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Directed evolution of CYP52A12 gene and application of gene in production of binaryacid

A CYP52A12, dibasic acid technology, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of shortening the fermentation cycle, significant cost advantages, and high concentration of acid production

Pending Publication Date: 2019-10-18
CATHAY R&D CENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] There is no report on the use of directed evolution to transform dibasic acid production strains to adapt to acidic fermentation conditions, and there is still a demand for strains and preparation methods adapted to acidic fermentation conditions in this field.

Method used

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  • Directed evolution of CYP52A12 gene and application of gene in production of binaryacid
  • Directed evolution of CYP52A12 gene and application of gene in production of binaryacid
  • Directed evolution of CYP52A12 gene and application of gene in production of binaryacid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1 culture medium, culture fermentation method and dibasic acid detection method

[0090] 1. YPD medium, the formula (w / v) is: 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP0021). 1.5-2% agar powder needs to be added to the solid medium.

[0091] During cultivation, a single colony can be placed in a 2ml centrifuge tube containing 1ml YPD liquid medium, and cultured on a shaker at 250RPM for 1 day at 30°C.

[0092] 2, seed medium, formula (w / v) is: sucrose 10~20g / L, yeast extract 3~8g / L, corn steep liquor (abbreviated as corn steep liquor, total nitrogen content 2.5wt%) 2~4g / L for industrial fermentation L, KH 2 PO 4 4-12g / L, 0.5-4g / L urea (sterilized separately at 115°C for 20min), and the fermentation substrate is n-dodecane 20mL / L.

[0093] When cultivating, insert the bacterial solution cultivated in step 1 into a 500mL shake flask containing 30mL seed medium, the inoculum amount is 3-5%, and cultivate to OD at 250rpm and 30°C on a shaker 620...

Embodiment 2

[0098] Example 2 Preparation of CYP52A12 Mutation Template

[0099] 1. Preparation of CYP52A12 mutation template.

[0100] Genomic DNA of Candida CCTCC M 2011192 was extracted using the Ezup Yeast Genomic DNA Rapid Extraction Kit (Sangon, Cat. No. 518257). In order to improve the efficiency of cell wall crushing, liquid nitrogen grinding was used to crush the cell wall. Error-prone PCR was performed using the genomic DNA obtained by this method as a template.

[0101] 2. Error-prone PCR

[0102] Adjust Mg 2+ Concentration (2-8mM), using Taq DNA polymerase (Takara, Cat. No. R001B) for error-prone PCR amplification of CYP52A12 gene. (PCR conditions are: step 1: 98°C for 30s, step 2: 98°C for 10s, 55°C for 30s, 72°C for 2m for 20s, a total of 35 cycles, step 3: 72°C for 5m), the primers are as follows:

[0103] CYP52A12-F: 5'-CAAAACAGCACTCCGCTTGT-3' (SEQ ID NO: 1),

[0104] CYP52A12-R: 5'-GGATGACGTGTGTGGCTTGA-3' (SEQ ID NO: 2),

[0105] After the PCR product was subjected ...

Embodiment 3

[0106] Example 3 Preparation of Homologous Recombination Template

[0107] All DNA fragments used in this example HS high-fidelity DNA polymerase (Takara, R040A) amplified. After 1% agarose gel electrophoresis, the purified DNA fragments were recovered with the Axygen gel recovery kit.

[0108] (1) Amplification of the resistance selection marker (HYG, hygromycin resistance gene), the amplification template is the vector pCIB2 (SEQ ID NO: 3) owned by our company, and the primer sequence is as follows:

[0109] CYP52A12_HYG-F:

[0110] 5'-TCAAGCCACACACGTCATCCGCATGCGAACCCGAAAATGG-3' (SEQ ID NO: 4),

[0111] CYP52A12_HYG-R:

[0112] 5'-GATGTGGTGATGGGTGGGCTGCTAGCAGCTGGATTTCACT-3' (SEQ ID NO: 5).

[0113] The PCR reaction conditions are as follows:

[0114] Step 1: 98°C for 30s,

[0115] Step 2: 98°C for 10s, 55°C for 30s, 72°C for 1m for 50s, 5 cycles,

[0116] Step 3: 98°C for 10s, 72°C for 2m, 25 cycles,

[0117] Step 4: 72°C for 5m.

[0118] The obtained product is c...

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Abstract

The present invention relates to a method for producing a long-chain binaryacid strain by a directed evolution and a homologous recombination method, and a strain obtained by the method for producingthe long-chain binaryacidunder an acidic condition and an application of the strain. Specifically, the invention relates to a method for preparing a long-chain binaryacid strain by directed evolutionof a CYP52A12 gene and a homologous recombination method, a strain obtained by the method for producing the long-chain binaryacid under an acidic condition and an application of the strain.

Description

technical field [0001] The present invention relates to a method for preparing long-chain dibasic acid strains by means of directed evolution and homologous recombination, a strain which can produce long-chain dibasic acids under acidic conditions obtained by the method, and the use of the strains. Specifically, the present invention relates to a method for preparing long-chain dibasic acid strains by directed evolution of CYP52A12 gene and homologous recombination method, the strains obtained by using this method that can produce long-chain dibasic acids under acidic conditions, and the strains the use of. Background technique [0002] Long-chain diacids (LCDA; also known as long-chain dicarboxylic acids or long-chain diacids) include the chemical formula HOOC (CH 2 ) n Dibasic acid of COOH, where n≥7. As an important monomer raw material, long-chain dibasic acids are widely used in the synthesis of nylon, resins, hot melt adhesives, powder coatings, preservatives, spice...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/77C12N15/81C12N1/19C12N1/21C12P7/64
CPCC12N9/0006C12N15/77C12N15/815C12N15/81C12P7/6409C12P7/6427
Inventor 刘文波徐敏周豪宏刘修才
Owner CATHAY R&D CENT CO LTD
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