Method for improving electric transformation efficiency of candida utilis

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.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, and in particular relates to a method for improving the electroconversion efficiency of Candida utilis. Background Technology

[0002] Candida utilis is a yeast strain that can be used as a food additive. It is rich in nutrients such as protein, nucleotides, enzymes, B vitamins, and oligosaccharides. Its diverse amino acid profile and protein content (approximately 50% of stem cell weight) make it an ideal protein supplement for animal feed. Candida utilis is a Clerbüchri-negative yeast that does not produce ethanol under aerobic conditions, exhibits high respiration capacity, and can be cultured at high density under efficient continuous culture conditions.

[0003] Candida utilis is a promising expression host capable of producing relatively high levels of recombinant products. Yeast is easily manipulated genetically, and strains can be optimized for specific protein expression. Candida utilis is safe, non-toxic, and easy to cultivate, making it a valuable microbial cell factory for high-value recombinant products. In recent years, Candida utilis genetic engineering technology has been used to express various important recombinant products, such as vitamins, carotenoids, proteins, glutathione, polysaccharides, and ribonucleic acid.

[0004] Currently, although electroconversion technology is widely used in the field of yeast, the electroconversion efficiency for Candida utilis is still at a low level, which seriously limits the progress of its metabolic engineering and functional gene research.

[0005] The cell wall of *Candida utilis* is composed of glucan, mannan, and protein, exhibiting high thickness and density. Its cell membrane has a more tightly packed phospholipid bilayer, resulting in stable electrical properties and a higher electric field threshold required for electroporation, making it difficult to form effective pores under conventional electroporation parameters. Furthermore, *Candida utilis* is a polyploid strain, inherently with low homologous recombination efficiency; this, coupled with insufficient electroporation efficiency, further exacerbates the difficulty of stable integration of exogenous genes.

[0006] Electroconversion, a core gene transfer technology in yeast genetic engineering, works by creating transient hydrophilic pores in the cell membrane using an external electric field, enabling efficient entry of exogenous DNA. It offers advantages such as ease of operation, no vector dependence, and wide applicability, and has become a routine transformation method for model yeasts like *Saccharomyces cerevisiae* and *Pichia pastoris*. Compared to chemical transformation methods (such as the LiAc / PEG method), electroconversion effectively overcomes the dual barriers of the yeast cell wall and cell membrane, making it particularly suitable for integrative vector-mediated homologous recombination, thus providing a technological foundation for the genetic modification of *Candida utilis*.

[0007] With the growing demand for efficient microbial cell factories in the biotechnology industry, *Candida utilis*' unique advantages make it an ideal host to replace *Saccharomyces cerevisiae* and *Pichia pastoris*. In the food industry, it can be used for the efficient production of functional peptides and probiotic preparations; in the pharmaceutical field, it can serve as a recombinant protein expression system; and in the bioenergy field, its efficient utilization of carbon sources provides a new pathway for the production of biofuels such as cellulosic ethanol. However, low electroconversion efficiency has become a core technological barrier restricting the implementation of these applications. Existing technologies cannot meet the industrial demands for large-scale strain screening, rapid metabolic engineering, and high-throughput gene editing, resulting in the application potential of *Candida utilis* not being fully realized.

[0008] Therefore, developing an efficient electroconversion method targeting the characteristics of Candida utilis, overcoming the dual barriers of its cell wall and cell membrane, and balancing conversion efficiency and cell survival rate has become an urgent technical problem to be solved in the field of biotechnology. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide a method for improving the electroconversion efficiency of Candida utilis, which has a high conversion rate, stable effect, and can efficiently convert large fragment plasmids (>9k bp) used for homologous recombination expression vectors.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for improving the electroconversion efficiency of Candida utilis includes the preparation of competent cells and electroconversion: (1) Preparation of competent cells: Candida utilis cells cultured to the logarithmic phase were resuspended in 45-50 mL of LiAc-DTT solution, and then 1.25 mL of 1 mol / L DTT was added. The cells were cultured in a shaker at 28 °C and 200 rpm for 30 min. After washing, competent cells were obtained. (2) Electroporation transformation: The linearized plasmid with a concentration of 1-1.2 µg was mixed with the competent cells and treated with ice bath. Electroporation was performed under the parameters of voltage 1.0 kV, capacitance 25 µF and resistance 1000 Ω. Immediately after electroporation, 1 mL of pre-cooled 1 mol / L sorbitol and 1 mL of YPD medium were added. The cells were then revived and cultured for 4-6 h. Transformants were obtained by plating and screening.

[0011] Preferably, in step (1), *Candida utilis* is cultured at 28°C until OD... 600 Collect bacterial cells when the temperature is 2.1-2.5℃.

[0012] Preferably, in step (1), the centrifugation conditions for collecting bacterial cells are 4°C, 5000g for 5 minutes, and repeated 3 times.

[0013] Preferably, in step (1), the reagent used to wash the bacterial cells is pre-cooled 1 mol / L sorbitol.

[0014] Preferably, in step (2), the plasmid is mixed with competent cells and then pre-cooled on ice for 5 minutes.

[0015] Preferably, in step (2), the conditions for resuscitation culture are 28°C and 200 rpm in a shaker.

[0016] Preferably, in step (2), the bacterial solution after resuscitation culture is centrifuged at 8000 rpm for 3 min, and 100 µL of bacterial solution is retained for coating.

[0017] Preferably, in step (2), the culture medium used for coating is YPD solid culture medium containing actinomycete ketone, the screening culture temperature is 28℃, and the culture time is 3-5 days.

[0018] Preferably, the preservation number of the *Candida utilis* is CICC31395.

[0019] Preferably, the plasmid is a pWG expression vector with the base sequence shown in SEQ ID No. 2.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention addresses the characteristics of Candida utilis cell walls, which are thick and densely packed with phospholipid bilayers in the cell membrane. It employs a combined approach of "LiAc-DTT resuspension + 1.25 mL 1 mol / L DTT treatment" to synergistically weaken the cross-linking structure of cell wall dextran and mannan, while simultaneously increasing cell membrane fluidity. This effectively lowers the electric field threshold required for electroporation, solving the problem of conventional electroporation methods failing to form effective hydrophilic pores and removing physical barriers to the efficient entry of exogenous DNA into cells.

[0021] This invention achieves a significant improvement in electroconversion efficiency through multi-dimensional parameter optimization, with strong reproducibility and high stability in experimental results. Furthermore, this method can efficiently convert large-fragment homologous recombination expression vectors (such as pWG expression vectors) with a length >9kbp, overcoming the limitations of existing technologies on plasmid fragment size. It solves the problem of stable integration of exogenous genes caused by the combined effects of low homologous recombination efficiency and insufficient electroconversion efficiency in Candida utilis polyploids, providing technical support for research on complex functional gene introduction and metabolic pathway reconstruction.

[0022] The optimized electroporation parameters and subsequent resuscitation medium of this invention create a synergistic effect. Sorbitol maintains cell osmotic pressure and reduces cell rupture after electroporation, while YPD medium provides sufficient nutrition for rapid cell recovery, significantly reducing cell damage from electroporation and ensuring cell viability while improving transformation efficiency. Combined with screening using YPD solid medium containing actinomycin, positive transformants can be efficiently enriched, further improving the success rate of exogenous gene integration. Compared to traditional chemical transformation methods, this invention better meets the needs of homologous recombination-mediated gene modification. Furthermore, this invention requires no complex equipment or special reagents; competent cells are prepared and used immediately, the transformation cycle is short, and strain construction can be achieved rapidly. Attached Figure Description

[0023] Figure 1 A schematic diagram of the construction of the WG expression vector; Figure 2 It is a recombinant Candida utilis strain; Figure 3 PCR identification of recombinant yeast genome. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1 1. Reagents, plasmids, and bacterial strains: (1) LiAc-DTT solution (1L): 0.1M LiAC, 0.01M Tris-HCl, 0.6M sucrose, 0.01M DTT; (2) YPD medium (1L): 10g yeast extract, 20g peptone, 20g glucose; (3) Construction of exogenous gene: pWG expression vector From differentially expressed genes related to secretion and folding, a protein similar to *Saccharomyces paradoxusflavodoxin*-like fold family protein (sequence number 1811) and a protein similar to NRRL Y-1542 heat shock protein 60 (sequence number 1036) were identified. Genes 1811 and 1036 were linked using the linker peptide RL, and the resulting WG sequence was synthesized by Nanjing GenScript Biotech Co., Ltd., as shown in SEQ ID No. 1.

[0026] The pGZM18-EGFP expression vector was digested with enzymes to replace the EGFP protein with the WG sequence, constructing the pWG expression vector, the base sequence of which is shown in SEQ ID No. 2. The WG sequence obtained from gel extraction was then recombinated with the linearized pGZM18-EGFP expression vector at a specific molar ratio. The mixture was gently mixed and incubated at 50°C for 15 min. The recombinant product was cooled on ice and then used for E. coli transformation experiments. The procedure was performed according to the Trans1-T1 E. coli competent cell instructions.

[0027] (4) Candida utilis: purchased from China Industrial Microbial Culture Collection Center, accession number CICC31395.

[0028] 2. Preparation of competent cells of Candida utilis: (1) Inoculate 50µL of Candida utilis stored at -80℃ into 5mL of YPD and incubate overnight at 28℃ and 200rpm.

[0029] (2) Transfer the overnight cultured yeast to liquid YPD medium and incubate at 28°C until OD. 600 =2.1-2.5.

[0030] (3) Transfer the cultured yeast culture to a pre-chilled 50 mL centrifuge tube and pre-chill on ice for 10 min. Centrifuge at 5000 g for 5 min at 4 °C and collect the cells. Repeat the collection 3 times.

[0031] (4) Discard the supernatant and keep the bacterial cells. Resuspend the bacterial cells in 45-50 mL of LiAc-DTT solution, then add 1.25 mL of 1 mol / L DTT. Incubate at 28℃, 200 rpm, and 30 min on a shaker. Centrifuge and discard the supernatant. Repeat this process 3 times.

[0032] (5) Resuspend the collected bacterial cells in 2.5 mL of pre-cooled 1 mol / L sorbitol, dispense 80 µL into each tube, and use immediately.

[0033] 3. Electroconversion of Candida utilis: (1) The linearization concentration of exogenous plasmid WG was 1.2 µg. The gel was recovered, and the exogenous gene was thoroughly mixed with the prepared yeast competent cells and transferred to a pre-cooled electric transfer cup. The mixture was then placed on ice for 5 min to pre-cool.

[0034] (2) Place the electroporation cup on the electroporation apparatus and set the electroporation parameters: 1.0 kV, 25 µF, 1000 Ω. After one electroporation, quickly add 1 mL of pre-cooled 1 mol / L sorbitol and 1 mL of YPD medium to the electroporation cup.

[0035] (3) Transfer the electroporation solution to a 2mL centrifuge tube and incubate for 4-6 hours at 28℃ and 200rpm on a shaker.

[0036] (4) Centrifuge the revived bacterial solution at 8000 rpm for 3 min, discard the supernatant, keep 100 µL of liquid, spread it on YPD solid medium containing actinomycete ketone, and incubate at 28℃ for 3-5 days.

[0037] 4. Results (1) Construction of expression carrier The pGZM18-EGFP expression vector was digested with enzymes to replace the EGFP protein with the WG sequence, constructing the pWG expression vector with a length of 11397 bp. (See schematic diagram). Figure 1 .

[0038] (2) C. utilis Identification of recombinant bacteria The prokaryotic expression cassette of the WG co-expression vector was removed using overlap extension PCR. The WG co-expression recombinant plasmid was then digested with ApaI. The digestion products were integrated into the recombinant *Candida utilis* genome to construct the WG yeast recombinant strain. The colony morphology of the recombinant *Candida utilis* strain is shown in the figure below. Figure 2 As shown.

[0039] The genome of the recombinant yeast strain was extracted, and the target gene sequence of the pWG recombinant yeast strain was identified by PCR and sequenced using primers WGS / WGAS. The results are as follows: Figure 3 The PCR results showed that the WG exogenous gene had been successfully integrated into the chromosome of Candida utilis.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the electroconversion efficiency of Candida utilis, characterized in that, This includes the preparation of competent cells and electroporation transformation: (1) Preparation of competent cells: Candida utilis cells cultured to the logarithmic phase were resuspended in 45-50 mL of LiAc-DTT solution, and then 1.25 mL of 1 mol / L DTT was added. The cells were cultured in a shaker at 28 °C and 200 rpm for 30 min. After washing, competent cells were obtained. (2) Electroporation transformation: The linearized plasmid with a concentration of 1-1.2 µg was mixed with the competent cells and treated with ice bath. Electroporation was performed under the parameters of voltage 1.0 kV, capacitance 25 µF and resistance 1000 Ω. Immediately after electroporation, 1 mL of pre-cooled 1 mol / L sorbitol and 1 mL of YPD medium were added. The cells were then revived and cultured for 4-6 h. Transformants were obtained by plating and screening.

2. The method according to claim 1, characterized in that, In step (1), *Candida utilis* was cultured at 28°C until OD500. 600 Collect bacterial cells when the temperature is 2.1-2.5℃.

3. The method according to claim 2, characterized in that, In step (1), the centrifugation conditions for collecting bacterial cells are 4℃, 5000g for 5min, and repeated 3 times.

4. The method according to claim 1, characterized in that, In step (1), the reagent used to wash the bacterial cells is pre-cooled 1 mol / L sorbitol.

5. The method according to claim 1, characterized in that, In step (2), the plasmid is mixed with competent cells and then pre-cooled on ice for 5 minutes.

6. The method according to claim 1, characterized in that, In step (2), the conditions for resuscitation culture are 28℃ and 200rpm shaking culture.

7. The method according to claim 1, characterized in that, In step (2), the bacterial culture after resuscitation is centrifuged at 8000 rpm for 3 min, and 100 µL of bacterial culture is retained for coating.

8. The method according to claim 1, characterized in that, In step (2), the culture medium used for coating is YPD solid medium containing actinomycete ketone, the screening culture temperature is 28℃, and the culture time is 3-5 days.

9. The method according to claim 1, characterized in that, The preservation number of the *Candida utilis* is CICC31395.

10. The method according to claim 1, characterized in that, The plasmid is a pWG expression vector, and its base sequence is shown in SEQ ID No. 2.

Images