Method for high-throughput flow Raman sorting of high-content docosahexaenoic acid strain

Abstract

The invention provides a method for screening a high-content DHA (docosahexaenoic acid) strain at high throughput. According to the screening method, characteristic peaks related to the DHA content and characteristic peaks related to the saturated fatty acid grease content in cells are detected based on a high-flux flow-type Raman sorting technology. Wherein the characteristic peak related to the DHA content is a Raman peak position at 3008 cm <-1 >, and the characteristic peak related to the saturated fatty acid grease content is a Raman peak position at 2844 cm <-1 >. According to the method, a high-throughput flow-type Raman sorting (FlowRACS) technology is adopted, and on the basis of characteristic Raman peak positions related to the DHA content and a corresponding culture medium, firstly, on the basis of realizing accurate identification of the DHA content in the cells, the screening accuracy of the high-yield DHA cells is greatly improved; secondly, high-throughput screening of the high-yield DHA cells is achieved, the screening throughput reaches 300,000 cells per day, the time cost and the labor cost of screening of the high-yield DHA cells are reduced, the problems in the prior art are solved, and the method has important application value.

Description

Technical Field

[0001] This invention relates to the field of microbial breeding technology, and to a method for screening strains, specifically a method for high-throughput screening of strains with high docosahexaenoic acid content. Background Technology

[0002] Over the past few decades, microbial cell factories have provided products and innovations in the fields of chemistry, food, and materials. On the one hand, for many industrial strains, random mutation breeding and adaptive laboratory evolution (ALE) can improve strain performance, but these methods are inefficient, time-consuming, and require significant manpower. On the other hand, with the advancement of synthetic biology, a vast library of strains has been generated. Therefore, there is an urgent need to combine high-throughput screening methods to achieve rapid identification and isolation of mutant strains with ideal titers and yields.

[0003] Docosahexaenoic acid (DHA) is an important omega-3 polyunsaturated fatty acid with vital physiological functions, including promoting brain cell development, lowering blood lipids, protecting vision, anti-cancer properties, and enhancing immunity. It is widely used in infant formula and the pharmaceutical industry. Because Schizochytrium can efficiently accumulate DHA-rich lipids, these lipids have become a widely used industrial substitute for the polyunsaturated fatty acids in traditional marine fish oil. Meanwhile, to improve the DHA production capacity of Schizochytrium, many strategies, including mutant breeding, ALE (Alternating Lesion Extraction), and metabolic engineering, have been used to obtain large libraries of mutant strains. Against this backdrop, high-throughput screening of cells with high DHA content is imperative.

[0004] As is well known, standard lipid analysis methods involve gas chromatography / gas chromatography-mass spectrometry (GC / MS), a process that includes several time-consuming and labor-intensive steps, such as cell culture, biomass collection and quantification, lipid extraction, fatty acid methylation, and GC / MS analysis. Compared to mass spectrometry, non-invasive spectroscopic techniques, such as infrared and Raman spectroscopy, offer advantages in rapidly and directly identifying single-cell components because they do not require additional cell culture and preservation steps. Raman Activated Cell Sorting (RACS), in particular, has been developed as a label-free, culture-free, and non-invasive method that can simultaneously screen cellular metabolites and classify target cells, making it a promising high-throughput lipid composition analysis strategy.

[0005] Currently, there are few reports on the application of Raman spectroscopy in the detection of intracellular lipids in microorganisms. Samek et al. established a method for estimating lipid unsaturation by analyzing the Raman spectra of cells immobilized on plates using specific peak ratios, but their cell processing steps were cumbersome and required cell fixation, preventing subsequent separation (Sensors 2010, 10: 8635-8651). Invention patent CN103592283B establishes a method for rapid detection of microalgal energy production processes, which uses Raman analysis of cells, specifically targeting cells at 1056 cm⁻¹. -1 1075cm -1 1116cm -1 1260cm -1 1302cm -1 1441cm -1 1656cm -1 1735cm -1 2850-2930cm -1 3023cm -1 The comparison of lipid-related Raman peak positions enables the assessment of total intracellular lipid content. Currently, there are no reports on the accurate identification of specific fatty acid types, such as DHA. Summary of the Invention

[0006] To address the current limitations of high-throughput screening of high-DHA-content lipid cells in existing technologies, this invention provides a method for high-throughput screening of high-DHA-content bacterial strains. The method employs high-throughput flow cytometry Raman sorting (FlowRACS) technology, utilizing characteristic Raman peak positions related to DHA content to achieve high-throughput screening of high-DHA-producing cells. This reduces the time and labor costs associated with high-DHA-producing cell screening, solves the problems of existing technologies, and has significant application value.

[0007] The technical solution of this invention:

[0008] A method for high-throughput screening of DHA-rich bacterial strains, wherein the screening method is based on high-throughput flow cytometry Raman sorting technology to detect characteristic peaks related to intracellular DHA content and characteristic peaks related to saturated fatty acid lipid content. The characteristic peak related to DHA content is 3008 cm⁻¹. -1 The Raman peak position at [location missing], the characteristic peak related to the saturated fatty acid oil content is 2844 cm⁻¹. -1 The Raman peak position is shown. This application achieves high-throughput screening of high-DHA-producing cells by rapidly identifying intracellular DHA content.

[0009] The method for high-throughput screening of DHA-rich strains specifically includes the following steps:

[0010] (1) Cell culture: Cells from the cell bank to be screened were cultured in a basic culture medium for 12-24 hours. The basic culture medium consisted of 10-20 g / L glucose, 10-20 g / L yeast extract, and 10-20 g / L sea salt. The cells to be screened included wild-type cells or mutant cells. The bacterial strains were oil-rich microbial strains, specifically including Schizochytrium, Lystropheus, and oil-producing yeast strains. By using the basic culture medium, rapid accumulation of intracellular lipids was achieved, significantly increasing the signal intensity during Raman detection and improving the accuracy of the results.

[0011] (2) Raman spectra acquisition: Raman spectra of each cell were acquired using high-throughput flow cytometry Raman sorting technology, and 3008 cm⁻¹ of samples were obtained. -1 Raman peak intensity I 3008 and 2844cm -1 Raman peak intensity I at the location 2844 The excitation source wavelength is 532nm, the power range is 10mW-100mW, and the Raman spectroscopy acquisition time is 0.01 seconds-0.5 seconds.

[0012] (3) Screening strains: According to the formula M=I 3008 / (I 3008 +I 2844 Calculate the M value and set the screening range for M to obtain the screened strains. In the cells of the screened strains, the DHA content is greater than 65% of the total fatty acid content.

[0013] Preferably, step (3) includes two rounds of screening: the first round screens cells with an M value greater than or equal to 0.5, which are then placed in wells of a plate containing induction medium and induced at 25℃-30℃ for 12-24 hours; then the second round screens cells with an M value greater than or equal to 0.6, thus obtaining cells that produce high levels of DHA. Only one cell is placed in each well of the plate; the induction medium consists of 15-20 g / L glucose and 10-20 g / L sea salt. Using this induction medium further facilitates the rapid accumulation of intracellular lipids, improving the signal intensity and accuracy of Raman detection.

[0014] The beneficial effects of the present invention: Compared with the prior art, the present invention has the following advantages:

[0015] (1) This application clarifies the Raman peak positions related to DHA content and the Raman peak positions related to saturated fatty acid oil content, and provides a method for high-throughput screening of strains with high DHA content based on the aforementioned Raman peak positions.

[0016] (2) The screening method described in this application, combined with the aforementioned Raman peak position and corresponding culture medium, achieves accurate identification of intracellular DHA content, thereby significantly improving the accuracy of screening high-DHA-producing cells.

[0017] (3) The screening method described in this application has a screening throughput of 300,000 cells / day, which significantly improves screening efficiency and reduces the time and labor costs of cell screening. Attached Figure Description

[0018] Appendix Figure 1 This is a schematic diagram of the process for high-throughput screening of cells with high DHA content as described in this application;

[0019] Appendix Figure 2 Raman spectra of Schizochytrium cells and extracted oils (2a) and linear relationships of DHA content were identified (2b and 2c).

[0020] Appendix Figure 3 The image shows the gas chromatographic analysis of intracellular lipid fatty acids in the high-DHA Schizochytrium strain (E3) obtained in Example 2. Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments.

[0022] Example 1: DHA Raman signal detection

[0023] First, the inventors analyzed the Raman spectra of Schizochytrium cells and the extracted oil, as detailed below. Figure 2 a. By Figure 2 As can be seen from point a, the Raman spectral peaks obtained by direct Raman observation of Schizochytrium cells are basically consistent with the Raman spectral peaks of the extracted oil. The main spectral peaks observed in the oil include, for example, 1445 cm⁻¹. -1 1665cm -1 2844cm -1 and 3008cm -1 The peaks observed in situ during Raman spectroscopy of Schizochytrium cells all appeared. This indicates that the spectral peaks obtained using Raman spectroscopy can be used to describe the accumulation of lipids in microbial cells.

[0024] The applicant unexpectedly discovered that 3008cm was used. -1 The peak value of Raman Peak is 2844 cm. -1 Raman Peak and 3008cm -1 The ratio of the sum of Raman peak values ​​can accurately determine the DHA content in oils, solving the problem of high-throughput screening of high-DHA-content oil cells in existing technologies, which is of great significance. Figure 2 As shown in b, the traditional 1665 cm is adopted. -1 and 1445cm -1 Peak ratio method (I 1665 / I 1445When determining the DHA content in oils, this value showed a correlation of only 0.93 with the DHA content value obtained by the traditional gold standard method (gas chromatography-GC determination method). 2 =0.93). But when using I 3008 / (I 3008 +I 2844 When calculating the DHA content in oils using the ratio of α to β, the correlation between the ratio and the DHA content obtained by the traditional gold standard method is as high as 0.99 (R). 2 =0.99) Figure 2 c). Therefore, compared to the traditional gold standard method, this application uses I... 3008 / (I 3008 +I 2844 This method significantly improves the accuracy of DHA content determination in oils and fats, laying the foundation for high-throughput screening of oil cells with high DHA content.

[0025] Example 2: Screening for DHA-rich Schizochytrium cells using this method

[0026] Wild-type Schizochytrium cells were cultured for 12 h in a basic medium containing 20 g / L glucose, 20 g / L yeast extract and 15 g / L sea salt.

[0027] High-throughput flow cytometry Raman sorting (FlowRACS) was used to acquire Raman spectra for each cell. The Raman excitation source had a wavelength of 532 nm and a power range of 10 mW. The Raman spectroscopy acquisition time was 0.01 seconds. Data was collected from a 3008 cm⁻¹ cell. -1 2844cm -1 Raman peak position. Set the M value to equal I. 3008 / (I 3008 +I 2844 Cells with an M value greater than or equal to 0.5 were selected and placed into 96-well plates containing induction medium (20 g / L glucose, 15 g / L sea salt). Induction was performed at 25°C for 12 h. The induced cells were then subjected to a second round of selection using flow cytometry Raman sorting with an M value greater than or equal to 0.6. The selected cells were then placed into 96-well plates containing basal culture medium.

[0028] The results showed that after the first round of screening of approximately 370,000 *Schizochytrium* cells, 2,168 cell strains with an M value greater than 0.5 were obtained. After the second round of screening, 8 cell strains with an M value greater than 0.6 were obtained. After culturing these 8 *Schizochytrium* cell strains, the DHA content in their oils was analyzed by gas chromatography, and it was found that the DHA content in the oils of all strains was greater than 65% (Table 1). Figure 3This is a gas chromatographic analysis of intracellular lipid fatty acids in Schizochytrium strain (E3). After 5 days of fed-batch fermentation in a 5L fermenter, strain E3, which had the highest DHA content, showed DHA and DPA contents of 66.9% and 15.7%, respectively, with yields of 50.2 g / L and 11.8 g / L.

[0029] Table 1. Analysis of DHA content in the oils of the selected Schizochytrium strains.

[0030] strain number DHA content in oils (% of total lipids) A10 66.4% B2 68.6% B9 66.4% B10 67.9% C1 65.2% E3 69.9% G3 67.3% H1 66.0%

[0031] Example 3: Screening for DHA-rich *Cyclophorus* cells using this method

[0032] The cells of *Cyclotridium perfringens* were cultured for 24 h in a basic medium containing 20 g / L glucose, 20 g / L yeast extract and 15 g / L sea salt.

[0033] High-throughput flow cytometry Raman sorting (FlowRACS) was used to acquire Raman spectra for each cell. The Raman excitation source had a wavelength of 532 nm and a power range of 100 mW. The Raman spectroscopy acquisition time was 0.5 seconds. Data was collected from a 3008 cm⁻¹ cell. -1 2844cm -1 Raman peak position. Set the M value to equal I. 3008 / (I 3008 +I 2844 Cells with an M value greater than or equal to 0.5 were selected and placed into 96-well plates containing induction medium (20 g / L glucose, 15 g / L sea salt). Induction was performed at 28°C for 24 h. The induced cells were then subjected to a second round of selection using flow cytometry Raman sorting with an M value greater than or equal to 0.6. The selected cells were then placed into 96-well plates containing basal culture medium.

[0034] The results showed that after the first round of screening of approximately 210,000 *Tetranychus spp.* cells, 584 cell strains with an M value greater than 0.5 were obtained. After the second round of screening, 3 cell strains with an M value greater than 0.6 were obtained. After culturing these 3 *Tetranychus spp.* cells, the DHA content in their oils was analyzed by gas chromatography. It was found that the DHA content in the oils of all strains was greater than 65% (Table 2).

[0035] Table 2. Analysis of DHA content in the oils of the selected *Cyclochytrium* strains.

[0036] strain number DHA content in oils (% of total lipids) T1 67.5% T2 65.1% T3 66.4%

[0037] Example 4: Screening for high-DHA-content oil-producing yeast cells using this method

[0038] The mutant oil-producing yeast cells were cultured for 14 h in a basic medium containing 20 g / L glucose and 20 g / L yeast extract.

[0039] High-throughput flow cytometry Raman sorting (FlowRACS) was used to acquire Raman spectra for each cell. The Raman excitation source had a wavelength of 532 nm and a power range of 70 mW. The Raman spectroscopy acquisition time was 0.05 seconds. Data was collected from a 3008 cm⁻¹ cell. -1 2844cm -1 Raman peak position. Set the M value to equal I. 3008 / (I 3008 +I 2844 Cells with an M value greater than or equal to 0.5 were selected and placed into 96-well plates containing induction medium (20 g / L glucose). Induction was performed at 30°C for 18 h. The induced cells were then subjected to a second round of selection using flow cytometry Raman sorting with an M value greater than or equal to 0.6. The selected cells were then placed into 96-well plates containing basal culture medium.

[0040] The results showed that after the first round of screening of approximately 630,000 oil-producing yeast cells, 353 cell lines with an M value greater than 0.5 were obtained. After the second round of screening, one cell line with an M value greater than 0.6 was obtained. Gas chromatography analysis of the DHA content in its oil revealed that the DHA content was greater than 65% (Table 3).

[0041] Table 3. Analysis of DHA content in the oil of the screened oil-producing yeast strains.

[0042] strain number DHA content in oils (% of total lipids) Y1 65.8%

[0043] In summary, this application provides I using Raman spectroscopy. 3008 / (I 3008 +I 2844 This method for detecting DHA content in oils significantly improves detection accuracy and achieves unexpected technical results. Furthermore, based on this detection method and corresponding culture medium, this application enables high-throughput screening of oil cells with high DHA content, which has significant practical application value.

Claims

1. A method for high-throughput screening of DHA-rich bacterial strains, characterized in that: The screening method is based on high-throughput flow cytometry Raman sorting technology to detect characteristic peaks related to intracellular DHA content and characteristic peaks related to saturated fatty acid lipid content.

2. The method for high-throughput screening of DHA-rich strains according to claim 1, characterized in that: The characteristic peak related to DHA content is 3008 cm⁻¹. -1 The Raman peak position at [location missing], the characteristic peak related to the saturated fatty acid oil content is 2844 cm⁻¹. -1 The Raman peak at that location.

3. The method for high-throughput screening of DHA-rich strains according to claim 2, characterized in that: Specifically, the following steps are included: (1) Cell culture: The cells to be screened were cultured in a basic culture medium for 12-24 hours; (2) Raman spectra acquisition: Raman spectra of each cell were acquired using high-throughput flow cytometry Raman sorting technology, and 3008 cm⁻¹ of the sample was obtained. -1 Raman peak intensity I 3008 and 2844cm -1 Raman peak intensity I at the location 2844 (3) Screening strains: According to the formula M=I 3008 / (I 3008 +I 2844 Calculate the M value, set the screening range of M, and you can obtain the screened strains.

4. The method for high-throughput screening of DHA-rich strains according to claim 3, characterized in that: The basic culture medium in step (1) consists of 10-20 g / L glucose, 10-20 g / L yeast extract and 10-20 g / L sea salt.

5. The method for high-throughput screening of DHA-rich strains according to claim 3, characterized in that: Step (3) includes two rounds of screening: the first round of screening for cells with M greater than or equal to 0.5, followed by induction; then the second round of screening for cells with M greater than or equal to 0.6, which yields cells that produce high levels of DHA.

6. The method for high-throughput screening of DHA-rich strains according to claim 5, characterized in that: The induction process specifically involves placing the cells selected in the first round into well plates containing induction culture medium and inducing them at 25℃-30℃ for 12-24 hours.

7. The method for high-throughput screening of DHA-rich strains according to claim 6, characterized in that: Only one cell is placed in each well of the plate; the induction culture medium consists of 15-20 g / L glucose and 10-20 g / L sea salt.

8. The method for high-throughput screening of DHA-rich strains according to any one of claims 1-7, characterized in that: When collecting Raman spectral peaks, the excitation light source wavelength is 532nm, the power range is 10mW-100mW, and the Raman spectral acquisition time is 0.01 seconds-0.5 seconds.

9. The method for high-throughput screening of DHA-rich strains according to claim 8, characterized in that: The cells to be screened include wild-type cells or mutant cells; the strains are oil-rich microbial strains.

10. The method for high-throughput screening of DHA-rich strains according to claim 8, characterized in that: The oil-rich microbial strains include Schizochytrium, Cynochytrium, or oil-producing yeast strains.

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