgtaatg ctattctgct gccgcacgtg tgccgtttta acctgatcag caaggtagaa 900
cgttatgccg agatcgcggc tttcctgggt gaaaacgtgg atggtcttag cacctatgaa 960
gcggctgaga aagcgattaa ggccatcgag cgtatggcgc gtgatttaaa tattccgaag 1020
ggtttcaaag agctgggtgc gaaagaggaa gatatcgaga cattggctaa aaacgctatg 1080
aatgatgcct gcgcactgac caatcctcgt aaaccgaagc tggaagaggt gattcaaatt 1140
atcaagaacg ccatg 1155
<210> 3
<211> 639
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaaaaatt ggaagacatc agctgaaagt atcctgacga ccggtccggt cgttccggtt 60
attgtggtga agaagttgga gcacgctgtg ccgatggcca aggctctggt ggccggtggt 120
gttcgtgttc tggaggtgac gctgcgtacc gagtgcgcag tcgatgccat ccgcgcaatt 180
gctaaggagg tgccggaagc gatcgttggt gctggcaccg ttctcaaccc gcagcaactg 240
gcagaagtaa ctgaggcggg cgcgcagttt gcaatctctc cgggtttgac cgagccgttg 300
ctcaaggccg caaccgaggg caccattccg ctgattccgg ggatctcgac cgtgagcgaa 360
ctgatgctgg gtatggacta cggcctgaaa gaatttaaat tcttcccggc ggaagcgaat 420
ggtggcgtga aagcgctgca agcgatcgcg ggccctttta gccaggttcg cttctgcccg 480
acgggcggca tctccccggc gaactataga gactacctgg cgttaaaaag cgtcctgtgt 540
attggtggta gctggctggt tccagcggat gcgttggagg ctggcgacta tgatcgtatt 600
accaaacttg cgcgtgaagc ggtggaaggt gccaagttg 639
<210> 4
<211> 1152
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggctacag tactatgtgt tttatatccc gacccggttg acggctaccc gcctcactac 60
gtgcgcgaca ccataccggt catcacccgt tacgcggacg gccagaccgc gccgacccca 120
gccggcccac cgggttttcg tccgggtgag ttggtgggca gcgtttccgg gcttggtctg 180
cgtggctatt tggaggccca cggtcacacc ctgatcgtta ccagcgacaa ggacggtccg 240
gacagcgaat ttgaacgtcg tctgccggat gctgatgtgg tgatttccca gcctttctgg 300
cctgcctact tgactgccga gcgcattgca cgcgcgccaa agctgcgctt ggcgctgacc 360
gcaggtattg gtagcgatca tgttgatctg gacgctgccg cgcgcgcgca catcaccgtg 420
gcggaggtga cgggtagcaa tagcattagt gttgctgagc acgttgttat gaccactctg 480
gcgttggttc gcaactattt accgtcccat gccatcgccc aacagggtgg catgaatatt 540
gcggattgcg tttctagatc ctacgacgtg gaaggtatgc attttggtac ggtcggggca 600
ggccgtatcg gccttgcggt actgcgtcgt ttaccgttcg gtctgcactt gcactatacc 660
caacgtcatc gtcttgatgc ggcgattgaa caagagctgg gtctgacgta tcatgcagat 720
ccggctagcc tggctgcggc ggtagacatc gtgaacctgc agattccgct gtatccgtcg 780
accgaacacc tattcgacgc ggcaatgatt gcccgtatga aacgtggtgc gtacctgatc 840
aacaccgctc gcgcgaaact ggtggatcgt gatgcggtcg tgagagcggt cacgtcaggt 900
catctcgctg gttacggcgg tgatgtttgg ttcccgcagc cggctccggc ggaccacccg 960
tggcgtgcga tgccgttcaa cggtatgacc ccgcatatct ctggcacttc tctgagcgca 1020
caggcacgct acgcagctgg caccctggag atcctgcaat gttggtttga tggccgtccg 1080
attcgtaatg aatatctgat cgtggacggc ggaacattgg cgggcacggg tgcacaaagc 1140
tatcgtttga cc 1152
<210> 5
<211> 777
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgacacagc aaagatggag gctagatgga caaaccgcgc tgatcaccgg tgcctccgct 60
ggcatcggcc tggcgattgc ccacgaatta gcaggtttcg gtgctgatct gatgatcgtt 120
ggtcgtgaca tcgatatgct ggaaaccgct agagacgagc tcttggacgt gtacccgcag 180
atgcaggttc atgcgctggc tgcggatgtt tccgacgacg aggatcgtcg ccaaattctg 240
gactgggtcg aggaccactc cgacggcctg cacatcttgg tcaacaacgc tggcggtaat 300
gttaccaaag cagccaccga atatagcgaa gatgagtggc gtaaaatctt tgagacaaat 360
ttgtttagcg cgtttgagtt gtctcgttac gcgcacccgc tgctggcgcg ccacgcgagc 420
agcagcattg tgaacgtagg tagcgtttct ggtttgaccc atgttcgttc tggcgttgtt 480
tatggcatga gcaaggcggc tatgcatcag atgacccgta atctggcggt ggagtgggca 540
gaagacggta ttcgtgttaa cgcagtggca ccgtggtata tccgcacgcg tcgcacgagc 600
ggcccactga gcgatccgga ttactacgag gaagtgatca accgcacccc gatgcgtcgt 660
attggtgaac cggaagaggt cgcggcggcg gtgggcttcc tttgcctgcc ggcagcgagc 720
tatgtgactg gtgaatgtat tgcggtggat ggtggtttcc tgcgttacgg cttctaa 777
<210> 6
<211> 471
<212> PRT
<213> Proteus mirabilis
<400> 6
Met Ala Ile Ser Arg Arg Lys Phe Ile Leu Gly Gly Thr Val Val Ala
1 5 10 15
Val Ala Ala Gly Ala Gly Val Leu Thr Pro Met Leu Thr Arg Glu Gly
20 25 30
Arg Phe Val Pro Gly Thr Pro Arg His Gly Phe Val Glu Gly Thr Gly
35 40 45
Gly Pro Leu Pro Lys Gln Asp Asp Val Val Val Ile Gly Ala Gly Ile
50 55 60
Leu Gly Ile Met Thr Ala Ile Asn Leu Ala Glu Arg Gly Leu Ser Val
65 70 75 80
Thr Ile Val Glu Lys Gly Asn Ile Ala Gly Glu Gln Ser Ser Arg Phe
85 90 95
Tyr Gly Gln Ala Ile Ser Tyr Lys Met Pro Asp Glu Thr Phe Leu Leu
100 105 110
His His Leu Gly Lys His Arg Trp Arg Glu Met Asn Ala Lys Val Gly
115 120 125
Ile Asp Thr Thr Tyr Arg Thr Gln Gly Arg Val Glu Val Pro Leu Asp
130 135 140
Glu Glu Asp Leu Glu Asn Val Arg Lys Trp Ile Asp Ala Lys Ser Lys
145 150 155 160
Asp Val Gly Ser Asp Ile Pro Phe Arg Thr Lys Met Ile Glu Gly Ala
165 170 175
Glu Leu Lys Gln Arg Leu Arg Gly Ala Thr Thr Asp Trp Lys Ile Ala
180 185 190
Gly Phe Glu Glu Asp Ser Gly Ser Phe Asp Pro Glu Val Ala Thr Phe
195 200 205
Val Met Ala Glu Tyr Ala Lys Lys Met Gly Ile Lys Ile Phe Thr Asn
210 215 220
Cys Ala Ala Arg Gly Leu Glu Thr Gln Ala Gly Val Ile Ser Asp Val
225 230 235 240
Val Thr Glu Lys Gly Pro Ile Lys Thr Ser Arg Val Val Val Ala Gly
245 250 255
Gly Val Gly Ser Arg Leu Phe Met Gln Asn Leu Asn Val Asp Val Pro
260 265 270
Thr Leu Pro Ala Tyr Gln Ser Gln Gln Leu Ile Ser Ala Ala Pro Asn
275 280 285
Ala Pro Gly Gly Asn Val Ala Leu Pro Gly Gly Ile Phe Phe Arg Asp
290 295 300
Gln Ala Asp Gly Thr Tyr Ala Thr Ser Pro Arg Val Ile Val Ala Pro
305 310 315 320
Val Val Lys Glu Ser Phe Thr Tyr Gly Tyr Lys Tyr Leu Pro Leu Leu
325 330 335
Ala Leu Pro Asp Phe Pro Val His Ile Ser Leu Asn Glu Gln Leu Ile
340 345 350
Asn Ser Phe Met Gln Ser Thr His Trp Asp Leu Asn Glu Glu Ser Pro
355 360 365
Phe Glu Lys Tyr Arg Asp Met Thr Ala Leu Pro Asp Leu Pro Glu Leu
370 375 380
Asn Ala Ser Leu Glu Lys Leu Lys Lys Glu Phe Pro Ala Phe Lys Glu
385 390 395 400
Ser Thr Leu Ile Asp Gln Trp Ser Gly Ala Met Ala Ile Ala Pro Asp
405 410 415
Glu Asn Pro Ile Ile Ser Asp Val Lys Glu Tyr Pro Gly Leu Val Ile
420 425 430
Asn Thr Ala Thr Gly Trp Gly Met Thr Glu Ser Pro Val Ser Ala Glu
435 440 445
Ile Thr Ala Asp Leu Leu Leu Gly Lys Lys Pro Val Leu Asp Ala Lys
450 455 460
Pro Phe Ser Leu Tyr Arg Phe
465 470
<210> 7
<211> 385
<212> PRT
<213> Bacillus methanolicus
<400> 7
Met Lys Asn Thr Gln Ser Ala Phe Tyr Met Pro Ser Val Asn Leu Phe
1 5 10 15
Gly Ala Gly Ser Val Asn Glu Val Gly Thr Arg Leu Ala Gly Leu Gly
20 25 30
Val Lys Lys Ala Leu Leu Val Thr Asp Ala Gly Leu His Ser Leu Gly
35 40 45
Leu Ser Glu Lys Ile Ala Gly Ile Ile Arg Glu Ala Gly Val Glu Val
50 55 60
Ala Ile Phe Pro Lys Ala Glu Pro Asn Pro Thr Asp Lys Asn Val Ala
65 70 75 80
Glu Gly Leu Glu Ala Tyr Asn Ala Glu Asn Cys Asp Ser Ile Val Thr
85 90 95
Leu Gly Gly Gly Ser Ser His Asp Ala Gly Lys Ala Ile Ala Leu Val
100 105 110
Ala Ala Asn Gly Gly Thr Ile His Asp Tyr Glu Gly Val Asp Val Ser
115 120 125
Lys Lys Pro Met Val Pro Leu Ile Ala Ile Asn Thr Thr Ala Gly Thr
130 135 140
Gly Ser Glu Leu Thr Lys Phe Thr Ile Ile Thr Asp Thr Glu Arg Lys
145 150 155 160
Val Lys Met Ala Ile Val Asp Lys His Val Thr Pro Thr Leu Ser Ile
165 170 175
Asn Asp Pro Glu Leu Met Val Gly Met Pro Pro Ser Leu Thr Ala Ala
180 185 190
Thr Gly Leu Asp Ala Leu Thr His Ala Ile Glu Ala Tyr Val Ser Thr
195 200 205
Gly Ala Thr Pro Ile Thr Asp Ala Leu Ala Ile Gln Ala Ile Lys Ile
210 215 220
Ile Ser Lys Tyr Leu Pro Arg Ala Val Ala Asn Gly Lys Asp Ile Glu
225 230 235 240
Ala Arg Glu Gln Met Ala Phe Ala Gln Ser Leu Ala Gly Met Ala Phe
245 250 255
Asn Asn Ala Gly Leu Gly Tyr Val His Ala Ile Ala His Gln Leu Gly
260 265 270
Gly Phe Tyr Asn Phe Pro His Gly Val Cys Asn Ala Ile Leu Leu Pro
275 280 285
His Val Cys Arg Phe Asn Leu Ile Ser Lys Val Glu Arg Tyr Ala Glu
290 295 300
Ile Ala Ala Phe Leu Gly Glu Asn Val Asp Gly Leu Ser Thr Tyr Glu
305 310 315 320
Ala Ala Glu Lys Ala Ile Lys Ala Ile Glu Arg Met Ala Arg Asp Leu
325 330 335
Asn Ile Pro Lys Gly Phe Lys Glu Leu Gly Ala Lys Glu Glu Asp Ile
340 345 350
Glu Thr Leu Ala Lys Asn Ala Met Asn Asp Ala Cys Ala Leu Thr Asn
355 360 365
Pro Arg Lys Pro Lys Leu Glu Glu Val Ile Gln Ile Ile Lys Asn Ala
370 375 380
Met
385
<210> 8
<211> 213
<212> PRT
<213> Escherichia coli
<400> 8
Met Lys Asn Trp Lys Thr Ser Ala Glu Ser Ile Leu Thr Thr Gly Pro
1 5 10 15
Val Val Pro Val Ile Val Val Lys Lys Leu Glu His Ala Val Pro Met
20 25 30
Ala Lys Ala Leu Val Ala Gly Gly Val Arg Val Leu Glu Val Thr Leu
35 40 45
Arg Thr Glu Cys Ala Val Asp Ala Ile Arg Ala Ile Ala Lys Glu Val
50 55 60
Pro Glu Ala Ile Val Gly Ala Gly Thr Val Leu Asn Pro Gln Gln Leu
65 70 75 80
Ala Glu Val Thr Glu Ala Gly Ala Gln Phe Ala Ile Ser Pro Gly Leu
85 90 95
Thr Glu Pro Leu Leu Lys Ala Ala Thr Glu Gly Thr Ile Pro Leu Ile
100 105 110
Pro Gly Ile Ser Thr Val Ser Glu Leu Met Leu Gly Met Asp Tyr Gly
115 120 125
Leu Lys Glu Phe Lys Phe Phe Pro Ala Glu Ala Asn Gly Gly Val Lys
130 135 140
Ala Leu Gln Ala Ile Ala Gly Pro Phe Ser Gln Val Arg Phe Cys Pro
145 150 155 160
Thr Gly Gly Ile Ser Pro Ala Asn Tyr Arg Asp Tyr Leu Ala Leu Lys
165 170 175
Ser Val Leu Cys Ile Gly Gly Ser Trp Leu Val Pro Ala Asp Ala Leu
180 185 190
Glu Ala Gly Asp Tyr Asp Arg Ile Thr Lys Leu Ala Arg Glu Ala Val
195 200 205
Glu Gly Ala Lys Leu
210
<210> 9
<211> 384
<212> PRT
<213> burkholderia stabilis
<400> 9
Met Ala Thr Val Leu Cys Val Leu Tyr Pro Asp Pro Val Asp Gly Tyr
1 5 10 15
Pro Pro His Tyr Val Arg Asp Thr Ile Pro Val Ile Thr Arg Tyr Ala
20 25 30
Asp Gly Gln Thr Ala Pro Thr Pro Ala Gly Pro Pro Gly Phe Arg Pro
35 40 45
Gly Glu Leu Val Gly Ser Val Ser Gly Leu Gly Leu Arg Gly Tyr Leu
50 55 60
Glu Ala His Gly His Thr Leu Ile Val Thr Ser Asp Lys Asp Gly Pro
65 70 75 80
Asp Ser Glu Phe Glu Arg Arg Leu Pro Asp Ala Asp Val Val Ile Ser
85 90 95
Gln Pro Phe Trp Pro Ala Tyr Leu Thr Ala Glu Arg Ile Ala Arg Ala
100 105 110
Pro Lys Leu Arg Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val
115 120 125
Asp Leu Asp Ala Ala Ala Arg Ala His Ile Thr Val Ala Glu Val Thr
130 135 140
Gly Ser Asn Ser Ile Ser Val Ala Glu His Val Val Met Thr Thr Leu
145 150 155 160
Ala Leu Val Arg Asn Tyr Leu Pro Ser His Ala Ile Ala Gln Gln Gly
165 170 175
Gly Met Asn Ile Ala Asp Cys Val Ser Arg Ser Tyr Asp Val Glu Gly
180 185 190
Met His Phe Gly Thr Val Gly Ala Gly Arg Ile Gly Leu Ala Val Leu
195 200 205
Arg Arg Leu Pro Phe Gly Leu His Leu His Tyr Thr Gln Arg His Arg
210 215 220
Leu Asp Ala Ala Ile Glu Gln Glu Leu Gly Leu Thr Tyr His Ala Asp
225 230 235 240
Pro Ala Ser Leu Ala Ala Ala Val Asp Ile Val Asn Leu Gln Ile Pro
245 250 255
Leu Tyr Pro Ser Thr Glu His Leu Phe Asp Ala Ala Met Ile Ala Arg
260 265 270
Met Lys Arg Gly Ala Tyr Leu Ile Asn Thr Ala Arg Ala Lys Leu Val
275 280 285
Asp Arg Asp Ala Val Val Arg Ala Val Thr Ser Gly His Leu Ala Gly
290 295 300
Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Ala Asp His Pro
305 310 315 320
Trp Arg Ala Met Pro Phe Asn Gly Met Thr Pro His Ile Ser Gly Thr
325 330 335
Ser Leu Ser Ala Gln Ala Arg Tyr Ala Ala Gly Thr Leu Glu Ile Leu
340 345 350
Gln Cys Trp Phe Asp Gly Arg Pro Ile Arg Asn Glu Tyr Leu Ile Val
355 360 365
Asp Gly Gly Thr Leu Ala Gly Thr Gly Ala Gln Ser Tyr Arg Leu Thr
370 375 380
<210> 10
<211> 258
<212> PRT
<213> Stenotrophomonas
<400> 10
Met Thr Gln Gln Arg Trp Arg Leu Asp Gly Gln Thr Ala Leu Ile Thr
1 5 10 15
Gly Ala Ser Ala Gly Ile Gly Leu Ala Ile Ala His Glu Leu Ala Gly
20 25 30
Phe Gly Ala Asp Leu Met Ile Val Gly Arg Asp Ile Asp Met Leu Glu
35 40 45
Thr Ala Arg Asp Glu Leu Leu Asp Val Tyr Pro Gln Met Gln Val His
50 55 60
Ala Leu Ala Ala Asp Val Ser Asp Asp Glu Asp Arg Arg Gln Ile Leu
65 70 75 80
Asp Trp Val Glu Asp His Ser Asp Gly Leu His Ile Leu Val Asn Asn
85 90 95
Ala Gly Gly Asn Val Thr Lys Ala Ala Thr Glu Tyr Ser Glu Asp Glu
100 105 110
Trp Arg Lys Ile Phe Glu Thr Asn Leu Phe Ser Ala Phe Glu Leu Ser
115 120 125
Arg Tyr Ala His Pro Leu Leu Ala Arg His Ala Ser Ser Ser Ile Val
130 135 140
Asn Val Gly Ser Val Ser Gly Leu Thr His Val Arg Ser Gly Val Val
145 150 155 160
Tyr Gly Met Ser Lys Ala Ala Met His Gln Met Thr Arg Asn Leu Ala
165 170 175
Val Glu Trp Ala Glu Asp Gly Ile Arg Val Asn Ala Val Ala Pro Trp
180 185 190
Tyr Ile Arg Thr Arg Arg Thr Ser Gly Pro Leu Ser Asp Pro Asp Tyr
195 200 205
Tyr Glu Glu Val Ile Asn Arg Thr Pro Met Arg Arg Ile Gly Glu Pro
210 215 220
Glu Glu Val Ala Ala Ala Val Gly Phe Leu Cys Leu Pro Ala Ala Ser
225 230 235 240
Tyr Val Thr Gly Glu Cys Ile Ala Val Asp Gly Gly Phe Leu Arg Tyr
245 250 255
Gly Phe

Claims (10)

1. A recombinant plasmid comprising:
a first recombinant plasmid containing an L-amino acid deaminase-encoding gene;
and/or, a second recombinant plasmid comprising a methanol dehydrogenase-encoding gene and an aldolase-encoding gene;
and/or, a third recombinant plasmid comprising a formate dehydrogenase-encoding gene and a ketopantoate reductase-encoding gene.
2. The recombinant plasmid of claim 1, wherein the L-amino acid deaminase encoding gene is derived from proteus mirabilis, and the encoded amino acid sequence is shown as SEQ ID NO. 6; preferably, the L-amino acid deaminase coding gene is subjected to codon optimization to obtain an L-amino acid deaminase optimized gene sequence, and the nucleotide sequence of the L-amino acid deaminase optimized gene sequence is shown as SEQ ID NO. 1; more preferably, the L-amino acid deaminase optimized gene sequence is artificially synthesized and added with enzyme cutting sites to obtain the target gene 1.
And/or the methanol dehydrogenase encoding gene is derived from bacillus, and the encoded amino acid sequence is shown as SEQ ID NO. 7; preferably, the methanol dehydrogenase encoding gene is subjected to codon optimization to obtain a methanol dehydrogenase optimized gene sequence, and the nucleotide sequence of the methanol dehydrogenase optimized gene sequence is shown as SEQ ID NO. 2; more preferably, the methanol dehydrogenase optimized gene sequence is artificially synthesized and added with enzyme cutting sites to obtain a target gene 2;
and/or the aldolase encoding gene is derived from escherichia coli, and the encoded amino acid sequence is shown as SEQ ID NO. 8; preferably, the aldolase encoding gene is subjected to codon optimization to obtain an aldolase optimized gene sequence, and the nucleotide sequence of the aldolase optimized gene sequence is shown as SEQ ID NO. 3; more preferably, the aldolase optimized gene sequence is artificially synthesized and added with enzyme cutting sites to obtain a target gene 3;
and/or the formate dehydrogenase encoding gene is derived from Burkholderia, and the encoded amino acid sequence is shown as SEQ ID NO. 9; preferably, the formate dehydrogenase encoding gene is subjected to codon optimization to obtain a formate dehydrogenase optimized gene sequence, and the nucleotide sequence of the formate dehydrogenase optimized gene sequence is shown as SEQ ID NO. 4; more preferably, the formate dehydrogenase optimized gene sequence is artificially synthesized and added with enzyme cutting sites to obtain a target gene 4;
and/or the ketopantoate reductase coding gene is derived from stenotrophomonas maltophilia, and the coded amino acid sequence is shown as SEQ ID NO. 10; preferably, the ketopantoate reductase encoding gene is subjected to codon optimization to obtain a ketopantoate reductase optimized gene sequence, and the nucleotide sequence of the ketopantoate reductase optimized gene sequence is shown as SEQ ID NO. 5; more preferably, the ketopantoate reductase optimized gene sequence is artificially synthesized and added with enzyme cutting sites to obtain the target gene 5.
3. A recombinant engineered bacterium comprising:
a first recombinant engineered bacterium capable of converting valine to alpha-ketoisovalerate;
and/or, a second recombinant engineered bacterium capable of converting alpha-ketoisovalerate to ketopantoate;
and/or a third recombinant engineered bacterium capable of converting ketopantoate to D-pantoate.
4. The recombinant engineering bacterium of claim 3, wherein the first recombinant engineering bacterium is obtained by a method comprising: the coding gene or the target gene 1 of the L-amino acid deaminase or the gene shown as SEQ ID NO:1 into host cell to obtain the first recombinant engineering bacterium;
and/or the second recombinant engineering bacterium is obtained by the following method: and (2) carrying out gene recombination on a methanol dehydrogenase encoding gene and an aldolase encoding gene, or a target gene 2 and a target gene 3, or a gene sequence shown as SEQ ID NO:2 and SEQ ID NO:3 into host cell to obtain second recombinant engineering bacterium;
and/or the third recombinant engineering bacterium is obtained by the following method: the formate dehydrogenase encoding gene and the ketopantoate reductase encoding gene, or the target gene 4 and the target gene 5, or the nucleotide sequence shown in SEQ ID NO:4 and SEQ ID NO:5 into host cell to obtain the third recombinant engineering bacterium.
5. An application of recombinant engineering bacteria in preparing D-pantoic acid, which comprises the following steps: the first recombinant engineering bacterium of any one of claims 3 to 4 is induced to express to obtain a first thallus, the second recombinant engineering bacterium is induced to express to obtain a second thallus, the third recombinant engineering bacterium is induced to express to obtain a third thallus, and the first thallus, the second thallus and the third thallus are used for preparing D-pantoic acid.
6. A preparation method of D-pantoic acid comprises the following steps: mixing the first thallus, the second thallus and the third thallus, valine and methanol with water, adding ammonium formate, and reacting at 30-40 ℃ and pH 4-7 for 10-60h to obtain D-pantoic acid.
7. The process according to claim 6, wherein the OD value of the first bacterial cell in the reaction system is 1 to 15, preferably 5 to 15, more preferably 8 to 12;
according to the preferable technical scheme of the invention, the OD value of the second thallus in the reaction system is 1-15, preferably 8-15, and more preferably 10-12;
in a preferred embodiment of the present invention, the OD of the third bacterial cell in the reaction system is 1 to 15, preferably 8 to 15, and more preferably 10 to 12.
8. The production method according to any one of claims 6 to 7, wherein the ratio of valine: the molar ratio of methanol is (0.9-1.1): 0.9-1.1, preferably (0.95-1.05): 0.95-1.05.
9. The process according to any of claims 6 to 8, wherein the ammonium formate is added in a fed-batch manner, preferably at a concentration of 1.5g/L to 2.5g/L, more preferably 1.8g/L to 2.2 g/L.
10. Use of D-pantoic acid produced by the process according to any one of claims 6 to 9 for the preparation of panto-compounds selected from any one of D-pantolactone, calcium D-pantothenate, D-panthenol, pantethine.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105506014A (en) * 2015-12-23 2016-04-20 湖南宝利士生物技术有限公司 Biosynthesis method of L-homoserine with high optical purity and L-homoserine derivatives
CN108456701A (en) * 2018-03-23 2018-08-28 精晶药业股份有限公司 A kind of preparation method of D-pantoyl lactone
CN110423717A (en) * 2019-05-05 2019-11-08 杭州鑫富科技有限公司 Multienzyme recombinant cell and multienzyme cascade the method for catalyzing and synthesizing D-pantoyl lactone
CN111876404A (en) * 2020-07-30 2020-11-03 浙大宁波理工学院 Aldolase mutant and coding gene and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105506014A (en) * 2015-12-23 2016-04-20 湖南宝利士生物技术有限公司 Biosynthesis method of L-homoserine with high optical purity and L-homoserine derivatives
CN108456701A (en) * 2018-03-23 2018-08-28 精晶药业股份有限公司 A kind of preparation method of D-pantoyl lactone
CN110423717A (en) * 2019-05-05 2019-11-08 杭州鑫富科技有限公司 Multienzyme recombinant cell and multienzyme cascade the method for catalyzing and synthesizing D-pantoyl lactone
CN111876404A (en) * 2020-07-30 2020-11-03 浙大宁波理工学院 Aldolase mutant and coding gene and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MICHELE SUGANTINO 等: "Mycobacterium tuberculosis Ketopantoate Hydroxymethyltransferase: Tetrahydrofolate-Indepen dent Hydroxymethyltransferase and Enolization Reactions with R-Keto Acids", 《BIOCHEMISTRY》, vol. 42, no. 1 *
应向贤等: "氧化还原酶在 多酶级联反应中的应用 进展", 《发酵科技通讯》, vol. 47, no. 3 *
杨延辉等: "泛酸的功能和 生物合成", 《生命的化学》, no. 4 *

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