Method for improving electric transformation efficiency of candida utilis

By combining LiAc-DTT solution with optimized electroporation parameters, the electroconversion efficiency of Candida utilis was improved, overcoming the cell wall and membrane barriers, achieving efficient transformation and high survival rate of large plasmid fragments, solving the problem of insufficient electroconversion efficiency, and supporting efficient gene modification and functional gene research.

CN121780587APending Publication Date: 2026-04-03INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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Patent Information

Application Number
CN202610133215.3
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-01-30
Publication Date
2026-04-03

AI Technical Summary

Technical Problem

The low electroconversion efficiency of Candida utilis makes it difficult to overcome the dual barriers of its cell wall and cell membrane, limiting its progress in genetic modification and functional gene research, especially in the application of high-efficiency microbial cell factories.

Method used

The cells were resuspended in LiAc-DTT solution and treated with 1.25 mL of 1 mol/L DTT. The electroporation parameters were optimized to be 1.0 kV, 25 µF, and 1000 Ω. The cells were then revived and cultured in sorbitol and YPD medium to synergistically weaken the cell wall and increase membrane fluidity, thereby reducing the electroporation threshold and improving DNA entry efficiency.

Benefits of technology

It significantly improves electroconversion efficiency, enabling efficient conversion of large-fragment homologous recombination expression vectors, ensuring cell survival, solving the problem of low efficiency in polyploid homologous recombination, and meeting the industrial demand for high-throughput gene editing.

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Abstract

The invention provides a method for improving the electric transformation efficiency of candida utilis, and belongs to the technical field of microbial genetic engineering. The method comprises two steps of competent cell preparation and electric shock transformation: resuspending candida utilis yeast cultured to a logarithmic phase by using 45-50 mL of a LiAc-DTT solution, then adding 1.25 mL of 1 mol / L DTT, carrying out shake cultivation for 30 minutes at 28 DEG C and 200 rpm, and washing to obtain competent cells; the method comprises the following steps: uniformly mixing plasmids with linear concentration of 1-1.2 g with competent cells, carrying out ice bath treatment, carrying out electric shock under the parameters of voltage of 1.0 kv, capacitance of 25F and resistance of 1000 omega, immediately adding 1mL of precooled sorbitol with the concentration of 1mol / L and 1mL of a YPD culture medium after the electric shock, carrying out resuscitation culture for 4-6 hours, and carrying out coating screening to obtain a transformant. The candida utilis genetic transformation method disclosed by the invention is high in transformation rate, stable in effect and capable of efficiently transforming large-fragment plasmids (gt; 9k bp) for homologous recombinant expression vectors.
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