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Recombinant Pichia pastoris engineering strain and its application in synthesizing levodopa

A technology of Pichia pastoris and levodopa, applied in application, genetic engineering, recombinant DNA technology, etc., can solve problems such as bacterial infection, increased process steps and energy consumption, fermentation failure, etc., to save production costs and reduce process treatment degree, the effect of continuous culture

Active Publication Date: 2019-10-08
ZHEJIANG UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the use of bacteria to express TPL has begun to be applied in industry, it is still limited in many ways.
First of all, bacteria such as Escherichia coli expressing TPL need to undergo wall-breaking treatment to release intracellular TPL, which increases the process steps and energy consumption, and the enzyme activity is easily reduced during the wall-breaking process
Second, bacteria are susceptible to phage infection, which can easily cause fermentation failure

Method used

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  • Recombinant Pichia pastoris engineering strain and its application in synthesizing levodopa
  • Recombinant Pichia pastoris engineering strain and its application in synthesizing levodopa
  • Recombinant Pichia pastoris engineering strain and its application in synthesizing levodopa

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Embodiment 1

[0026] Embodiment 1 Construction of Pichia pastoris recombinant strain YZ001

[0027] This example is based on the amino acid sequence of Kluyvera intermedia tyrosine phenol lyase shown in SEQ ID NO.2. The DNA sequence of tyrosine phenol lyase (TPL) shown in SEQ ID NO.1 is optimized according to the codon preference of Pichia pastoris to obtain the TPL gene after codon optimization, and its optimized sequence is as SEQ ID NO. 3 shown. The whole gene synthesis was carried out by a biotechnology company (Jinweizhi Biotechnology Company), and the synthesized gene was cloned on the pETDuet-1 vector to obtain the plasmid pETDuet-TPL. By designing primers YW500 and YW504, the plasmid pETDuet-TPL was used as a template to amplify TPL by PCR. The amplification system is 50 μL (2x PrimeSTAR Max DNA polymerase (purchased from Beijing Baoriyi Biological Co., Ltd.) 25 μL, 1 μL YW500+1 μL YW504, 0.5 μL pETDuet-TPL plasmid, distilled water to 22.5 μL), pre-denaturation at 95°C for 3 minut...

Embodiment 2

[0030] The construction of embodiment 2 Pichia pastoris recombinant strain YZ002

[0031] Using the genome of Pichia pastoris GS115 as a template, PCR amplification of HAC1 (its nucleotide sequence is shown in SEQ ID NO.4) was performed by designing primers YW507 and YW508. The amplification system is 50 μL (2×PrimeSTAR Max DNA polymerase 25 μL, 1 μL YW507+1 μL YW508, 0.5 μL GS115 genome, distilled water to 22.5 μL), pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for 10 seconds, annealing at 58°C for 15 seconds, Extend at 72°C for 2 minutes (30 cycles), and extend again at 72°C for 10 minutes.

[0032]After recovering the HAC1 fragment gel obtained above, digest it with restriction endonucleases EcoR I and Not I, and connect it to the pPICZαA vector that was also digested with EcoR I and Not I to construct the pPICZαA-HAC1 plasmid, such as figure 2 shown. The cloning system is 20 μL (2 μL 10x T4 ligase buffer, 6 μL digested TPL fragment, 2 μL digested pPICZαA ...

Embodiment 3

[0037] The SDS-PAGE analysis of embodiment 3 Pichia pastoris recombinant strain YZ002

[0038] BMGY medium: 1% yeast powder, 2% peptone, 1.34% YND, 0.1mol / L pH 6.0 phosphate buffer, 1% glycerol, 4×10-5% biotin.

[0039] BMMY medium: 1% yeast powder, 2% peptone, 1.34% YND, 0.1mol / L pH 6.0 phosphate buffer, 2% methanol, 4×10-5% biotin.

[0040] The SDS-PAGE analysis of Pichia recombinant bacteria is as follows: inoculate Pichia recombinant bacteria in BMGY medium and culture overnight at 30°C, then transfer to 50mL fresh BMGY medium to make the initial OD600 0.1, and cultivate to OD600 at 30°C 6.0, centrifuged at 4000rpm for 10min, collected bacteria, resuspended with 50mL BMMY medium to OD600 of 1.0 for culture, and added 100% methanol to the medium every 24h to a final concentration of 1%. After induction of expression for 72 hours, the supernatant was taken to analyze the protein expression by SDS-PAGE. Gel preparation and electrophoresis are as follows, use 10% gel formula...

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Abstract

The invention discloses a recombinant Pichia pastoris engineering strain and its application in synthesizing levodopa. The recombinant Pichia pastoris engineering strain of the present invention is obtained by the following method: a DNA sequence of tyrosine phenol lyase (TPL) shown in SEQ ID NO. 1 is codon-optimized and transferred to Pichia pastoris GS115 to construct recombinant Pichia pastorisYZ001, then HAC1 shown in SEQ ID NO. 4 is overexpressed in Pichia pastoris YZ001, and the recombinant Pichia pastoris engineering strain YZ002 is constructed. A supernatant of the recombinant Pichiapastoris engineering bacteria fermented by centrifugation can be effectively used for the synthesis of levodopa, and the thalline after centrifugation can be used for the next batch of fermentation, which not only improves the synthesis efficiency and utilization rate of levodopa, saves production costs, but also opens up a new industrialization route for the production and processing of levodopa.

Description

technical field [0001] The invention relates to a recombinant Pichia pastoris engineering bacterium and its application in synthesizing levodopa, belonging to the field of bioengineering. Background technique [0002] Levodopa is a common drug used in the treatment of Parkinson's disease, hepatic encephalopathy, neuralgia, hyperprolactinemia and other diseases, and has great application value in the pharmaceutical market. The current methods for preparing levodopa mainly include plant extraction, chemical synthesis and biological enzyme conversion. However, both the plant extraction method and the chemical synthesis method have disadvantages such as complex processes, high cost, and environmental pollution. The bioenzyme-catalyzed synthesis of levodopa is environmentally friendly, intensive and efficient, and has become the main method for the industrialization of levodopa. [0003] The bioenzyme conversion method mainly uses tyrosine phenol lyase (TPL) as a catalyst, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/60C12N15/31C12P13/22C12R1/84
CPCC07K14/39C12N9/88C12N15/815C12N2800/22C12P13/225C12Y401/99002
Inventor 袁围钟爽肖延铭华超汪钊祁瑛梁阿朋
Owner ZHEJIANG UNIV OF TECH
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