Method for producing levodopa through microbial fermentation and application

[0047] Example 1

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

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

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

[0051] 5'-GGGAATTCCATATGAAAACCAGAAGATTTCCGC-3',

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

[0053] 5'-CGGAATTCATTATTTCAGCAGCTTATCCAGCAT-3'; wherein, the italicized letter parts are the enzyme cleavage sites NdeI and EcoRI, respectively. The PCR reaction was carried out in a total system of 50 μl. The reaction conditions were: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 1 min, extension at 72°C for 2 min, a total of 30 cycles; extension at 72°C for 10 min. Take 5 μl of PCR amplification product for verification by agarose gel electrophoresis. The target product is about 1.6 kb in size.

[0054] Example 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 PCR product after double digestion with restriction enzymes NdeI and EcoRI, and ligate it with hpaC/pET-24a vector digested with NdeI and EcoRI endonucleases (hpaC is 4-hydroxyphenylacetic acid 3-monooxygenase). C, previously inserted into the pET-24a vector), the ligation product mixture was transformed into E. coli Top10, 20 clones were picked, and primers T7hpaB-F and T7hpaB-R were used for PCR identification. Select PCR-identified positive clones for sequence determination, save the vector with correct sequencing, and name it: pET-hpaBC.

[0057] Example 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 high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (wherein dATP and dGTP concentrations are 1.5mmol/L, dTTP and dCTP concentrations are 3.0 mmol/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 was 1000 ng/mL, the enzyme concentration was 5 U/μL, and the primer concentration was 100 μM.

[0060] Error-prone PCR reaction system (50 μl): 10× PCR reaction buffer 5 μl, dNTP (2.5 nM) 5 μl, 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-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min. PCR products were recovered by gel recovery method. Take 5 μl of the product for 1% agarose gel electrophoresis test, and store it at -20°C for later use.