A method for rapid screening of high-yield triglyceride-type polyunsaturated fatty acid filamentous fungi

Abstract

This invention relates to a method for rapidly screening filamentous fungi that produce high levels of triglycerides and polyunsaturated fatty acids, belonging to the field of microbial technology. The method employs a two-stage screening strategy of "resistance primary screening – fluorescence secondary screening." In the primary screening stage, strains with strong lipid synthesis capabilities are initially screened by adding a combination of phytocyanin and quizalofop-p-ethyl inhibitors to the culture medium. In the secondary screening stage, cellophane culture technology and Nile red fluorescence staining are combined to quantitatively assess the triglyceride content of the colonies, achieving rapid and non-destructive screening. This method is simple to operate, highly efficient, and shows a significant positive correlation between fluorescence intensity and triglyceride content. It is suitable for mutagenesis breeding and industrial strain selection of oil-producing filamentous fungi such as *Morchella alpineensis*.

Description

Technical Field

[0001] This invention relates to a method for rapidly screening filamentous fungi that produce high levels of triglycerides and polyunsaturated fatty acids, belonging to the field of microbial technology. Background Technology

[0002] Polyunsaturated fatty acids (PUFAs), as important functional lipids, cannot meet the growing market demand through traditional sources. Microbial fermentation for PUFA production has become a research hotspot due to its short cycle, low cost, and high sustainability. Among these, oleophobic filamentous fungi have attracted widespread attention for their efficient accumulation of triglyceride (TAG)-type PUFAs. In oleophobic filamentous fungi, PUFAs are mainly stored in lipid droplets in the form of TAGs. Unlike phospholipids, TAG-type PUFAs are the primary target form for industrial extraction and utilization, and their content directly determines the production value of the strain and the efficiency of downstream extraction.

[0003] Obtaining high-yielding strains through mutagenesis breeding is an effective strategy for increasing microbial lipid production, but its success highly depends on efficient high-throughput screening methods. Currently, rapidly and accurately screening strains with significantly enhanced lipid synthesis capabilities from mutagenized oil-producing microbial libraries still faces significant technical bottlenecks. On the one hand, traditional primary screening methods rely on colony morphology observation, which cannot apply effective lipid synthesis pressure, leading to high screening blindness and low throughput. While conventional Nile Red fluorescence staining can indicate lipid content, direct staining on solid culture media results in high background fluorescence and uneven staining. Furthermore, traditional methods are destructive, making it impossible to continuously track or subsequently validate the same colony, hindering non-destructive, quantitative assessment at the colony level. On the other hand, more precise detection methods, such as gas chromatography-mass spectrometry (GC-MS), while accurate, are cumbersome, costly, and time-consuming, failing to meet the need for high-throughput screening of thousands or even tens of thousands of mutant strains generated by mutagenesis.

[0004] Therefore, there is an urgent need in this field to develop a novel screening method that can simultaneously assess the growth and lipid production capacity of strains at the plate stage, and is high-throughput, non-destructive, quantitative, and easy to operate, so as to overcome the technical bottleneck in the breeding of high triglyceride-producing polyunsaturated fatty acid strains. Summary of the Invention

[0005] [Technical Issues] The purpose of this invention is to provide a culture medium and its application method for rapidly screening oleoresin-producing filamentous fungal mutant strains, aiming to rapidly and accurately screen for oleoresin-producing filamentous fungi with high triglyceride production, and to address the technical challenge of efficiently screening strains with significantly enhanced lipid synthesis capabilities from a large mutant library.

[0006] [Technical Solution] This invention provides a rapid screening method for filamentous fungal mutant strains that produce high levels of triglycerides and polyunsaturated fatty acids. The method employs a two-stage screening process of "resistance primary screening - fluorescence secondary screening," and is particularly suitable for *Morchella alpineensis* strains that have undergone physical or chemical mutagenesis treatment. Mortierella alpina Highly efficient screening of oil-producing filamentous fungi such as ( ).

[0007] The initial screening stage employs an resistance screening method. The mutagenized spore suspension is spread onto a primary screening solid medium lined with sterile cellophane and cultured. The primary screening medium is PDA medium supplemented with specific lipid synthesis inhibitors, including but not limited to phytocyanin (a fatty acid synthase inhibitor) and quizalofop-p-ethyl (an acetyl-CoA carboxylase inhibitor), with a preferred final concentration of 10 g / L. -8 mol / L and 10 -6 mol / L. This inhibitor combination can effectively inhibit the growth of strains with weak lipid synthesis capabilities, thereby initially screening out strains with good growth and high lipid production potential.

[0008] The secondary screening stage employs Nile Red fluorescence staining. Colonies grown on the initial screening medium, along with the attached cellophane, are removed and sequentially fixed, permeabilized, stained, and destained. Specifically, the treatment steps include: fixing the bacterial morphology with paraformaldehyde, permeating the cell wall with Triton solution, staining the intracellular triglycerides with Nile Red solution in the dark, and finally destaining with isopropanol to remove background fluorescence. After these treatments, the fluorescence intensity of each colony is detected using an inverted fluorescence microscope at an excitation wavelength of 543 nm. Fluorescence intensity is positively correlated with intracellular triglyceride content, allowing for rapid and quantitative assessment of the strain's ability to accumulate triglyceride-type polyunsaturated fatty acids, and screening for target colonies with high fluorescence intensity.

[0009] In one embodiment of the present invention, the method further includes a step of performing ARTP mutagenesis on the starting strain to increase genetic diversity. The working gas for the ARTP mutagenesis is helium, the power is 100 W, the processing distance is 2 mm, the gas flow rate is 10 L / min, the mutagenesis time is 90 s, and the plasma jet temperature is controlled within the range of 25-35°C.

[0010] In one embodiment of the present invention, the preparation method of the primary screening culture medium is as follows: based on PDA culture medium, after high-temperature autoclaving at 115-120℃ for 20-30 min, phytocyanin and quizalofop-p-ethyl are immediately added to the required final concentration through filtration sterilization. To maintain the inhibitor activity, the prepared culture medium should be stored in the dark and used within 2 weeks.

[0011] In one embodiment of the present invention, the cellophane is moistened before use, sandwiched between filter papers, and sterilized at 115-120°C for 20-30 minutes. The use of this cellophane facilitates the complete transfer of bacterial colonies and subsequent batch staining operations.

[0012] In one embodiment of the present invention, the method further includes verifying the high fluorescence intensity strains obtained by secondary screening through shake-flask fermentation, and analyzing their biomass, triglyceride content and fatty acid composition by GC-MS, so as to finally confirm the improvement in the production capacity of triglyceride-type polyunsaturated fatty acids by the screened strains.

[0013] In one embodiment of the present invention, the fixation refers to placing the cultured cellophane in paraformaldehyde for at least 30 minutes.

[0014] In one embodiment of the present invention, the permeability refers to being placed in a 1% Triton solution for at least 5 minutes.

[0015] In one embodiment of the present invention, the staining refers to staining in a light-protected solution of at least 1 mg / L Nile Red for at least 15 min.

[0016] In one embodiment of the present invention, the decolorization refers to decolorization in at least 60% w / v isopropanol.

[0017] [Beneficial Effects] The high-throughput screening method constructed in this invention has many significant advantages, mainly reflected in: (1) combining "cellophane culture-transfer technology" with Nile red staining to achieve non-destructive, complete, and quantitative assessment of triglyceride content in solid plate colonies; (2) designing a two-stage screening of "resistance primary screening-fluorescence secondary screening" to greatly improve the directionality and efficiency of screening. By organically combining resistance primary screening with Nile red fluorescence secondary screening, rapid and accurate screening of high triglyceride-producing polyunsaturated fatty acid strains is achieved. This method uses a combination of light blue bromide and quizalofop-p-ethyl as a primary screening agent, which can effectively inhibit the growth of strains with weak lipid synthesis ability, thereby efficiently enriching potential high-yielding strains in the early stage, greatly improving the targeting and efficiency of screening. In the secondary screening stage, the innovative combination of cellophane culture and Nile red staining technology is used to successfully overcome the technical problem of difficult complete transfer and uniform staining of colonies on traditional solid culture media, and achieve non-destructive and quantitative assessment of lipid content at the colony level. By optimizing staining conditions and fluorescence detection parameters, a high positive correlation between fluorescence signal and intracellular triglyceride content was ensured. Furthermore, to verify and establish this quantitative relationship, this study obtained cell samples at different lipid accumulation stages through liquid shake-flask fermentation and systematically constructed a standard curve between Nile Red fluorescence intensity and total lipid content measured by GC-MS. Experiments confirmed a very strong linear positive correlation between the two (coefficient of determination R² > 0.96).

[0018] Compared to traditional methods, this method applies directional selection through resistance pressure, achieving high screening accuracy while enabling non-destructive testing. It allows for subsequent validation of the same colony, and produces more uniform staining with lower background interference. Compared to direct GC-MS analysis, this method offers high throughput, low cost, and high speed, making it suitable for screening the massive mutant libraries generated through mutagenesis breeding.

[0019] Furthermore, this method can be efficiently integrated with mutagenesis techniques such as ARTP, providing strong technical support for creating genetically diverse mutant strain libraries and rapidly selecting target strains from them. Compared to traditional methods such as oil extraction and weighing, and chromatographic analysis, this method significantly reduces the time and material costs for single-colony screening while maintaining high accuracy, achieving high-throughput operation.

[0020] This method is not only applicable to alpine spores, but can also be extended to the screening of other oil-producing filamentous fungi or yeasts. It provides an efficient, economical and reliable general solution for microbial oil breeding, especially for the selection of high-triglyceride-type polyunsaturated fatty acid industrial strains, and has broad application prospects. Attached Figure Description

[0021] Figure 1 Standard curve of Nile red fluorescence intensity of *Morchella alpineensis* and triglyceride fatty acid content.

[0022] Figure 2 M.alpina Biomass and lipid composition analysis of ATCC32222 after ARTP mutagenesis; A: Fatty acid composition of the starting strain and the mutant strain; B: Biomass, total lipids, and ARA content of the starting strain and mutant strain.

[0023] Figure 3 Images of similar locations of nine different bacterial colonies obtained from mixed bacterial culture under the same parameters.

[0024] Figure 49 different bacterial colonies are respectively compared with M.alpina ATCC32222(A), M.alpina Analysis of the difference in red fluorescence intensity of LS-2(B) (P<0.0001) (ns represent no significant difference).

[0025] Figure 59 shows the analysis of the differences in red fluorescence intensity among different colonies (P<0.05) (different letters represent significant differences). Detailed Implementation

[0026] The initial screening medium consisted of (1 L): 200 g peeled potatoes, 20 g glucose, 15-20 g agar, and the remainder was water (PDA medium). After autoclaving at 115℃ for 20 min, phytocyanin and quizalofop-p-ethyl were immediately added through a 0.22 μm aqueous filter to a final concentration of 10 g / L. -8 mol / L and 10 -6 The concentration is mol / L, which completes the preparation of the primary screening medium. To ensure the effectiveness of the inhibitor, the primary screening medium must be stored in the dark, especially avoiding ultraviolet light, and used within 2 weeks.

[0027] The cellophane preparation method is as follows: cut the cellophane into appropriate sizes, wet it with water, sandwich it alternately between filter paper, and then place them together in a petri dish. Sterilize at 115℃ for 20 minutes. Prepare primary screening plates using aseptic technique. After cooling, use sterile tweezers to pick up sterile cellophane and attach it to the surface of the plate.

[0028] The preparation method of spore suspension is as follows: Rinse the *Morchella alpineensis* spore solution preserved on GY solid slant with 6-8 mL of 0.9% physiological saline, filter through a 45 μm filter membrane to allow spores to pass through while retaining hyphae, centrifuge at 9000 rpm for 20-30 min, discard the supernatant, reconstitute with 30% sterile glycerol, and mix thoroughly in a vortex mixer. The final spore concentration should be at least 10. 6 The spores were dispensed at a concentration of 1 / mL and stored at -80℃ for later use. The spore solution could be a starting strain of *Morchella alpineensis* or a mixed spore solution after ARTP mutagenesis.

[0029] The conditions for ARTP mutagenesis treatment were as follows: 10 μL of fresh spore suspension (concentration 10) was taken. 6 Plasma particles (per mL) were placed on a sterile metal slide and mutagenized using an ARTP mutagen. Helium was used as the working gas, with a power of 100 W, a processing distance of 2 mm, a gas flow rate of 10 L / min, a mutagenization time of 90 s, and the plasma jet temperature controlled within the range of 25-35℃.

[0030] The specific steps for primary screening and secondary screening are as follows: (1) Take 100 μL of the mutagenized spore suspension and spread it on a primary screening solid culture medium lined with cellophane. Incubate at 28°C in the dark for 48 h. (2) After the culture is completed, select the top 40% of single colonies by diameter and number them. Remove the cellophane from the culture medium and fix it in a petri dish containing about 8 mL of paraformaldehyde for 30 min; (3) After the fixed cellophane is removed, it is placed in 1% Triton solution for 5 min to permeate the cell wall, and then stained in 1 mg / L Nile red staining solution for 15 min in the dark. (4) After staining, decolorize with 60% isopropanol. Prepare slides for each colony according to its number, and measure the fluorescence intensity using an inverted fluorescence microscope (excitation wavelength 543 nm, magnification 20x, exposure time 4 s). Select a region for each colony using the five-point method to ensure the accuracy of the data. (5) Screen colonies with high fluorescence intensity, number them and retain them for subsequent GC-MS validation analysis.

[0031] Method validation: The high-fluorescence-intensity strains obtained from the secondary screening were subjected to shake-flask fermentation. Biomass, total lipid content, and fatty acid composition were determined according to the aforementioned GC-MS analysis method to verify the correlation between fluorescence intensity and triglyceride-type polyunsaturated fatty acid content. To further solidify the quantitative basis of this method, a standard curve of Nile Red fluorescence intensity versus lipid content was systematically constructed. Specifically, a series of bacterial samples with broad-spectrum lipid content were obtained through liquid fermentation. Standardized Nile Red staining fluorescence measurement and precise GC-MS quantification were performed on parallel samples, and then a quantitative relationship model between the two was established using linear regression analysis. Figure 1 This standard curve can be used to quantify the fluorescence intensity of colonies on primary screening plates, thereby more accurately predicting and screening high-lipid-producing strains.

[0032] Regarding the optimization and universality of the method: In constructing the above quantitative model, in-depth exploration and optimization were conducted on technical details such as the dense mycelium of filamentous fungi, the easy loss of fungal powder during post-staining processing, and the difficulty in controlling the uniformity of slide preparation. This practical experience further enriched and improved the technical details of this method, ensuring its effectiveness in dealing with different microbial morphologies. Although there is room for optimization in the absolute quantification of extremely fine fungal powder, this does not affect the core advantages and industrial application value of the strategy described in this invention in achieving rapid, non-destructive, and quantitative comparison and screening at the plate colony level.

[0033] The strains involved in the following examples M. alpina LS-2, M. alpina TSM-3-1 has been published in the literature "Mutation Breeding Study of High-Yielding Arachidonic Acid Mold".

[0034] Example 1: High-throughput screening of strains producing high levels of triglycerides and polyunsaturated fatty acids (1) Strains and mutagenesis treatment: Select M.alpina Using ATCC32222 as the starting strain, a spore suspension was prepared. 10 μL of fresh spore suspension (concentration 10) was taken. 6 (Spores / mL) were placed on a sterile metal slide and mutagenized using an ARTP mutagenizer. The working gas was helium, the power was set to 100 W, the treatment distance was 2 mm, the gas flow rate was 10 L / min, the mutagenization time was 90 s, and the plasma jet temperature was controlled within the range of 25–35 °C. After mutagenization, the spores were eluted and serially diluted, and stored at -80 °C for later use.

[0035] (2) Initial screening: 100 μL of the mutagenized spore suspension was spread onto a primary screening solid culture medium lined with cellophane and incubated at 28°C in the dark for 48 h. The primary screening culture medium consisted of (1 L): 200 g peeled potato, 20 g glucose, 15–20 g agar, and the remainder water. Immediately after sterilization, phytocyanin and quizalofop-p-ethyl were added to a final concentration of 10 g / L. -8 mol / L and 10 - 6 mol / L.

[0036] (3) Secondary screening: After incubation, the top 40% of single colonies by diameter were selected and numbered. Cellophane was removed, and the colonies were fixed in a petri dish containing 8 mL of paraformaldehyde for 30 min. They were then permeated with 1% Triton solution for 5 min, followed by staining in 1 mg / L Nile red solution in the dark for 15 min. After staining, the colonies were destained with 60% isopropanol. The fluorescence intensity of each colony was measured using an inverted fluorescence microscope (excitation wavelength 543 nm, magnification 20x, exposure time 4 s). Each colony was photographed using a five-point method. The fluorescence intensity represented the lipid content of the colony. By comparing the fluorescence intensity with that of the starting strain (standard sample), colonies with lipid contents close to or higher than the standard sample could be effectively screened.

[0037] (4) Verification analysis: The strains obtained from the secondary screening were subjected to shake-flask fermentation. Specifically, the selected colonies were dispersed and inoculated into 100 mL of Broth fermentation medium (Broth fermentation medium (1L): 30.0 g glucose, 5.0 g yeast extract, 10.0 g potassium nitrate, 0.25 g magnesium sulfate heptahydrate, 1.0 g potassium dihydrogen phosphate). The cultures were incubated at 28°C and 200 rpm for 48 h. The biomass, total lipid content, and fatty acid composition were then analyzed by GC-MS.

[0038] A standard curve was prepared using the fluorescence intensity of the screened colonies and the total lipid content after validation. The results are as follows: Figure 1 As shown, fluorescence intensity is significantly positively correlated with total lipid content (R0). 2 >0.85). Figure 2 The results showed that the ARA content in the selected strains was close to that of the starting strain.

[0039] Therefore, this method can efficiently and accurately screen target strains with excellent triglyceride-type polyunsaturated fatty acid synthesis ability from a large number of mutagenic strains, and is suitable for industrial strain breeding.

[0040] Example 2 Assessment of lipid accumulation in mixed bacterial culture (1) Strains and experimental design: Select Mortierella alpina ATCC32222 M. alpina LS-2, M. alpina Three different subtypes of TSM-3-1 strains were used to prepare spore suspensions (using the same method as in Example 1), and the following two sets of experiments were set up: ① Mixed culture group: Equal volumes of spore suspensions of the three strains were mixed to prepare a mixed spore suspension; ② Individual culture group: The three strains were individually plated on different plates.

[0041] (2) Initial screening and culture: Take 100 μL of the mixed spore suspension and each single spore suspension respectively, spread them on the primary screening solid culture medium (prepared as in Example 1) with cellophane attached, and incubate at 28°C in the dark for 48 h.

[0042] (3) Secondary screening and fluorescence determination: After incubation, the top 40% of single colonies by diameter in each group were selected and numbered. Cellophane, along with the colonies, was removed from the culture medium and subjected to fixation, permeation, staining, and destaining in sequence. The fluorescence intensity of each colony was then measured using an inverted fluorescence microscope. Each colony was photographed using a five-point method (the procedures were the same as in Example 1). Nine colonies were selected for further analysis and named H1 to H9.

[0043] (4) Results analysis: By comparing the fluorescence intensity of each strain in the mixed culture group and the single culture group ( Figure 4 The study found that under mixed culture conditions, the fluorescence intensity of some strains was significantly higher than that of their individual strains (P<0.05), indicating that mixed growth may promote lipid accumulation; the fluorescence intensity of other strains decreased slightly in mixed culture, suggesting possible nutrient competition or metabolic inhibition.

[0044] like Figure 3 , 5 As shown, this result indicates that different subtypes of *Morchella alpineensis* exhibit different lipid accumulation behaviors in a mixed culture environment, and this method can effectively identify and assess such population effects.

[0045] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for rapidly screening filamentous fungi that produce high levels of triglycerides and polyunsaturated fatty acids, characterized in that, Includes the following steps: (1) Initial screening: Spread the suspension of filamentous fungi to be tested on the initial screening solid culture medium with cellophane attached, and incubate at 27-29℃ in the dark for 40-48 h to screen out strains with good growth and normal colony morphology. (2) Secondary screening: Take off the cellophane after cultivation in step (1), and perform paraformaldehyde fixation, Triton permeation treatment, Nile red staining, and isopropanol decolorization in sequence. Then, measure the fluorescence intensity of each colony and screen colonies with high fluorescence intensity.

2. The method according to claim 1, characterized in that, The filamentous fungi to be tested include filamentous fungi obtained through natural selection or mutagenesis; the filamentous fungi include alpine spores of *Morchella esculenta*.

3. The method according to claim 1, characterized in that, In step (1), the composition of the initial screening solid culture medium is: 180-200 g / L peeled potato, 19-20 g / L glucose, 15-20 g / L agar, 1-3 × 10 -8 mol / L cyanobacterium, 1-3×10 -6 mol / L quizalofop-P-ethyl, with the remainder being water.

4. The method according to claim 3, characterized in that, The method for preparing the primary screening solid culture medium is as follows: Sterilize the culture medium containing 180-200 g / L peeled potato, 19-20 g / L glucose, 15-20 g / L agar, and the remainder water at 115-120℃ for 20-30 min, then immediately add chlorpyrifos and quizalofop-p-ethyl to a final concentration of 1-3 × 10⁻⁶ g / L. -8 mol / L, 1-3×10 -6 mol / L.

5. The method according to claim 1, characterized in that, Step (2) refers to fixing the paraformaldehyde, which means placing the cultured cellophane in paraformaldehyde for at least 30 minutes.

6. The method according to claim 1, characterized in that, The permeation treatment in step (2) refers to placing the solution in a 1% Triton solution for at least 5 minutes.

7. The method according to claim 1, characterized in that, The staining in step (2) refers to staining in a light-protected solution of at least 1 mg / L Nile Red for at least 15 min.

8. The method according to claim 1, characterized in that, The decolorization in step (2) refers to decolorization in at least 60% w / v isopropanol.

9. The method according to claim 1, characterized in that, Step (2) Measure the fluorescence intensity of each colony using an inverted fluorescence microscope.

10. The method according to claim 1, characterized in that, The strains obtained from the secondary screening were subjected to shake-flask fermentation, and the biomass, triglyceride content, and fatty acid composition of the strains were verified by GC-MS.

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