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Directed evolution of cyp52a12 gene and its use in dicarboxylic acid production

a technology of cyp52a12 and dicarboxylic acid, which is applied in the field of preparing a long-chain dicarboxylic acid producing strain, can solve the problems of huge obstacles to serious affecting the development of long-chain dicarboxylic acid in the industry, and many challenges in chemical synthesis. achieve the effect of significantly shortening the fermentation cycl

Active Publication Date: 2021-06-17
CIBT AMERICA INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0028]By directed evolution of CYP52A12 gene, the invention screened out one strain which has a base mutation at the promoter region of said gene and is capable of producing a long chain dicarboxylic acid under an acidic condition. The establishment of an acidic culture condition does not require addition of a large amount of alkali during the acid producing process to maintain an alkaline environment, which simplifies the subsequent extraction process of the dicarboxylic acid product, and avoids the production of a large amount of high-salt wastewater during the fermentation process, greatly reducing adverse effects on the environment. In addition, the strain produces the acid in high concentration and shortens the fermentation cycle significantly. Compared to the prior production process, it not only has significant cost advantages, but also can effectively reduce the pressure on resources and environment. Therefore, it has very remarkable industrialization advantages.

Problems solved by technology

However, the chemical synthesis faces many challenges.
The dicarboxylic acids obtained by the chemical synthesis are a mixture of long chain dicarboxylic acids and short-chain dicarboxylic acids, and therefore subsequent complicated extraction and purification steps are required, which are huge obstacles for production processes and production costs.
The existence of high salt wastewater has brought great challenges to the environment, which seriously affects the development of the biosynthesis of a long chain dicarboxylic acid in industry.
However, the activity of the P450 enzyme is lower at a low pH, the metabolism is slow, and the production efficiency is low.
Due to the randomness of mutagenesis, there is a high requirement for screening throughput, and a new round of mutagenesis screening is required for each trait change, which has become an important limiting factor in technology.
Too high or too low mutation rate will affect the effect of constructing a mutant library.

Method used

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  • Directed evolution of cyp52a12 gene and its use in dicarboxylic acid production
  • Directed evolution of cyp52a12 gene and its use in dicarboxylic acid production
  • Directed evolution of cyp52a12 gene and its use in dicarboxylic acid production

Examples

Experimental program
Comparison scheme
Effect test

example 1

edia and Methods for Culture and Fermentation as Well as for Detecting a Dicarboxylic Acid

[0086]1. YPD medium formula (w / v) was: 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP0021). 1.5-2% agar powder was added to form a solid media.

[0087]During culturing, a single colony was picked in a 2 ml centrifuge tube containing 1 ml YPD liquid medium, incubated at 30° C. in a 250 RPM shaker for 1 day.

[0088]2. Seed medium formula (w / v): sucrose 10 to 20 g / L, yeast extract 3 to 8 g / L, industrial fermentation corn syrup (for short, corn syrup, with total nitrogen content 2.5 wt %) 2 to 4 g / L, KH2PO4 4 to 12 g / L, urea 0.5 to 4 g / L (separately sterilized at 115° C. for 20 min), and the substrate for fermentation was n-dodecane 20 mL / L.

[0089]During culturing, the inoculum obtained in step 1 was inoculated into a 500 mL shake flask containing 30 mL seed medium, wherein the amount of inoculum was 3-5%, and incubated at 30° C. in a 250 RPM shaker until OD620 reached 0.8 (after 30-fold dilutio...

example 2

on of CYP52A12 Mutation Template

[0094]1. Preparation of the CYP52A12 mutation template.

[0095]The genomic DNA of Candida CCTCC M 2011192 was extracted by using Ezup Yeast Genomic DNA Extraction Kit (Sangon, Cat No. 518257). A method with liquid nitrogen grinding was used in favor of increasing the cell wall disruption efficiency. Genomic DNA obtained by this method was used as template for error-prone PCR.

[0096]2. Error-prone PCR

[0097]The concentration of Mg2+ was adjusted (2-8 mM) and the CYP52A12 gene was amplified by error-prone PCR using Taq DNA Polymerase (Takara, Cat No. R00113).

[0098](PCR condition was: Step 1: 98° C. for 30 s, step 2: 98° C. for 10 s, 55° C. for 30 s, 72° C. for 2 m 20 s, 35 cycles in total, Step 3: 72° C. for 5 m).

[0099]The primers were as follows:

CYP52A12-F:(SEQ ID NO: 1)5′-CAAAACAGCACTCCGCTTGT-3′,CYP52A12-R:(SEQ ID NO: 2)5′-GGATGACGTGTGTGGCTTGA-3′,

[0100]The PCR product was subjected to electrophoresis on a 1% agarose gel and recovered and purified by using...

example 3

on of Homologous Recombination Template

[0101]All DNA fragments in this example were obtained by amplification using PrimeSTAR® HS High Fidelity DNA polymerase (Takara, R040A). The DNA fragments were subjected to electrophoresis on a 1% agarose gel, followed by recovery and purification by using the Axygen Gel Recovery Kit.

[0102](1) Amplification of the resistance selection marker (HYG, the hygromycin resistance gene). The amplification template was the vector pCIB2 (SEQ ID NO: 3) owned by our company. The primer sequences were as follows:

CYP52A12_HYG-F:(SEQ ID NO: 4)5′-TCAAGCCACACACGTCATCCGCATGCGAACCCGAAAATGG-3′,CYP52A12_HYG-R:(SEQ ID NO: 5)5′-GATGTGGTGATGGGTGGGCTGCTAGCAGCTGGATTTCACT-3′.

[0103]The PCR reaction condition was as follows:

[0104]Step 1: 98° C. for 30 s,

[0105]Step 2: 98° C. for 10 s, 55° C. for 30 s, 72° C. for 1 m 50 s, 5 cycles,

[0106]Step 3: 98° C. for 10 s, 72° C. for 2 m, 25 cycles,

[0107]Step 4: 72° C. for 5 m.

[0108]The resulting product, named HYG, was verified by seq...

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Abstract

The invention relates to the directed evolution of CYP52A12 gene and the use thereof for the production of a dicarboxylic acid. In particular, it relates to a method of preparing a long chain dicarboxylic acid producing strain by using directed evolution and homologous recombination, a strain obtained by the method that is capable of producing a long chain dicarboxylic acid under an acidic condition and the use thereof. In particular, the invention relates to a method of preparing a long chain dicarboxylic acid producing strain by directed evolution of CYP52A12 gene and homologous recombination, a strain obtained by the method that is capable of producing a long chain dicarboxylic acid under an acidic condition and the use thereof. By directed evolution of CYP52A12 gene, one strain which has a base mutation at the promoter region of said gene and is capable of producing a long chain dicarboxylic acid under an acidic condition in a shortened fermentation time is screened out in the invention.

Description

FIELD OF THE INVENTION[0001]The invention relates to a method of preparing a long chain dicarboxylic acid producing strain by using directed evolution and homologous recombination, a strain obtained by this method that is capable of producing a long chain dicarboxylic acid under an acidic condition and the use thereof. In particular, the invention relates to a method of preparing a long chain dicarboxylic acid producing strain by using directed evolution of CYP52A12 gene and homologous recombination, a strain obtained by this method that is capable of producing a long chain dicarboxylic acid under an acidic condition and the use thereof.BACKGROUND[0002]A long chain dicarboxylic acid (LCDA; also referred to as long chain diacid) is a diacid having the formula HOOC(CH2)nCOOH, n≥7. As an important monomer raw material, long chain dicarboxylic acids are widely used in the synthesis of nylon, resin, hot-melt adhesive, powder coating, preservative, perfume, lubricant, plasticizer, and the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/02C12P7/64
CPCC12N9/0071C12P7/6409C12Y114/14C12N9/001C12N15/52C12P7/44
Inventor LIU, WENBOXU, MINCHOU, HOWARDLIU, XIUCAI
Owner CIBT AMERICA INC
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