CN112941123A - Method for biologically synthesizing droxidopa based on L-threonine aldolase - Google Patents
Method for biologically synthesizing droxidopa based on L-threonine aldolase Download PDFInfo
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Abstract
The invention discloses a method for biologically synthesizing droxidopa based on L-threonine aldolase, which takes BL21DE3 as an initial strain, overexpresses an ItaE gene which is derived from coding the L-threonine aldolase in pseudomonas aeruginosa, a P-TA gene which is derived from coding the L-threonine aldolase in pseudomonas putida, an LTA gene which is derived from coding the L-threonine aldolase in escherichia coli, or an STA gene which is derived from coding the L-threonine aldolase in streptomyces, obtains engineering bacteria through the operation, and obtains the yield of the droxidopa which is 3.3-4.15g/L after the wet cells obtained by centrifugal collection are reacted. Compared with the prior art, the biosynthesis method of the invention has the advantages of green, high yield, mild conditions and the like, and particularly has few byproducts and high environmental friendliness.
Description
Technical Field
The invention belongs to the technical field of droxidopa preparation, and particularly relates to a method for biologically synthesizing droxidopa based on L-threonine aldolase.
Background
Droxidopa (L-threo-DOPS) is a novel anti-parkinson drug for improving gait rigidity and erect dizziness caused by parkinson's disease; ameliorating orthostatic hypotension, orthostatic dizziness and fainting caused by Shy-Drager syndrome or familial amyloid neuropathy; improve dizziness and hypodynamia of hemodialysis patients caused by orthostatic hypotension. The existing method for preparing droxidopa is mainly a chemical method and faces the problems of more byproducts, difficult resolution and separation and the like, so the preparation cost is high.
L-Threonine Aldolases (TAs) can catalyze different types of aldehydes to perform aldol condensation reaction with alpha-amino acids to generate core fragments in a plurality of active medical components such as chloramphenicol, florfenicol, thiamphenicol, epinephrine, droxidopa, vancomycin, sphingolipid and the like. Therefore, TAs have important application value in biosynthesis of droxidopa.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for biologically synthesizing droxidopa based on L-threonine aldolase, which has the advantages of mild reaction conditions, easy control and low cost, avoids the problems of more byproducts and high resolution cost in the chemical synthesis process, and reduces the preparation cost.
A method for biosynthesis of droxidopa based on L-threonine aldolase, comprising the steps of:
When the exogenous gene is ItaE, the obtained genetically engineered bacterium is BL21(DE3)/pET28 a-ItaE;
when the exogenous gene is STA, the obtained genetically engineered bacterium is BL21(DE3)/pET28 a-STA;
when the exogenous gene is LTA, the obtained genetically engineered bacterium is BL21(DE3)/pET28 a-LTA;
when the exogenous gene is P-TA, the obtained genetically engineered bacterium is BL21(DE3)/pET28 a-P-TA;
Selecting single colony of Escherichia coli MG1655, inoculating in LB culture medium, culturing at 37 deg.C for 12 hr, collecting Escherichia coli cell, and extracting its genome;
step 4, resuspending the gene engineering bacteria collected in the step 3 by using PBS buffer solution with pH 6-11;
and 6, after the catalytic reaction is finished, adding 1N diluted hydrochloric acid with the same volume, centrifuging at 12000rpm for 2min, and taking supernate to detect the generation condition of the product.
The improvement is that the exogenous gene ItaE in the step 1 is subjected to codon optimization according to the reported amino acid sequence of the L-threonine aldolase from the pseudomonas aeruginosa, and the optimized sequence is synthesized in a whole gene manner; after the exogenous gene STA is subjected to codon optimization according to the reported amino acid sequence of the streptomyces source L-threonine aldolase, the optimized sequence is synthesized by the whole gene; after the exogenous gene P-TA is subjected to codon optimization according to the reported amino acid sequence of the L-threonine aldolase derived from the pseudomonas putida, the optimized sequence is synthesized in a whole gene manner; the exogenous gene LTA is cloned according to the reported amino acid sequence of L-threonine aldolase derived from escherichia coli, and the sequence is obtained after heterologous expression.
Further improved, the specific steps for constructing the genetically engineered bacterium BL21(DE3)/pET28a-LTA are as follows:
designing an upstream primer with a BamH I enzyme cutting site: cgcggatccATGATTGATTTACGCAGTG, respectively;
designing a downstream primer with HindIII enzyme cutting sites: cccaagcttACGCGCCAGGAATGCACGCC, respectively;
amplifying the genome DNA of Escherichia coli MG1655 as a template to obtain a segment LTA, extracting pET28a plasmid preserved in Escherichia coli T1, selecting enzyme cutting sites BamH I and Hind III, carrying out double enzyme cutting operation on the segment and the plasmid, respectively carrying out gel recovery after the enzyme cutting is finished, detecting the concentration of the plasmid after the completion, connecting the plasmid and the segment after the gel recovery by using T4 DNA Ligase (Takara), transforming competence, coating the plate on a plate with kanamycin resistance for overnight culture, carrying out colony PCR verification by using a plasmid universal primer after a single bacterium grows out on the plate, simultaneously scribing on the plate with the same resistance for 12-16h, selecting a correct positive result after the verification of nucleic acid gel electrophoresis, carrying out the plasmid extraction and the company sequencing to confirm that an insertion sequence is the plasmid Pet28a-LTA which is successfully constructed, and obtaining the recombinant plasmid pET28a-LTA, the constructed recombinant plasmid pET28a-LTA is transformed into an escherichia coli expression host BL21(DE3) by a calcium chloride method to obtain an expression L-threonine aldolase strain BL21(DE3)/pET28 a-LTA.
Further improved, the specific steps for constructing genetically engineered bacteria BL21(DE3)/pET28a-ItaE, BL21(DE3)/pET28a-STA or BL21(DE3)/pET28a-P-TA in the step 1 are as follows: after codon optimization is carried out according to the reported amino acid sequence of the L-threonine aldolase, the sequence is consigned to the plasmid pET28a after the complete gene synthesis optimization of Onychoideae Biotech Co., Ltd (Nanjing), the recombinant plasmid pET28a-ItaE, pET28a-STA or pET28a-P-TA is obtained, the constructed recombinant plasmid pET28a-ItaE, pET28a-STA or pET28a-P-TA is transformed into an Escherichia coli expression host BL21(DE3) by a calcium chloride method, and the L-threonine aldolase strain BL21(DE3)/pET28a-ItaE, BL21(DE3)/pET28a-STA or BL21(DE3)/pET28a-P-TA is obtained.
As a modification, the final concentration of 3, 4-dihydroxybenzaldehyde in step 5 was 100mM/L and the final concentration of glycine was 1M/L.
In a further improvement, the wet cell sap OD of the genetically engineered bacteria in the step 5600Is 50.
In a further improvement, the reaction temperature in the step 5 is 30 ℃, and the reaction pH is 7.5.
Has the advantages that:
compared with the prior art, the method for biologically synthesizing droxidopa based on L-threonine aldolase has the following advantages: the method screens out four new sources of L-threonine aldolase, can synthesize droxidopa, provides more new choices for synthesizing droxidopa by a biological enzyme method, has the advantages of greenness, high yield, mild conditions and the like, and is particularly few in byproducts and high in environmental friendliness.
Drawings
FIG. 1 is a high performance liquid chromatogram of a droxidopa standard product of the invention;
FIG. 2 is a quantitative standard curve diagram of droxidopa of the invention;
FIG. 3 is a high performance liquid chromatogram of a substrate 3, 4-dihydroxybenzaldehyde standard according to the present invention;
FIG. 4 is a high performance liquid chromatogram of the product and the substrate in the L-threonine aldolase reaction solution derived from Pseudomonas aeruginosa according to the present invention;
FIG. 5 is a high performance liquid chromatogram of a substrate and a product in a Pseudomonas putida-derived L-threonine aldolase reaction solution according to the present invention;
FIG. 6 is a high performance liquid chromatogram of a substrate and a product in an Escherichia coli-derived L-threonine aldolase reaction liquid according to the present invention;
FIG. 7 is a high performance liquid chromatogram of a product and a substrate in a Streptomyces-derived L-threonine aldolase reaction liquid.
Detailed description of the preferred embodiments
In the following examples, the formulation of LB medium: 10g/l peptone, 5g/l yeast powder and 5g/l sodium chloride; the reagents used in the examples are all conventional.
Example 1 expression and catalysis of E.coli-derived L-threonine aldolase
Escherichia coli MG1655 single colonies were picked, inoculated in LB medium, cultured at 37 ℃ for 12 hours, Escherichia coli cells were collected, and the collected Escherichia coli genome was extracted using a bacterial genome DNA extraction kit (DP302) (Kyoto Biochemical (Beijing) science and technology Co., Ltd.) according to the instructions.
Designing an upstream primer (shown as SEQ NO. 4) with a BamH I enzyme cutting site: cgcggatccATGATTGATTTACGCAGTG
Designing a downstream primer with HindIII enzyme cutting site (shown as SEQ NO. 5): cccaagcttACGCGCCAGGAATGCACGCC
Using genome DNA of Escherichia coli MG1655 as a template to obtain a fragment LTA through PCR amplification, using a plasmid miniprep kit (DP103) (Tiangen Biochemical (Beijing) science and technology limited company) to extract pET28a plasmid preserved in Escherichia coli T1, selecting enzyme cutting sites BamH I and Hind III, carrying out double enzyme cutting operation on the fragment and the plasmid, respectively recovering the fragments through a general DNA purification recovery kit (DP214) (Tiangen Biochemical (Beijing) science and technology limited company) after the enzyme cutting is finished, detecting the concentration of the plasmid after the completion, using T4 DNA Ligase (Takara) to connect the plasmid and the fragment after the glue recovery, transforming the plasmid and the fragment into a competence of Escherichia coli expression engineering bacterium BL21(DE3), coating the plate on a plate with kanamycin resistance for overnight culture, using pET28a plasmid for colony PCR verification after the single bacterium grows out on the plate, and simultaneously scribing the plate with the same resistance for culture for 12-16h, the correct positive result is selected for plasmid extraction after nucleic acid gel electrophoresis verification, sequencing is carried out by Scotteraceae biotechnology limited company, the plasmid Pet28a-LTA is successfully constructed if the insertion sequence is confirmed to be correct, the recombinant plasmid pET28a-LTA is obtained, the constructed recombinant plasmid pET28a-LTA is converted into an escherichia coli expression host BL21(DE3) competence by a calcium chloride method, and the expression L-threonine aldolase strain BL21(DE3)/pET28a-LTA is obtained;
Selecting a single colony of genetically engineered bacteria BL21(DE3)/pET28a-LTA for expressing LTA, inoculating the single colony to an LB culture medium, culturing at 37 ℃ until the OD value reaches above 0.6, then adding 0.25mM-1mM I PTG, culturing at 15-30 ℃ for 12-18 hours, and centrifugally collecting genetically engineered bacteria BL21(DE3)/pET28a-LTA for expressing L-threonine aldolase;
step 4, collecting genetically engineered bacteria BL21(DE3)/pET28a-LTA for expressing L-threonine aldolase, and resuspending the bacteria with PBS buffer solution with the pH of 6-11;
Wherein the final concentration of 3, 4-dihydroxybenzaldehyde is 100mM/L, the final concentration of glycine is 1M/L, the final concentration of pyridoxal-5-phosphate is 5 μ M/L, and L-threonine aldolase whole cell liquid OD is added60050, the reaction temperature of the reaction system is 30 ℃, and the reaction pH is 7.5.
Catalyzing to produce droxidopa, the specific operation steps are that the reaction system is 1ml, the final concentration of 3, 4-dihydroxy benzaldehyde is 100mM/L, the final concentration of glycine is 1M/L, the final concentration of 5-pyridoxal phosphate is 5 mu M/L, and OD is added60050 wet cell liquid, adjusting the system to 1ml with PBS buffer solution, pH 7.5, reacting for 2-12h at 30 ℃.
After the reaction, an XSe lect HSS T3 type chromatographic column (4.6X 250mm, 5 μm) with a mobile phase of 23.3g sodium chloride (Shanghai Michelle chemical technology Co., Ltd.) and 1ml glacial acetic acid was diluted with water to 1L and mixed with 2% pure acetonitrile (analytical grade) solution at 30 ℃ and a flow rate of 5ml/min with an ultraviolet detection wavelength of 280 nm. Adopting a gradient elution method:
after detection by high performance liquid chromatography, a result graph shown in FIG. 6 is obtained, wherein the peak at 6.313min is the detection peak of the droxidopa product, and the calculated product amount is 4.15 g/L.
Example 2 expression and catalysis of Pseudomonas aeruginosa-derived L-threonine aldolase
After codon optimization is carried out according to the reported amino acid sequence of the L-threonine aldolase derived from the pseudomonas aeruginosa, the sequence is shown as SEQ NO. 1:
ATGACCGATCACACCCAACAGTTCGCCAGCGACAACTATTCCGGCATCTGCCCCGAAGCCTGGGCGGCGATGGCCGAAGCCAACCGCGGCCACGAACGCGCCTATGGCGACGACCAGTGGACCGCGCGTGCTTCGGACTACTTCCGCCAGTTGTTCGAGACCGATTGCGAAGTGTTCTTCGCCTTCAACGGCACCGCCGCCAACTCCCTGGCCCTGGCTGCGCTGTGCCAGAGCTACCACAGCGTGATCTGCTCGGAGACCGCCCACGTCGAGACCGACGAGTGCGGCGCGCCGGAGTTCTTCTCCAATGGCTCCAAGCTGCTGCTGGCGCAGACCGAGGTCGGCAAGCTGACCCCGGCGTCGATCCGCGACATCGCTCTCAAGCGCCAGGATATCCACTATCCGAAGCCGCGCGTGGTGACTCTCACCCAGGCCACCGAGGTCGGCACGGTGTACCGCCCGGACGAGCTGAAGGCGATCAGCGCCACCTGCAAGGAGCTCGGCCTGCACCTGCACATGGACGGCGCGCGCTTCTCCAACGCCTGCGCCTTCCTCGGCTGCTCGCCGGCGGAGCTGAGCTGGAAGGCCGGGGTCGACGTGCTCTGCTTCGGCGGCACCAAGAACGGCATGGCGGTGGGCGAGGCGATCCTGTTCTTCAACCGCGACCTGGCCGAGGACTTCGACTACCGCTGCAAGCAGGCCGGCCAACTGGCCTCGAAGATGCGCTTCCTCGCCGCGCCCTGGGTCGGCGTACTGCAGGACGATGCTTGGCTGCGCTACGCCGATCACGCCAACCGCTGCGCGCGGCTGTTGGCCGAACTGGTGGCCGACGTTCCCGGCGTCAGCCTGATGTTCCCGGTAGAAGCCAACGGCGTGTTCCTGCAGCTTTCCGAGCCGGCGATCGAAGCCTTGCGCGCGCGCGGCTGGCGCTTCTACACCTTCATCGGTGAAGGTGGCGCGCGTTTCATGTGTTCCTGGGATACCGATATCGAGCGGGTCCGCGAACTGGCGCGGGACATCCGCCTGGTGATGGGCGCC, entrusting the sequence after the whole gene synthesis optimization of Onychosantha scientific and technology Co., Ltd (Nanjing), subcloning the sequence onto a vector pET28a to obtain a recombinant plasmid pET28a-ItaE, transforming the constructed recombinant plasmid pET28a-ItaE into an Escherichia coli expression host BL21(DE3) by a calcium chloride method to obtain an expression L-threonine aldolase strain BL21(DE3)/pET28a-ItaE, and the rest steps are the same as the steps 3,4 and 5 of the embodiment 1.
After detection by high performance liquid chromatography, a result graph shown in FIG. 4 is obtained, wherein a peak at 6.311min is a detection peak of the droxidopa product, and the calculated product amount is 3.4 g/L.
Example 3 and example 4 except that the source of L-threonine aldolase was different from that of example 2, example 3 was a streptomyces source (codon-optimized sequence shown in SEQ No. 2:
GTTAACCCGCCTAAAACCGATGCACGTCGTCATCATGATCCTGAAGTGCGTGGTTTTGCAAGTGATAATTATGCAGGTGCACATCCGGAAGTGCTGGCAGCACTGGCACTGGCAAATGGTGGCCATCAGGTAGCATACGGTGAAGATGATTATACCGAAAATCTGCAGCGTGTTATTCGTAGCCATTTTGGTAGCGGTGCAGAAGCATTTCCTGTTTTTAATGGTACCGGTGCAAATGTTGTTGCTCTGCAGGCCGTTACAGATCGTTGGGGTGCAGTGATTTGTGCCGAAAGCGCACATATTAATGTTGATGAAGGTGGTGCACCGGAACGTATGGGCGGTCTGAAACTGCTGACCGTTCCAACCCCGGATGGTAAACTGACACCGGAACTGATTGATCGCCAGGCATACGGTTGGGATGATGAACATCGTGCGATGCCGCAGGTTGTTAGTATTACCCAGTCCACCGAACTGGGTACCCTGTATACCCCGGATGAAATTCGTGCAGTTTGTGAACATGCACATGCCCATGGTATGAAAGTGCATCTGGATGGTAGCCGTATTGCAAATGCCGCAGCAAGCCTGGATGTCCCGATGCGTACCTTTACCAATGCAGTTGGTGTTGATCTGCTGAGCCTGGGTGGTACCAAAAATGGCGCGCTGTTTGGTGAAGCAGTTGTTGTTCTGAATCAGGATGCAGTTCGTCACATGAAACATCTGCGTAAACTGAGTATGCAGCTGGCAAGCAAAATGCGTTTTGTGTCAGTGCAGCTGGAAGCCCTGCTGGCAAAAGATCTGTGGCTGCGCAATGCGCGTCATAGCAATGAAATGGCACAGCGTCTGGCCGAAGGTGTTCGTGCAGTTCATGGTGTTGAAATTCTGTATCCTGTTCAGGCAAATGCAGTATTTGCGCGTCTGCCTCATGAAGTTAGCGAACGCCTGCAGAAACGTTTTCGTTTTTATTTTTGGGATGAAGCAGCAGGTGATGTGCGTTGGATGTGTGCATTTGATACCACCGAAAATGATGTTGATGCATTTGTTAGTGCACTGAAAGAAGAAATGGCACGT), example 4 was of pseudomonas putida origin (codon optimized sequence shown in SEQ No. 3:
ATGACCGATCAGAGCCAGCAGTTTGCGAGCGATAATTATAGCGGTATTTGTCCGGAAGCATGGGCAGCAATGGAAAAAGCGAACCACGGTCACGAACGCGCATACGGTGATGATCAGTGGACCGCACGTGCAGCAGATCATTTTCGTAAACTGTTTGAAACAGATTGTGAAGTATTCTTTGCATTTAACGGTACCGCAGCAAACAGTCTGGCACTGAGTAGCCTGTGTCAGAGCTATCACAGCGTTATTTGTAGCGAAACCGCACATGTTGAAACAGATGAATGTGGAGCACCGGAGTTTTTCAGCAATGGCAGTAAACTGCTGACCGCACGTAGCGAAGGTGGTAAACTGACCCCTGCAAGTATTCGTGAAGTGGCACTGAAACGTCAGGATATTCATTATCCGAAACCTCGTGTTGTTACCATCACCCAGGCCACCGAAGTTGGTAGCGTTTATCGTCCGGATGAACTGAAAGCAATTAGCGCAACATGTAAAGAACTGGGTCTGAATCTGCACATGGATGGCGCACGTTTTAGTAACGCATGCGCATTTCTGGGTTGTACACCGGCAGAGCTGACTTGGAAAGCAGGAATCGATGTGCTGTGCTTTGGTGGAACTAAAAATGGCATGGCAGTTGGTGAGGCAATTCTGTTTTTTAATCGTAAACTGGCTGAAGATTTCGATTATCGTTGTAAACAGGCAGGTCAGCTGGCAAGTAAAATGCGTTTTCTGAGCGCGCCGTGGGTTGGTCTGCTGGAAGATGGTGCTTGGCTGCGTCATGCAGCACATGCAAATCATTGTGCGCAGCTGCTGAGCAGCCTGGTTGCTGATATTCCGGGTGTTGAGCTGATGTTTCCTGTTGAGGCAAATGGTGTTTTTCTGCAGATGTCTGAACCGGCACTGGAAGCACTGCGTAATAAAGGTTGGCGTTTTTATACCTTTATCGGTTCAGGTGGTGCACGTTTTATGTGTTCATGGGATACCGAGGAAGCCCGTGTTCGTGAGCTGGCAGCAGATATTCGTGCGGTTATGTCTGCA) as in example 2.
After detection by high performance liquid chromatography, example 3 obtains a result graph as shown in fig. 5, wherein a peak at 6.269min is a detection peak of the droxidopa product, and the calculated product amount is 3.3 g/L.
Example 4 a graph of the results shown in fig. 7 was obtained, in which the peak at 6.252min was the detected peak of the product droxidopa, which was calculated to give a product amount of 3.9 g/L. In conclusion, the droxidopa is prepared by the biological method, four new sources of L-threonine aldolase are screened out, the droxidopa can be synthesized, more new choices are provided for synthesizing the droxidopa by the biological enzyme method, and the biological method has the advantages of greenness, high yield, mild conditions and the like, and particularly has few byproducts and high environmental friendliness.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Sequence listing
<110> Nanjing university of industry
<120> method for biologically synthesizing droxidopa based on L-threonine aldolase
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgcggatcca tgattgattt acgcagtg 28
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cccaagctta cgcgccagga atgcacgcc 29
<210> 3
<211> 1038
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaccgatc acacccaaca gttcgccagc gacaactatt ccggcatctg ccccgaagcc 60
tgggcggcga tggccgaagc caaccgcggc cacgaacgcg cctatggcga cgaccagtgg 120
accgcgcgtg cttcggacta cttccgccag ttgttcgaga ccgattgcga agtgttcttc 180
gccttcaacg gcaccgccgc caactccctg gccctggctg cgctgtgcca gagctaccac 240
agcgtgatct gctcggagac cgcccacgtc gagaccgacg agtgcggcgc gccggagttc 300
ttctccaatg gctccaagct gctgctggcg cagaccgagg tcggcaagct gaccccggcg 360
tcgatccgcg acatcgctct caagcgccag gatatccact atccgaagcc gcgcgtggtg 420
actctcaccc aggccaccga ggtcggcacg gtgtaccgcc cggacgagct gaaggcgatc 480
agcgccacct gcaaggagct cggcctgcac ctgcacatgg acggcgcgcg cttctccaac 540
gcctgcgcct tcctcggctg ctcgccggcg gagctgagct ggaaggccgg ggtcgacgtg 600
ctctgcttcg gcggcaccaa gaacggcatg gcggtgggcg aggcgatcct gttcttcaac 660
cgcgacctgg ccgaggactt cgactaccgc tgcaagcagg ccggccaact ggcctcgaag 720
atgcgcttcc tcgccgcgcc ctgggtcggc gtactgcagg acgatgcttg gctgcgctac 780
gccgatcacg ccaaccgctg cgcgcggctg ttggccgaac tggtggccga cgttcccggc 840
gtcagcctga tgttcccggt agaagccaac ggcgtgttcc tgcagctttc cgagccggcg 900
atcgaagcct tgcgcgcgcg cggctggcgc ttctacacct tcatcggtga aggtggcgcg 960
cgtttcatgt gttcctggga taccgatatc gagcgggtcc gcgaactggc gcgggacatc 1020
cgcctggtga tgggcgcc 1038
<210> 4
<211> 1068
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gttaacccgc ctaaaaccga tgcacgtcgt catcatgatc ctgaagtgcg tggttttgca 60
agtgataatt atgcaggtgc acatccggaa gtgctggcag cactggcact ggcaaatggt 120
ggccatcagg tagcatacgg tgaagatgat tataccgaaa atctgcagcg tgttattcgt 180
agccattttg gtagcggtgc agaagcattt cctgttttta atggtaccgg tgcaaatgtt 240
gttgctctgc aggccgttac agatcgttgg ggtgcagtga tttgtgccga aagcgcacat 300
attaatgttg atgaaggtgg tgcaccggaa cgtatgggcg gtctgaaact gctgaccgtt 360
ccaaccccgg atggtaaact gacaccggaa ctgattgatc gccaggcata cggttgggat 420
gatgaacatc gtgcgatgcc gcaggttgtt agtattaccc agtccaccga actgggtacc 480
ctgtataccc cggatgaaat tcgtgcagtt tgtgaacatg cacatgccca tggtatgaaa 540
gtgcatctgg atggtagccg tattgcaaat gccgcagcaa gcctggatgt cccgatgcgt 600
acctttacca atgcagttgg tgttgatctg ctgagcctgg gtggtaccaa aaatggcgcg 660
ctgtttggtg aagcagttgt tgttctgaat caggatgcag ttcgtcacat gaaacatctg 720
cgtaaactga gtatgcagct ggcaagcaaa atgcgttttg tgtcagtgca gctggaagcc 780
ctgctggcaa aagatctgtg gctgcgcaat gcgcgtcata gcaatgaaat ggcacagcgt 840
ctggccgaag gtgttcgtgc agttcatggt gttgaaattc tgtatcctgt tcaggcaaat 900
gcagtatttg cgcgtctgcc tcatgaagtt agcgaacgcc tgcagaaacg ttttcgtttt 960
tatttttggg atgaagcagc aggtgatgtg cgttggatgt gtgcatttga taccaccgaa 1020
aatgatgttg atgcatttgt tagtgcactg aaagaagaaa tggcacgt 1068
<210> 5
<211> 1038
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgaccgatc agagccagca gtttgcgagc gataattata gcggtatttg tccggaagca 60
tgggcagcaa tggaaaaagc gaaccacggt cacgaacgcg catacggtga tgatcagtgg 120
accgcacgtg cagcagatca ttttcgtaaa ctgtttgaaa cagattgtga agtattcttt 180
gcatttaacg gtaccgcagc aaacagtctg gcactgagta gcctgtgtca gagctatcac 240
agcgttattt gtagcgaaac cgcacatgtt gaaacagatg aatgtggagc accggagttt 300
ttcagcaatg gcagtaaact gctgaccgca cgtagcgaag gtggtaaact gacccctgca 360
agtattcgtg aagtggcact gaaacgtcag gatattcatt atccgaaacc tcgtgttgtt 420
accatcaccc aggccaccga agttggtagc gtttatcgtc cggatgaact gaaagcaatt 480
agcgcaacat gtaaagaact gggtctgaat ctgcacatgg atggcgcacg ttttagtaac 540
gcatgcgcat ttctgggttg tacaccggca gagctgactt ggaaagcagg aatcgatgtg 600
ctgtgctttg gtggaactaa aaatggcatg gcagttggtg aggcaattct gttttttaat 660
cgtaaactgg ctgaagattt cgattatcgt tgtaaacagg caggtcagct ggcaagtaaa 720
atgcgttttc tgagcgcgcc gtgggttggt ctgctggaag atggtgcttg gctgcgtcat 780
gcagcacatg caaatcattg tgcgcagctg ctgagcagcc tggttgctga tattccgggt 840
gttgagctga tgtttcctgt tgaggcaaat ggtgtttttc tgcagatgtc tgaaccggca 900
ctggaagcac tgcgtaataa aggttggcgt ttttatacct ttatcggttc aggtggtgca 960
cgttttatgt gttcatggga taccgaggaa gcccgtgttc gtgagctggc agcagatatt 1020
cgtgcggtta tgtctgca 1038
Claims (7)
1. A method for biologically synthesizing droxidopa based on L-threonine aldolase is characterized by comprising the following steps:
step 1, constructing gene engineering bacteria for expressing exogenous genes
When the exogenous gene is ItaE, the obtained genetically engineered bacterium is BL21(DE3)/pET28a-ItaE,
when the exogenous gene is STA, the obtained genetically engineered bacterium is BL21(DE3)/pET28a-STA,
when the exogenous gene is LTA, the obtained genetically engineered bacterium is BL21(DE3)/pET28a-LTA, or
When the exogenous gene is P-TA, the obtained genetically engineered bacterium is BL21(DE3)/pET28 a-P-TA;
step 2, extracting the genome of Escherichia coli MG1655
Selecting single colony of Escherichia coli MG1655, inoculating in LB culture medium, culturing at 37 deg.C for 12 hr, collecting Escherichia coli cell, and extracting its genome;
step 3, selecting a single colony of the genetic engineering bacteria, inoculating the single colony in an LB culture medium, culturing at 37 ℃ until the OD value reaches above 0.6, adding 0.25mM-1mM IPTG, culturing at 15-30 ℃ for 12-18 hours, and centrifugally collecting the genetic engineering bacteria;
step 4, resuspending the gene engineering bacteria collected in the step 3 by using PBS buffer solution with pH 6-11;
step 5, selecting 3, 4-dihydroxybenzaldehyde asTaking glycine as a substrate, taking pyridoxal 5-phosphate as a cofactor, adding a buffer solution to adjust the pH value of the reaction system to 6-11, and adding the wet cell sap OD of the genetically engineered bacteria resuspended in the PBS buffer solution in the step 4600Reacting at 10-200 deg.C for 0.5-24 h; wherein the final concentration of 3, 4-dihydroxybenzaldehyde is 20-200mM/L, the final concentration of glycine is 0.1-2M/L, and the final concentration of pyridoxal-5-phosphate is 1-20 μ M/L;
and 6, after the catalytic reaction is finished, adding 1N diluted hydrochloric acid with the same volume, centrifuging at 12000rpm for 2min, and taking supernate to detect the generation condition of the product.
2. The method for biosynthesis of droxidopa based on L-threonine aldolase of claim 1, wherein the exogenous gene ItaE in step 1 is codon-optimized according to the reported amino acid sequence of L-threonine aldolase derived from Pseudomonas aeruginosa, and the optimized sequence is synthesized in a whole gene; after the exogenous gene STA is subjected to codon optimization according to the reported amino acid sequence of the streptomyces source L-threonine aldolase, the optimized sequence is synthesized by the whole gene; after the exogenous gene P-TA is subjected to codon optimization according to the reported amino acid sequence of the L-threonine aldolase derived from the pseudomonas putida, the optimized sequence is synthesized in a whole gene manner; the exogenous gene LTA is cloned according to the reported amino acid sequence of L-threonine aldolase derived from escherichia coli, and the sequence is obtained after heterologous expression.
3. The method for biosynthesis of droxidopa based on L-threonine aldolase as claimed in claim 2, wherein the genetically engineered bacterium BL21(DE3)/pET28a-LTA is constructed by the following steps:
designing an upstream primer with a BamH I enzyme cutting site: cgcggatccATGATTGATTTACGCAGTG, respectively;
designing a downstream primer with HindIII enzyme cutting sites: cccaagcttACGCGCCAGGAATGCACGCC, respectively;
amplifying the genome DNA of Escherichia coli MG1655 as a template to obtain a segment LTA, extracting pET28a plasmid preserved in Escherichia coli T1, selecting enzyme cutting sites BamH I and Hind III, carrying out double enzyme cutting operation on the segment and the plasmid, respectively carrying out gel recovery after the enzyme cutting is finished, detecting the concentration of the plasmid after the completion, connecting the plasmid and the segment after the gel recovery by using T4 DNA Ligase, transforming competence, coating the plate on a plate with kanamycin resistance for overnight culture, carrying out colony PCR verification by using a plasmid universal primer after a single bacterium grows out on the plate, simultaneously scribing on the plate with the same resistance for 12-16h, selecting a correct positive result after the verification of nucleic acid gel electrophoresis, carrying out plasmid extraction and company sequencing, confirming that an insertion sequence is correct, namely successfully constructing plasmid Pet 28-28 a-LTA, obtaining recombinant plasmid pET28a-LTA, the constructed recombinant plasmid pET28a-LTA is transformed into an escherichia coli expression host BL21(DE3) by a calcium chloride method to obtain an expression L-threonine aldolase strain BL21(DE3)/pET28 a-LTA.
4. The method for biosynthesis of droxidopa based on L-threonine aldolase as claimed in claim 2, wherein the specific steps for constructing genetically engineered bacteria BL21(DE3)/pET28a-ItaE, BL21(DE3)/pET28a-STA or BL21(DE3)/pET28a-P-TA are as follows: after codon optimization is carried out according to the reported amino acid sequence of the L-threonine aldolase, the optimized sequence is synthesized in a whole gene and is subcloned to a carrier pET28a, a recombinant plasmid pET28a-ItaE, pET28a-STA or pET28a-P-TA is obtained, the constructed recombinant plasmid pET28a-ItaE, pET28a-STA or pET28a-P-TA is transformed into an expression host BL21(DE3) by a calcium chloride method, and an expression L-threonine aldolase strain BL21(DE3)/pET28a-ItaE, BL21(DE3)/pET28a-STA or BL21(DE3)/pET28a-P-TA is obtained.
5. The method for the biosynthesis of droxidopa based on L-threonine aldolase of claim 1, wherein the final concentration of 3, 4-dihydroxybenzaldehyde in step 5 is 100mM/L, the final concentration of glycine is 1M/L, and the final concentration of PLP is 5. mu.M/L.
6. The method for biosynthesis of droxidopa based on L-threonine aldolase according to claim 1Characterized in that the wet cell sap OD of the genetically engineered bacteria in the step 5600Is 50.
7. The method for the biosynthesis of droxidopa based on L-threonine aldolase as claimed in claim 1, wherein the reaction system in step 5 has a pH of 7.5 and a reaction temperature of 30 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114703169A (en) * | 2022-04-29 | 2022-07-05 | 重庆大学 | L-threonine aldolase mutant R318L/H128N and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03277282A (en) * | 1990-03-27 | 1991-12-09 | Mitsui Toatsu Chem Inc | New threonine aldolase, gene capable of coding the same and utilization thereof |
| JP2007054013A (en) * | 2005-08-26 | 2007-03-08 | Research Institute Of Innovative Technology For The Earth | Thermostable l-threonine aldolase and gene encoding the same |
| CN110592058A (en) * | 2019-05-30 | 2019-12-20 | 重庆大学 | Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis |
| CN111394343A (en) * | 2020-04-22 | 2020-07-10 | 重庆大学 | L-threonine aldolase mutant R318L and application thereof |
| CN111849951A (en) * | 2020-04-22 | 2020-10-30 | 重庆大学 | L-threonine aldolase mutant R318M and application thereof |
| CN111849952A (en) * | 2020-04-22 | 2020-10-30 | 重庆大学 | L-threonine aldolase mutant R318V and application thereof |
-
2021
- 2021-02-25 CN CN202110212026.2A patent/CN112941123A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03277282A (en) * | 1990-03-27 | 1991-12-09 | Mitsui Toatsu Chem Inc | New threonine aldolase, gene capable of coding the same and utilization thereof |
| JP2007054013A (en) * | 2005-08-26 | 2007-03-08 | Research Institute Of Innovative Technology For The Earth | Thermostable l-threonine aldolase and gene encoding the same |
| CN110592058A (en) * | 2019-05-30 | 2019-12-20 | 重庆大学 | Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis |
| CN111394343A (en) * | 2020-04-22 | 2020-07-10 | 重庆大学 | L-threonine aldolase mutant R318L and application thereof |
| CN111849951A (en) * | 2020-04-22 | 2020-10-30 | 重庆大学 | L-threonine aldolase mutant R318M and application thereof |
| CN111849952A (en) * | 2020-04-22 | 2020-10-30 | 重庆大学 | L-threonine aldolase mutant R318V and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703169A (en) * | 2022-04-29 | 2022-07-05 | 重庆大学 | L-threonine aldolase mutant R318L/H128N and application thereof |
| CN114703169B (en) * | 2022-04-29 | 2023-10-31 | 重庆大学 | L-threonine aldolase mutant R318L/H128N and application thereof |
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