Method for high-throughput screening of L-valine high-yield strain

Abstract

The invention discloses a method for high-throughput screening of a high-yield L-valine strain. The method comprises the following steps: activating a strain; carrying out compound mutagenesis on normal-pressure and room-temperature plasmas and diethyl sulfate; a step of mixed culture; performing mixed culture and preliminary screening; wherein the strain is corynebacterium glutamicum; in the step of mixed culture, 24-pore plate culture and screening are adopted. According to the method disclosed by the invention, the normal-pressure and room-temperature plasma and diethyl sulfate are adopted for compound mutation, and mixed culture is adopted for screening, so that the mutation rate and the positive mutation rate of the strain are increased, the screening flux is improved, the probability of obtaining the high-yield strain is increased, and the high-yield strain with good hereditary stability is further obtained.

Description

Technical Field

[0001] This invention provides a high-throughput screening method for strain selection, specifically a screening method for L-valine-glutamic acid Corynebacterium based on ARTP and diethyl sulfate combined mutagenesis. Background Technology

[0002] L-Valine, chemically known as 2-amino-3-methylbutyric acid, is a branched-chain amino acid. At room temperature, L-valine is a white crystalline powder or white crystals, odorless, readily soluble in water (88.5 g / L), slightly soluble in ethanol, and insoluble in ether. It is a natural essential amino acid for the human body. It promotes normal growth, repairs tissues, regulates blood sugar, and provides necessary energy. In the early 1960s, amino acids were mainly used in umami seasonings; in the late 1960s, they began to be used as beverage additives; in the 1970s and 1980s, they were used in nutritional preparations; and since the 1990s, they have been used in pharmaceuticals, health products, food additives, daily chemicals, and pesticides. Currently, amino acids are mainly used as food supplements, animal feed additives, clinical nutritional agents, and amino acid drugs, such as amino acid injections, oral amino acid solutions, and amino acid preparations.

[0003] Due to the increasing demand for these applications of L-valine, supply is always limited.

[0004] Traditionally, L-valine production has been achieved through extraction, processing, and purification from various protein sources. Microbial fermentation of L-valine offers the potential for high purity and large-scale industrial production, making it the mainstream industrial synthesis method. *Escherichia coli* and *Corynebacterium glutamicum* are promising choices for developing L-valine producers, but the former can produce endotoxins that contaminate the final product. *Corynebacterium glutamicum* has proven to be an attractive microorganism for industrial production of various amino acids, including L-valine.

[0005] To achieve optimal results in industrial fermentation production, a strain with excellent fermentation performance is first required, followed by optimization of fermentation conditions based on the strain's characteristics. Therefore, obtaining a high-performance production strain is a crucial step in the industrial production process. When wild-type strains are insufficient to meet production demands, superior strains can be obtained through mutagenesis. Without external intervention, the spontaneous mutation rate of conventionally cultured microorganisms is 10-1. -5 -10 -8 After mutagenesis treatment, its mutation rate increases significantly, reaching 10. -3 -10 -6 It is easier to build a mutation library.

[0006] Obtaining mutants through mutagenesis breeding is a prerequisite; the key is to screen out the target high-yielding strains from a large mutant library. Traditional screening techniques are highly random, time-consuming, and labor-intensive, significantly reducing the probability of obtaining high-yielding strains. Therefore, a screening technique capable of screening a large number of strains at once is needed. High-throughput screening technology, developed in the 1980s, is characterized by its miniaturization, accuracy, speed, efficiency, and low cost. Initially used for drug screening, it has recently been frequently applied to bacterial strain selection.

[0007] High-throughput screening is an efficient screening system based on plate culture and rapid detection, and it is an important means to quickly obtain high-yield strains from a large mutant library. The three important steps in high-throughput screening technology are: (1) establishment of mutant strain library, (2) development of high-throughput detection method, and (3) application of high-throughput culture technology.

[0008] By reviewing a large number of literatures, a 24-well plate and ninhydrin colorimetric method were used for screening, and optimization experiments were conducted to improve the accuracy of the screening. The feasibility of the method was confirmed through subsequent fermentation.

[0009] This method uses Corynebacterium glutamicum VAL4.6 as the research object. Based on the traditional high-throughput screening model, it optimizes a more efficient and stable screening method by introducing compound mutagenesis and mixed culture to perform high-throughput screening of the mutagenized strain, forming a more rational high-throughput screening system. Based on this optimized model, after primary screening, secondary screening, and fermentation verification, the desired L-valine-producing strain with higher yields compared to the starting strain was obtained. Summary of the Invention

[0010] The purpose of this invention is to provide a method for high-throughput screening of high-yield L-valine strains. By combining ARTP mutagenesis with diethyl sulfate to form a compound mutagenesis and introducing it into mixed culture, the screening efficiency of strains can be improved, and the probability of positively mutated strains during the screening process can be increased.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] This invention provides a method for high-throughput screening of high-yield L-valine-producing strains, the method comprising:

[0013] Steps for activating bacterial strains:

[0014] The steps of inducing mutagenesis by combining ambient pressure room temperature plasma (ARTP) with diethyl sulfate;

[0015] The steps of mixed culture;

[0016] The steps for initial screening using mixed culture;

[0017] The bacterial strain is Corynebacterium glutamicum; the mixed culture step uses a 24-well plate for culture and screening.

[0018] Furthermore, the overall lethality of the combined mutagenesis induced by the diethyl sulfate and ARTP is approximately 91.5%.

[0019] Furthermore, the activation steps of the strain include: first, selecting a single colony from the plate and inoculating it into a seed culture medium, and culturing it at 30°C and 200 rpm for 24 hours until it enters the logarithmic growth phase. The culture medium is then diluted 50-fold to obtain a target concentration with an OD of 0.5. 1 mL of the seed culture is taken and thoroughly shaken to obtain a homogeneous mutagenic starter culture.

[0020] Further, the steps of the atmospheric pressure room temperature plasma (ARTP) include: placing the autoclaved iron sheet in the outer flame of an alcohol lamp for about 30 seconds, placing it in a sterile glass petri dish, and after it cools down, taking 10 μL of the above-mentioned evenly shaken mutagenic starting solution and spreading it evenly on the mutagenic iron sheet; turning on the ARTP mutagenesis breeding instrument and turning on the condensate and nitrogen valves in sequence; turning on the ultraviolet lamp from the control panel to sterilize the mutagenesis chamber for 30 minutes; fixing a 2 mL EP tube containing 990 μL of sterile water into the slot of the ARTP breeding mutagenesis instrument; transferring the mutagenic iron sheet into the ARTP mutagenesis chamber using a sterile glass petri dish; placing the mutagenic iron sheet into the corresponding slot from left to right using autoclaved tweezers; and setting the ARTP mutagenesis conditions and time on the control panel. After mutagenesis was stopped, a 2 mL EP tube containing the mutagenic iron plate was vortexed for 1 min to wash out the mutagenic iron plate. The tube was then diluted with the appropriate amount and 100 μL was evenly spread on a nutrient broth solid medium. The tube was incubated at 30 °C for 24-36 h. The number of colonies on the plate was recorded and the ARTP mutagenesis lethality rate was calculated.

[0021] Further, the steps of the combined mutagenesis using ambient pressure room temperature plasma (ARTP) and diethyl sulfate include: placing the autoclaved mutagenesis iron sheet in the outer flame of an alcohol lamp for approximately 30 seconds, then allowing it to cool in a sterile glass petri dish. Taking 10 μL from the well-vibrated mutagenesis starter culture and spreading it evenly onto the mutagenesis iron sheet. Sequentially activating the condensate and nitrogen valves of the ARTP mutagenesis breeding instrument, and sterilizing the mutagenesis chamber with a UV lamp for 30 minutes. Fixing a 2 mL EP tube containing 990 μL of sterile water into the slot of the ARTP breeding mutagenesis instrument, and transferring the mutagenesis iron sheet into the mutagenesis chamber using a sterile glass petri dish. Using autoclaved tweezers, placing the mutagenesis iron sheet sequentially from left to right into the corresponding slots, and setting the ARTP mutagenesis time to 45 seconds on the control panel. Remove a sterilized 50mL centrifuge tube from a clean bench and add 100μL of ARTP mutagenic bacterial solution, 100mL of PTFE buffer, and 9.9mL of phosphate buffer (0.1mol / L, pH=7.0). Add 20μL of filtered and sterilized diethyl sulfate stock solution to bring the final concentration to 0.2%. Seal the tube and place it in a shaker at 30℃ and 200rpm in the dark for 20min. Then, add 500μL of 10% Na2S2O3 to the clean bench and incubate under the same conditions for 30min to terminate the reaction.

[0022] Further, the mixed culture step includes: centrifuging the mutagenic sample obtained in the previous step at 4℃ and 4000rpm for 10 minutes to collect all mutagenic bacteria, and resuspending them with an appropriate volume of 0.9% physiological saline. Inoculate 150μL per well into sterilized 24-well plates containing seed culture medium, and incubate at 30℃ and 200rpm on a shaker for 24h, then transfer to fermentation plates for further incubation for 48h. Simultaneously, dilute the combined mutagenic bacterial suspension and spread 150μL evenly onto LB agar plates, spreading three plates in parallel, and incubate at 30℃ for 24-36h. Count the colonies to determine the number of colonies to be inoculated into each well during subsequent mixed culture in the 24-well plates.

[0023] The steps of the mixed culture initial screening include: for the fermentation plate described in the mixed culture step, centrifuge at 4000 rpm for 10 min, collect the fermentation supernatant, dilute it appropriately, estimate the L-valine yield using the ninhydrin colorimetric method, and select the mixed culture wells with higher L-valine yield.

[0024] Furthermore, the method for high-throughput screening of high-yield L-valine strains also includes the steps of plate screening and shake-flask screening;

[0025] The steps of the plate secondary screening include: marking the fermentation wells in the mixed culture plate where the amino acid yield is 20% higher than that of the control group, tracing the corresponding seed wells in the seed plate stored at 4°C, diluting and spreading the mixture, sealing it with sealing film, and culturing it in a 30°C incubator for 24-36 hours. Selecting single colonies with good growth from the culture medium plate and culturing them in a 24-well plate containing seed medium for 24 hours, then transferring them to a 24-well plate fermentation medium and culturing them for 48 hours, estimating the L-valine yield using the ninhydrin colorimetric method, and selecting strains with high L-valine yield.

[0026] The shake-flask re-screening step includes: marking the wells in the re-screening plate where the yield is 20% higher than the yield of the starting strain, and tracing the corresponding seed wells in the seed plate stored at 4°C. The wells are then diluted, streaked, and incubated at 30°C for 24-36 hours to grow single colonies. Larger single colonies are selected and inoculated into 500mL shake flasks containing 100mL of seed culture medium, and incubated at 30°C and 200rpm for 24 hours. Then, a 10% inoculum is inoculated into 500mL shake flasks containing 100mL of fermentation culture medium and incubated at 30°C and 200rpm for 48 hours until fermentation ends. One mL of sample from all shake flasks at the fermentation endpoint is taken, centrifuged at 4000rpm for 5 minutes, and the fermentation supernatant is collected. After dilution to an appropriate factor, the L-valine yield is determined by HPLC.

[0027] Furthermore, the nutrient broth solid culture medium comprises: 5 g / L sodium chloride, 10 g / L beef extract, 10 g / L peptone, and 20 g / L agar.

[0028] Furthermore, the seed culture medium comprises: 25 g / L glucose, 1 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 5 g / L ammonium sulfate, 40 g / L corn steep liquor, 0.5 g / L magnesium sulfate, 0.0001 g / L biotin, and 0.0002 g / L vitamin B1;

[0029] Furthermore, the fermentation medium comprises: 90 g / L glucose, 2 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 20 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 30 g / L corn steep liquor, 10 g / L calcium carbonate, 0.5 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.00008 g / L biotin, and 0.0001 g / L vitamin B1.

[0030] In this invention, the mutation rate (Q) M ) and positive mutation rate (Q T The calculation formula for ) is as follows:

[0031]

[0032] In the formula, H represents the total number of single colonies; M represents the number of mutant strains whose L-valine production is significantly different from that of the starting strain; and H represents the number of mutant strains among the screening strains whose L-valine production is significantly higher than that of the starting strain. (Because there is an error in the yield calculation, and the error is within 3%, strains with yield changes outside the ±5% range are selected as mutant strains.)

[0033] In this invention, the L-valine content is determined as follows: During the initial screening in mixed culture and the secondary screening in well plates, the L-valine content is rapidly detected using the ninhydrin colorimetric method. Standard solutions of L-valine at different concentration gradients are prepared, and a standard curve is plotted. During the secondary screening in shake flasks, the L-valine content is determined using HPLC.

[0034] In this invention, data processing and analysis can be performed using Origin 2021 software.

[0035] The beneficial effects of this invention are as follows:

[0036] This invention employs a combination of ambient pressure, room temperature plasma, and diethyl sulfate for mutagenesis, along with co-culture, to increase the mutation rate and positive mutation rate of the strains, thereby increasing the screening efficiency for high-yielding strains and ultimately yielding high-yielding strains with good genetic stability. The combined mutagenesis exhibits a synergistic effect, effectively increasing the positive mutation rate of the strains and resulting in strains with good genetic stability and minimal yield variation between generations. Furthermore, co-culture significantly improves the processing efficiency of the high-throughput screening system, reduces labor costs, and allows for the processing of more mutagenic strains in a shorter time, while also ensuring the high-yielding strains exhibit good genetic stability. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 L-valine production of the strain in Experimental Example 1 of this invention Figure 1 .

[0039] Figure 2 L-valine production of the strain in Experimental Example 1 of this invention Figure 2 .

[0040] Figure 3 L-valine production of the strain in Experimental Example 2 of this invention Figure 1 .

[0041] Figure 4 L-valine production of the strain in Experimental Example 2 of this invention Figure 2 .

[0042] Figure 5 This is a comparison of L-valine production in shake-flask experiments between the high-yielding strain and the starting strain in this invention.

[0043] Figure 6 This is a schematic diagram illustrating the genetic stability of the high-yielding strain in this invention.

[0044] Figure 7 This is a comparison chart of L-valine production in a 5L fermenter experiment between the high-yield strain and the starting strain in this invention.

[0045] Figure 8 This is a schematic diagram of the process of high-throughput screening of high-yield L-valine strains in Example 2 of the present invention. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0047] Example 1

[0048] This embodiment provides a method for high-throughput screening of high-yield L-valine-producing strains, the method comprising:

[0049] The steps involved in activating bacterial strains;

[0050] The steps of ambient pressure room temperature plasma (ARTP);

[0051] The steps of perforated plate screening;

[0052] The bacterial strain is Corynebacterium glutamicum;

[0053] In this embodiment, the activation steps of the bacterial strain include: first, selecting a single colony from the plate and inoculating it into a seed culture medium, and culturing it at 30°C and 200 rpm for 24 hours until it enters the logarithmic growth phase. The culture medium is then diluted 50 times to obtain a target concentration with an OD of 0.5. 1 mL of the seed culture is taken and thoroughly shaken to obtain a homogeneous mutagenic starting culture.

[0054] In this embodiment, the steps of atmospheric pressure room temperature plasma (ARTP) mutagenesis include: placing the autoclaved iron sheet in the outer flame of an alcohol lamp for about 30 seconds, placing it in a sterile glass petri dish, and after it cools down, taking 10 μL of the uniformly shaken mutagenesis starter culture and spreading it evenly on the mutagenesis iron sheet; turning on the ARTP mutagenesis breeding instrument and opening the condensate and nitrogen valves in sequence; sterilizing the mutagenesis chamber with the ultraviolet lamp from the control panel for 30 minutes; fixing a 2 mL EP tube containing 990 μL of sterile water into the slot of the ARTP breeding mutagenesis instrument; transferring the mutagenesis iron sheet into the ARTP mutagenesis chamber using a sterile glass petri dish; placing the mutagenesis iron sheet into the corresponding slot from left to right using autoclaved tweezers; and setting the ARTP time to 40 seconds on the control panel. After mutagenesis was stopped, a 2 mL EP tube containing the mutagenic iron plate was vortexed for 1 min to wash out the mutagenic iron plate. The tube was then diluted with the appropriate amount and 100 μL was evenly spread on a nutrient broth solid medium. The tube was incubated at 30 °C for 24-36 h. The number of colonies on the plate was recorded and the ARTP mutagenesis lethality rate was calculated.

[0055] In this embodiment, the well plate screening step includes: picking single colonies with good growth from the culture medium plate obtained in the previous step and transferring them to a 24-well plate containing seed culture medium. After incubating at 30°C and 200 rpm for 24 hours on a shaker, the seed culture is transferred to a 24-well plate containing fermentation culture medium at a 1% inoculation rate and incubated at 30°C and 200 rpm for 48 hours on a shaker. After fermentation, the plate is centrifuged at 4000 rpm for 10 minutes, the fermentation supernatant is collected, diluted appropriately, and the L-valine yield is estimated using a chemical colorimetric method.

[0056] Furthermore, the nutrient broth solid culture medium comprises: 5 g / L sodium chloride, 10 g / L beef extract, 10 g / L peptone, and 20 g / L agar.

[0057] In this embodiment, the seed culture medium comprises: 25 g / L glucose, 1 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 5 g / L ammonium sulfate, 40 g / L corn steep liquor, 0.5 g / L magnesium sulfate, 0.0001 g / L biotin, and 0.0002 g / L vitamin B1.

[0058] In this embodiment, the fermentation medium comprises: 90 g / L glucose, 2 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 20 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 30 g / L corn steep liquor, 10 g / L calcium carbonate, 0.5 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.00008 g / L biotin, and 0.0001 g / L vitamin B1.

[0059] In this embodiment, the lethality of ARTP-induced mutagenesis is 90%-92%.

[0060] In this invention, the method of this embodiment is used to conduct multiple sets of parallel experiments.

[0061] Example 2

[0062] This embodiment provides a method for high-throughput screening of high-yield L-valine-producing strains, the method comprising:

[0063] The steps involved in activating bacterial strains;

[0064] The steps of inducing mutagenesis by combining ambient pressure room temperature plasma (ARTP) with diethyl sulfate;

[0065] The steps of mixed culture;

[0066] The steps for initial screening using mixed culture;

[0067] The bacterial strain is Corynebacterium glutamicum; the mixed culture step uses 24-well plate culture for screening.

[0068] In this embodiment, the activation steps of the bacterial strain include: first, selecting a single colony from the plate and inoculating it into a seed culture medium, and culturing it at 30°C and 200 rpm for 24 hours until it enters the logarithmic growth phase. The culture medium is then diluted 50 times to obtain a target concentration with an OD of 0.5. 1 mL of the seed culture is taken and thoroughly shaken to obtain a homogeneous mutagenic starting culture.

[0069] In this embodiment, the steps of combined mutagenesis using ambient pressure room temperature plasma (ARTP) and diethyl sulfate include: placing the autoclaved mutagenesis iron sheet in the outer flame of an alcohol lamp for approximately 30 seconds, then allowing it to cool in a sterile glass petri dish. Taking 10 μL from the well-vibrated mutagenesis starter culture and spreading it evenly onto the mutagenesis iron sheet. Sequentially activating the condensate and nitrogen valves of the ARTP mutagenesis breeding instrument, and sterilizing the mutagenesis chamber with a UV lamp for 30 minutes. Fixing a 2 mL EP tube containing 990 μL of sterile water into the slot of the ARTP breeding mutagenesis instrument, and transferring the mutagenesis iron sheet into the mutagenesis chamber using a sterile glass petri dish. Using autoclaved tweezers, placing the mutagenesis iron sheet sequentially from left to right into the corresponding slots, and setting the ARTP mutagenesis time to 45 seconds on the control panel. Remove a sterilized 50 mL centrifuge tube from a clean bench and add 100 μL of ARTP mutagenic bacterial solution and 9.9 mL of phosphate buffer (0.1 mol / L, pH = 7.0). Add 20 μL of filtered and sterilized diethyl sulfate stock solution to bring the final concentration to 0.8%. Seal the tube and place it in a shaker at 30°C and 200 rpm in the dark for 20 min. Then, add 500 μL of 10% Na₂S₂O₃ to the clean bench and incubate under the same conditions for 30 min to terminate the reaction.

[0070] In this embodiment, the mixed culture step includes: centrifuging the mutagenized sample obtained in the previous step at 4℃ and 4000rpm for 10 minutes to collect all the mutagenized bacteria, and resuspending them with an appropriate volume of 0.9% physiological saline. Inoculate 150μL per well into sterilized 24-well plates containing seed culture medium and incubate at 30℃ and 200rpm for 24h on a shaker. Simultaneously, dilute the combined mutagenized bacterial suspension and spread 150μL evenly onto LB agar plates, spreading three plates in parallel, and incubate at 30℃ for 24-36h. Count the colonies to determine the number of colonies to be inoculated into each well during subsequent mixed culture in the 24-well plates.

[0071] In this embodiment, the initial screening step of mixed culture includes: centrifuging the fermentation plate in the mixed culture step at 4000 rpm for 10 min, collecting the fermentation supernatant, diluting it appropriately, estimating the L-valine yield using the ninhydrin colorimetric method, and selecting the mixed culture wells with higher L-valine yield.

[0072] In this embodiment, the step of secondary screening of the well plate includes: marking the fermentation wells in the mixed culture well plate where the amino acid yield is 20% higher than that of the control group, tracing the corresponding seed wells in the seed well plate stored at 4°C, diluting and coating the wells, sealing them with sealing film, and culturing them in a 30°C incubator for 24-36 hours. Single colonies with good growth on the culture medium plate are picked and cultured in a 24-well plate containing seed culture medium for 24 hours, and then transferred to a 24-well plate fermentation medium for 48 hours. The L-valine yield is estimated using the ninhydrin colorimetric method, and strains with high L-valine yield are selected.

[0073] In this embodiment, the shake-flask re-screening step includes: marking the wells in the re-screening plate where the yield is 20% higher than the yield of the starting bacteria, tracing the corresponding seed wells in the seed plate stored at 4°C, diluting and streaking, and culturing at 30°C for 24-36 hours to grow single colonies. Selecting the larger single colonies, inoculate them into a 500mL shake flask containing 100mL of seed culture medium, and culturing at 30°C and 200rpm for 24 hours. Then, inoculate 10% of the culture medium into a 500mL shake flask containing 100mL of fermentation medium and culturing at 30°C and 200rpm for 48 hours until fermentation ends. Take 1mL of the sample from all shake flasks at the fermentation endpoint, centrifuge at 4000rpm for 5 minutes, collect the fermentation supernatant, dilute appropriately, and determine the L-valine yield using HPLC.

[0074] In this embodiment, the seed culture medium comprises: 25 g / L glucose, 1 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 5 g / L ammonium sulfate, 40 g / L corn steep liquor, 0.5 g / L magnesium sulfate, 0.0001 g / L biotin, and 0.0002 g / L vitamin B1.

[0075] In this embodiment, the fermentation medium comprises: 90 g / L glucose, 2 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 20 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 30 g / L corn steep liquor, 10 g / L calcium carbonate, 0.5 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.00008 g / L biotin, and 0.0001 g / L vitamin B1.

[0076] The lethality of the combined mutagenesis in this embodiment is approximately 91.5%.

[0077] In this invention, the method of this embodiment is used to conduct multiple sets of parallel experiments.

[0078] Comparative Example 1

[0079] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses ambient pressure room temperature plasma (ARTP) mutagenesis for 45 seconds.

[0080] Comparative Example 2

[0081] The only difference between Comparative Example 2 and Example 2 is that Comparative Example 2 uses ambient pressure room temperature plasma (ARTP) for mutagenesis alone.

[0082] Experimental Example 1

[0083] This experiment measured the mutagenesis results of the strains in Example 1 and Comparative Example 1, respectively.

[0084] Methods: After the mutagenesis step was completed, the mutagenized strains were measured and the results were recorded as follows:

[0085] ①See Figure 1 In Comparative Example 1, the results of ARTP mutagenesis alone for 40 seconds were as follows: a total of 192 strains were obtained, of which 59 were mutant strains with a mutation rate of 30.50%, of which 32 were positive mutations with a positive mutation rate of 16.90%.

[0086] ②See Figure 2 In Example 1, the results of ARTP-induced mutagenesis for 45 seconds were as follows: a total of 192 strains were obtained, of which 56 were mutant strains, with a mutation rate of 29.60%. Among these, 24 strains were positive mutations, with a positive mutation rate of 12.50%.

[0087] In this invention, compared with ARTP mutagenesis 45s, ARTP mutagenesis 40s can improve the mutation rate and positive mutation rate of strains when applied to high-throughput screening of L-valine-producing strains, laying a good foundation for establishing a composite mutagenesis method.

[0088] Experimental Example 2

[0089] This experiment measured the mutagenesis results of the strains in Example 2 and Comparative Example 1, respectively.

[0090] Methods: After the mutagenesis step was completed, the mutagenized strains were measured and the results were recorded as follows:

[0091] ①See Figure 3 In Comparative Example 2, the results of ARTP mutagenesis alone were as follows: a total of 216 strains were obtained, of which 64 were mutant strains with a mutation rate of 31.80%, of which 38 were positive mutations with a positive mutation rate of 15.70%.

[0092] ②See Figure 4 In Example 2, the results of combined mutagenesis with ARTP-diethyl sulfate were as follows: a total of 216 strains were identified, of which 75 were mutant strains, representing a mutation rate of 37.60%. Of these, 49 were positive mutations, representing a positive mutation rate of 24.50%.

[0093] In this invention, the application of compound mutagenesis in the high-throughput screening of L-valine-producing strains not only improves the mutation rate of the strains, but also greatly increases the positive mutation rate, laying a good foundation for establishing a high-throughput screening mutant strain library.

[0094] Experimental Example 3

[0095] This experimental example demonstrates the genetic stability verification experiment performed on the strain screened in Example 2 of the present invention. The specific steps are as follows:

[0096] Methods: The strains with high yields selected after the initial screening and rescreening steps of mixed culture in Example 2 were cultured in shake flasks. The content of L-valine was determined by HPLC. Finally, the strain with the highest L-valine yield was selected for fermentation in a fermenter to verify its genetic stability.

[0097] Fermentation in the fermenter: Fermentation was carried out in a 5L bioreactor at 30℃ for 72 hours with an inoculum of 10%. The dissolved oxygen in the fermenter was maintained at around 20% by adjusting the rotation speed, and the pH was controlled at 7.0 using ammonia water. During fermentation, the residual sugar concentration in the tank was measured using the DNS method, and the sugar replenishment rate was controlled according to the results to ensure that the residual sugar concentration in the tank was maintained at 10-20g / L.

[0098] The above method ultimately screened out a high-yielding strain, VAL6.0.

[0099] ①See Figure 5 A shake-flask experiment was conducted on the high-yielding strain VAL6.0, compared with the starting strain VAL4.6. The high-yielding strain was compared to the starting strain. The starting strain was VAL4.6, and the high-yielding strain was VAL6.0; the high-yielding strain showed a 47.6% increase in yield compared to the original strain.

[0100] ②See Figure 6 Genetic stability experiments were conducted on the high-yielding strain VAL6.0. The results showed that the yield of this high-yielding strain did not change significantly after five generations of subculturing, indicating good genetic stability.

[0101] It is evident that the high-yielding strain obtained in this invention is indeed a high-yielding strain, with a yield 47.6% higher than the original strain, and exhibits good genetic stability.

[0102] ③ The high-yield L-valine strain VAL6.0 was validated in a 5L fermenter and compared with the starting strain VAL4.6. The results are shown below.

[0103] See Figure 7 The results showed that the L-valine yield of the starting strain VAL4.6 was 24.36 g / L, while the L-valine yield of the high-yielding strain VAL6.0 was 30.61 g / L, representing a 24.65% increase in L-valine yield compared to the starting strain.

[0104] See Figure 7 The figure shows the yield results of the high-yielding strain VAL6.0 and the starting strain VAL4.6. It is clear from the figure that the yield of the high-yielding strain VAL6.0 is higher than that of the starting strain VAL4.6.

[0105] The validation results of the high-yielding L-valine strain on a 5L tank showed a significantly higher yield compared to the starting strain. The high-yielding strain VAL6.0 obtained in this invention had a fermentation yield of 30.61 g / L, a conversion rate of 0.34 g / g, and a yield of 0.43 g / L / h. Therefore, the fermentation performance of the high-yielding strain screened in this invention is much superior to that of the starting strain.

[0106] In summary, this invention employs a combined mutagenesis and mixed culture method, optimizes the high-throughput screening model, and improves the mutation rate and positive mutation rate of the strains, thereby obtaining high-yield L-valine strains. The high-throughput screening system based on combined mutagenesis and mixed culture increases the screening throughput of strains, efficiently expands the mutagenesis library of strains in a short period, and saves time and costs in screening high-yield strains, thus obtaining high-yield L-valine strains.

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications 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 high-throughput screening of high-yield L-valine strains, characterized in that, The method includes: The steps involved in activating bacterial strains; The steps of inducing mutagenesis by combining atmospheric pressure, room temperature plasma, and diethyl sulfate; The steps of mixed culture; The steps for initial screening using mixed culture; The bacterial strain is Corynebacterium glutamicum; The mixed culture step uses 24-well plates for culture and screening.

2. The method for high-throughput screening of high-yield L-valine strains according to claim 1, characterized in that, The overall lethality rate of the combined mutagenesis induced by diethyl sulfate and ambient pressure room temperature plasma is approximately 91.5%.

3. The method for high-throughput screening of high-yield L-valine strains according to claim 1 or 2, characterized in that, The steps for activating the strain include: first, selecting a single colony from the plate and inoculating it into a seed culture medium, and culturing it at 30°C and 200 rpm for 24 hours until it enters the logarithmic growth phase; diluting the culture medium 50 times to obtain a target concentration with an OD of 0.5; taking 1 mL of the seed culture and shaking it thoroughly to obtain a uniform mutagenic starter culture.

4. The method for high-throughput screening of high-yield L-valine strains according to claim 1 or 2, characterized in that, The steps of the combined mutagenesis using ambient pressure room temperature plasma and diethyl sulfate include: coating the activated bacterial solution onto a mutagenic iron sheet and mutagenizing it for 40 seconds on an ARTP instrument; mixing 100 μL of the mutagenic bacterial solution with 20 μL of diethyl sulfate solution and 9.9 mL of phosphate buffer (0.1 mol / L, pH = 7.0) to obtain a mixed bacterial solution containing 0.2% diethyl sulfate; then incubating it at 200 rpm and 30 °C for 20 min; then adding 500 μL of 10% Na2S2O3 sequentially on a clean bench and incubating under the same conditions with shaking for 30 min to terminate the reaction; finally, centrifuging the sample at 4 °C and 4000 rpm for 10 minutes, collecting all the mutagenic bacteria, and resuspending and diluting them with an appropriate volume of 0.9% physiological saline.

5. The method for high-throughput screening of high-yield L-valine strains according to claim 1, characterized in that, The mixed culture steps include: inoculating the combined mutagenic bacterial solution and the mutagenic bacterial solution diluted to an appropriate concentration into a sterilized 24-well plate containing seed culture medium at an inoculation rate of 150 μL per well, ensuring that the number of colonies per well is 10-12, and culturing on a shaker at 30°C and 200 rpm for 24 h; then transferring to a fermentation plate for further culture for 48 h.

6. The method for high-throughput screening of high-yield L-valine strains according to claim 1, characterized in that, The steps of the mixed culture initial screening include: for the fermentation plate described in the mixed culture step, centrifuge at 4000 rpm for 10 min, collect the fermentation supernatant, dilute it appropriately, estimate the L-valine yield using the ninhydrin colorimetric method, and select the mixed culture wells with higher L-valine yield.

7. The method for high-throughput screening of high-yield L-valine strains according to claim 1, characterized in that, The method also includes the steps of plate secondary screening and shake flask secondary screening. The plate secondary screening step includes: marking the fermentation wells in the mixed culture plate that have an amino acid yield higher than the control group by 20% in the initial screening, tracing the corresponding seed wells in the seed plate stored at 4°C, diluting and spreading the mixture, sealing it with sealing film, and culturing it in a 30°C incubator for 24-36 hours. Selecting the best-growing single colonies from the culture medium plate and culturing them in a 24-well plate containing seed medium for 24 hours, then transferring them to a 24-well plate fermentation medium and culturing them for 48 hours, estimating the L-valine yield using the ninhydrin colorimetric method, and selecting strains with high L-valine yield. The shake flask re-screening step includes: marking the wells in the re-screening plate where the yield is 20% higher than that of the starting strain, and tracing the corresponding seed wells in the seed plate stored at 4°C. The wells are then diluted, streaked, and incubated at 30°C for 24-36 hours to grow single colonies. Larger single colonies are selected and inoculated into 500mL shake flasks containing 100mL of seed culture medium. The flasks are incubated at 30°C and 200rpm for 24 hours. Then, a 10% inoculum is added to 500mL shake flasks containing 100mL of fermentation medium and incubated at 30°C and 200rpm for 48 hours until fermentation ends. One mL of the sample from all shake flasks at the fermentation endpoint is taken, centrifuged at 4000rpm for 5 minutes, and the fermentation supernatant is collected. After diluting appropriately, the L-valine yield is determined by HPLC.

8. The method for high-throughput screening of high-yield L-valine strains according to claim 3, 5, or 7, characterized in that, Both the activation culture medium and the well plate seed culture medium contain: 25 g / L glucose, 1 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 5 g / L ammonium sulfate, 40 g / L corn steep liquor, 0.5 g / L magnesium sulfate, 0.0001 g / L biotin, and 0.0002 g / L vitamin B1.

9. The method for high-throughput screening of high-yield L-valine strains according to claim 5, 6, or 7, characterized in that, The fermentation medium comprises: 90 g / L glucose, 2 g / L potassium dihydrogen phosphate, 2.5 g / L yeast extract, 20 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 30 g / L corn steep liquor, 10 g / L calcium carbonate, 0.5 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.00008 g / L biotin, and 0.0001 g / L vitamin B1.

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