Method for producing levodopa through microbial fermentation and application

Abstract

The invention discloses a method for producing levodopa through microbial fermentation. According to the method, levodopa is produced with higher efficiency and higher yield mainly by improving the effect of 4-hydroxyphenylacetic acid 3-monooxygenase B (hpaB) in microorganisms.

Description

technical field

[0001] The invention relates to a high-yield levodopa genetically engineered Escherichia coli strain and a construction method thereof, and a method for fermenting and producing levodopa by using the strain. The present invention also relates to a method for recovering levodopa from a fermentation process. It belongs to the field of medicine, food and feed. Background technique

[0002] Levodopa (L-DOPA), chemical name: 3-hydroxy-L-tyrosine. Chinese synonyms: L-3-(3,4-dihydroxyphenyl)alanine; L-B-(3,4-dihydroxyphenyl)alanine; L-β-(3,4-dihydroxyphenyl)alanine acid; levodopa; 3-(3,4-dihydroxyphenyl)-L-alanine; L-3-(3,4-dihydroxyphenyl)alanine. Levodopa is a white or off-white crystalline powder, easily soluble in dilute acid, slightly soluble in water, insoluble in ethanol, ether or chloroform, odorless, tasteless, and turns black in the air.

[0003] Levodopa is an important biologically active substance in organisms and an important intermediate product i...

Examples

Embodiment 1

[0048] This example describes the acquisition of the E. coli 4-hydroxyphenylacetic acid 3-monooxygenase B gene.

[0049] According to the NCBI Reference Sequence published by Pubmed: NC_012971.2: Escherichia coliBL21 (DE3) wild-type 4-hydroxyphenylacetic acid 3-monooxygenase B (the amino acid sequence is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4 shows) the nucleotide sequence SEQ ID NO: 4 design primers.

[0050] Forward primer (T7hpaB-F) SEQ ID NO: 5:

[0051] 5'-GGGAATTCCATATGAAACCAGAAGATTTCCGC-3',

[0052] Reverse primer (T7hpaB-R) SEQ ID NO: 6:

[0053] 5'-CGGAATTCATTATTTCAGCAGCTTTATCCAGCAT-3'; Among them, the parts in italics are the enzyme cutting sites NdeI and EcoRI respectively. The PCR reaction was carried out in 50 μl total system, and the reaction conditions were: denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles; extensi...

Embodiment 2

[0055] This example describes the construction of a wild-type 4-hydroxyphenylacetic acid 3-monooxygenase B gene expression vector.

[0056] The PCR product in Example 1 was subjected to agarose gel electrophoresis, and the target fragment was recovered according to the instructions of the gel recovery kit. Take 100 μl of the PCR product and digest it with restriction endonucleases NdeI and EcoRI, and then connect it to the hpaC / pET-24a vector digested with NdeI and EcoRI (hpaC is 4-hydroxyphenylacetic acid 3-monooxygenase C, previously inserted into the pET-24a vector), the ligation product mixture was transformed into Escherichia coli Top10, 20 clones were picked, and PCR identification was performed with primers T7hpaB-F and T7hpaB-R. The positive clones identified by PCR were selected for sequence determination, and the vectors with correct sequencing were saved and named: pET-hpaBC.

Embodiment 3

[0058] This example describes error-prone PCR amplification of the E. coli 4-hydroxyphenylacetic acid 3-monooxygenase B gene.

[0059] Utilizing the property that Taq DNA polymerase does not have 3'-5' proofreading function, under the concentration of high magnesium ion concentration (8mmol / L) and different concentration of dNTP (wherein the concentration of dATP and dGTP is 1.5mmol / L, the concentration of dTTP and dCTP 3.0mmol / L) to control the frequency of random mutations, introduce random mutations into the target gene, and construct a mutation library. Template concentration A 260 The value is 1000ng / mL, the enzyme concentration is 5U / μL, and the primer concentration is 100μM.

[0060] Error-prone PCR reaction system (50μl): 5μl of 10×PCR reaction buffer, 5μl of dNTP (2.5nM), MgCl 2 5 μl, forward primer (T7hpaB-F) 1 μl, reverse primer (T7hpaB-R) 1 μl, DNA template (PCR product of Example 1) 1 μl, Taq DNA polymerase 0.5 μl, ddH 2 O 31.5 μl.

[0061]PCR program: pre-den...

technical field

[0001] The invention relates to a high-yield levodopa genetically engineered Escherichia coli strain and a construction method thereof, and a method for fermenting and producing levodopa by using the strain. The present invention also relates to a method for recovering levodopa from a fermentation process. It belongs to the field of medicine, food and feed. Background technique

[0002] Levodopa (L-DOPA), chemical name: 3-hydroxy-L-tyrosine. Chinese synonyms: L-3-(3,4-dihydroxyphenyl)alanine; L-B-(3,4-dihydroxyphenyl)alanine; L-β-(3,4-dihydroxyphenyl)alanine acid; levodopa; 3-(3,4-dihydroxyphenyl)-L-alanine; L-3-(3,4-dihydroxyphenyl)alanine. Levodopa is a white or off-white crystalline powder, easily soluble in dilute acid, slightly soluble in water, insoluble in ethanol, ether or chloroform, odorless, tasteless, and turns black in the air.

[0003] Levodopa is an important biologically active substance in organisms and an important intermediate product i...

Examples

Embodiment 1

[0048] This example describes the acquisition of the E. coli 4-hydroxyphenylacetic acid 3-monooxygenase B gene.

[0049] According to the NCBI Reference Sequence published by Pubmed: NC_012971.2: Escherichia coliBL21 (DE3) wild-type 4-hydroxyphenylacetic acid 3-monooxygenase B (the amino acid sequence is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4 shows) the nucleotide sequence SEQ ID NO: 4 design primers.

[0050] Forward primer (T7hpaB-F) SEQ ID NO: 5:

[0051] 5'-GGGAATTCCATATGAAACCAGAAGATTTCCGC-3',

[0052] Reverse primer (T7hpaB-R) SEQ ID NO: 6:

[0053] 5'-CGGAATTCATTATTTCAGCAGCTTTATCCAGCAT-3'; Among them, the parts in italics are the enzyme cutting sites NdeI and EcoRI respectively. The PCR reaction was carried out in 50 μl total system, and the reaction conditions were: denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles; extensi...

Embodiment 2

[0055] This example describes the construction of a wild-type 4-hydroxyphenylacetic acid 3-monooxygenase B gene expression vector.

[0056] The PCR product in Example 1 was subjected to agarose gel electrophoresis, and the target fragment was recovered according to the instructions of the gel recovery kit. Take 100 μl of the PCR product and digest it with restriction endonucleases NdeI and EcoRI, and then connect it to the hpaC / pET-24a vector digested with NdeI and EcoRI (hpaC is 4-hydroxyphenylacetic acid 3-monooxygenase C, previously inserted into the pET-24a vector), the ligation product mixture was transformed into Escherichia coli Top10, 20 clones were picked, and PCR identification was performed with primers T7hpaB-F and T7hpaB-R. The positive clones identified by PCR were selected for sequence determination, and the vectors with correct sequencing were saved and named: pET-hpaBC.

Embodiment 3

[0058] This example describes error-prone PCR amplification of the E. coli 4-hydroxyphenylacetic acid 3-monooxygenase B gene.

[0059] Utilizing the property that Taq DNA polymerase does not have 3'-5' proofreading function, under the concentration of high magnesium ion concentration (8mmol / L) and different concentration of dNTP (wherein the concentration of dATP and dGTP is 1.5mmol / L, the concentration of dTTP and dCTP 3.0mmol / L) to control the frequency of random mutations, introduce random mutations into the target gene, and construct a mutation library. Template concentration A 260 The value is 1000ng / mL, the enzyme concentration is 5U / μL, and the primer concentration is 100μM.

[0060] Error-prone PCR reaction system (50μl): 5μl of 10×PCR reaction buffer, 5μl of dNTP (2.5nM), MgCl 2 5 μl, forward primer (T7hpaB-F) 1 μl, reverse primer (T7hpaB-R) 1 μl, DNA template (PCR product of Example 1) 1 μl, Taq DNA polymerase 0.5 μl, ddH 2 O 31.5 μl.

[0061]PCR program: pre-den...

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