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High-throughput screening method of epsilon-caprolactone high-yield strain

A technology for high-yielding strains and screening methods, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of cumbersome and time-consuming processes, and achieve simple operation steps, strong long-term stability, The effect of high catalytic efficiency

Pending Publication Date: 2022-04-15
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The screening of existing ε-caprolactone-producing strains generally uses shake flask catalysis followed by gas chromatography detection. The process is cumbersome and time-consuming. At present, no one has applied high-throughput screening technology to screen high-yielding ε-caprolactone-producing strains.

Method used

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  • High-throughput screening method of epsilon-caprolactone high-yield strain
  • High-throughput screening method of epsilon-caprolactone high-yield strain
  • High-throughput screening method of epsilon-caprolactone high-yield strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Cascade Reaction Intermediate Product Concentration and Product Concentration Correlation Discovery

[0053] The laboratory has constructed multiple strains of ε-caprolactone-producing bacteria BDR-2, BDR-5~BDR-13 in the early stage, and used the above 10 strains to carry out the whole-cell catalytic reaction of 40mM and 60mM cyclohexanol, and found that each The yield of ε-caprolactone varies among the strains (Xiong Jinghui. The construction and catalytic performance of Escherichia coli whole-cell high-efficiency synthesis of ε-caprolactone [D]. South China University of Technology, 2020.). Through further analysis of the whole-cell catalysis results of the above-mentioned series of strains, we found that after different strains catalyzed the same concentration of substrate cyclohexanol, the concentration of the product ε-caprolactone in the reaction system was higher than that of the intermediate product cyclohexanone. A good negative correlation ( fig...

Embodiment 2

[0054] Example 2 Construction of a library of mutant strains producing ε-caprolactone based on RBS engineering technology

[0055] The laboratory has constructed a strain containing the recombinant vector pRSF-RBS in the early stage 10 -ADH-RBS 20 -CHMO (pRSF-A10C20) ε-caprolactone-producing strain BDR-3 (Xiong Jinghui. Construction and catalytic performance of Escherichia coli whole cell efficient synthesis of ε-caprolactone [D]. South China University of Technology, 2020. ). In the present invention, the pRSF-RBS 10 -ADH-RBS 20 -CHMO renamed to pRSF-RBS A0 -ADH-RBS C0 -CHMO(RBS A0 and RBS C0 shown in SEQ ID NO.3 and SEQ ID NO.4, respectively); BDR-3 was renamed RCL0. pRSF-RBS A0 -ADH-RBS C0 -CHMO is the starting vector for library construction, and the SD sequence in the original ribosome binding site sequence of the adh and chmo genes is mutated, and the degenerate SD sequence of the two genes is designed using the RBS LibraryCalculator web software and the RedLib...

Embodiment 3

[0066] Example 3 High-throughput screening of ε-caprolactone high-yielding strains

[0067] 1. Well-plate culture and whole-cell catalysis of mutant library strains

[0068] Pick 1743 single colonies from the mutant strain library in Example 2 and inoculate them in 150 μL LB medium (containing kanamycin) in a 96-well plate. In fresh LB medium (containing kanamycin), cultivate under the same conditions until OD 600After reaching 0.5-0.7, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce protein expression. After induction at 30°C and 900rpm for 7h, the fermentation broth was centrifuged at 4°C and 2000g for 10min to collect E. 7.5) Resuspend the bacteria, and carry out the whole-cell catalytic reaction at 25° C. and 800 rpm for 16 hours.

[0069] 2. High-throughput screening of mutant library strains

[0070] After the catalytic reaction for 16 hours, high-throughput detection of the concentration of cyclohexanone ...

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Abstract

The invention discloses a high-throughput screening method of epsilon-caprolactone high-yield strains, and belongs to the technical field of high-throughput screening. According to the invention, correlation analysis on the concentration of an ADH and CHMO double-enzyme cascade reaction product epsilon-caprolactone and the concentration of an intermediate product cyclohexanone shows that after strains with different catalytic performances are subjected to a whole-cell catalytic reaction, the concentration of the intermediate product cyclohexanone and the concentration of the product epsilon-caprolactone are in a negative correlation relationship, so that cyclohexanone is taken as a screening marker, and the screening efficiency is improved. The strain with high epsilon-caprolactone yield is obtained through high-throughput screening of the strain with low cyclohexanone concentration. A mutation library of epsilon-caprolactone producing bacteria is constructed by utilizing an RBS engineering technology, seven mutant strains obtained by screening are subjected to shake flask experiment verification, and compared with an original strain RCL0, the yield of epsilon-caprolactone for catalyzing 70mM cyclohexanol is remarkably improved. The method is simple in operation steps, and can greatly reduce the experiment cost in the strain screening process and shorten the screening period.

Description

technical field [0001] The invention relates to a high-throughput screening method for high-yield ε-caprolactone strains, and belongs to the technical field of high-throughput screening. Background technique [0002] ε-caprolactone is a colorless transparent oily liquid with aromatic odor, easily soluble in water, ethanol, benzene and other solvents, insoluble in petroleum ether, is a widely used organic chemical intermediate, and can be used as a monomer to synthesize high-performance Polymer materials such as polycaprolactone. Due to the unique biocompatibility, excellent biodegradability and good permeability of polycaprolactone, it is widely used in the field of biomedical engineering and biodegradation: it can be used as various biodegradable medical materials, such as Surgical sutures, carriers with controlled drug release, bone splints, etc.; can be used to synthesize environmentally friendly materials, such as degradable plastics, fully degradable membrane materials...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/10C12N15/31C12P7/62C12R1/19
Inventor 李爽刘然陈和锋余皓
Owner SOUTH CHINA UNIV OF TECH
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