Corynebacterium glutamicum for producing L-isoleucine and application of corynebacterium glutamicum

Abstract

The invention discloses corynebacterium glutamicum for producing L-isoleucine and application of the corynebacterium glutamicum. The strain is obtained by performing ultraviolet mutagenesis and structural analogue resistance screening on corynebacterium glutamicum ATCC13869. According to the method, the L-isoleucine can be produced through direct fermentation of glucose, the yield of the L-isoleucine can reach 51.69 g / L, the conversion rate can reach 28.5%, the production efficiency is improved, meanwhile, the main raw material glucose used for fermentation is low in price and easy to obtain, the method conforms to the green food safety production concept, and industrial production of the L-isoleucine is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Corynebacterium glutamicum capable of producing L-isoleucine and its application. Background Art

[0002] As one of the eight essential amino acids, L-isoleucine plays an important role in protein synthesis, muscle growth and repair, maintaining liver function, blood circulation, immune homeostasis, and energy supply. Therefore, it is widely used in industries such as sports nutrition, medicine, and functional foods. In addition, L-isoleucine can improve the growth performance, muscle mass, and immune function of animals, enhance the production efficiency and disease resistance of livestock and poultry, and is also widely used in the feed and livestock industries, with a large demand and broad market application prospects.

[0003] Currently, the main production methods of L-isoleucine include extraction method, chemical synthesis method, microbial transformation method, and direct fermentation method. Among them, the products obtained by the chemical synthesis method are all racemates, which must be subjected to racemic resolution and have many by-products, and high-purity L-isoleucine cannot be obtained cheaply. The extraction method can obtain high-purity L-isoleucine, but the production process is time-consuming, seriously polluting, low in yield, and the product quality cannot be guaranteed. The microbial transformation method uses glucose as the fermentation carbon source and energy source, and then adds specific precursor substances (aminobutyric acid, hydroxybutyric acid, ketobutyric acid, or D-threonine), which are effectively converted into L-isoleucine by the action of microorganisms. However, due to the scarcity or high price of its precursor substances, it is rarely used for industrial production of L-isoleucine at present. The direct fermentation method is that microorganisms directly ferment to produce L-isoleucine using cheap glucose as the carbon source, which has the advantages of cheap and easily available raw materials, high yield, short cycle, and easy control. Currently, it has become the mainstream industrial method for producing L-isoleucine.

[0004] Corynebacterium glutamicum has been the main production bacterium for the global amino acid fermentation industry in the past forty years and has currently been safely applied to the production of most amino acids, including threonine, lysine, valine, leucine, isoleucine, etc. In Corynebacterium glutamicum strains, the activities of multiple key enzymes in the L-isoleucine biosynthesis pathway are feedback-inhibited by the end product L-isoleucine. Therefore, relieving feedback inhibition is the key to obtaining high-yield L-isoleucine strains. Most of the existing high-yield L-isoleucine bacteria are obtained through genetic modification, and there are disadvantages such as easy loss of plasmids and unstable fermentation production. With the continuous maturity of fermentation technology and the development of genetic engineering technology, a large number of studies have shown that by taking advantage of the ability of microorganisms to synthesize the amino acids they need, through mutagenesis treatment of specific microorganisms and screening for auxotrophic and / or amino acid structural analog-resistant mutants to relieve feedback inhibition and feedback repression in metabolic regulation, the purpose of excessive accumulation of a certain amino acid can be achieved. For example, Xie Xixian et al. mutagenized Corynebacterium glutamicum - YI strain CGMCC No. 12153 and screened for resistance to L-isoleucine structural analogs, obtaining a mutant strain with high-yield isoleucine, and its isoleucine yield can reach 28 g / L. However, compared with genetically engineered bacteria, its yield still needs to be improved. Therefore, screening for food-safe strains that can de novo synthesize and have high-yield L-isoleucine remains an urgent problem to be solved. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned prior art, the present invention uses Corynebacterium glutamicum ATCC13869 as the parental strain, and through ultraviolet mutagenesis and screening for resistance to structural analogs, obtains a food-safe strain GIL-018 that can produce high-yield L-isoleucine; this strain can directly ferment glucose to produce L-isoleucine, and the conversion rate can reach 28.5%, the yield can reach 51.69 g / L, the production efficiency is high, and the main raw material glucose used in fermentation is cheap and easy to obtain, which conforms to the concept of green food safety production.

[0006] In the first aspect, the present invention provides a Corynebacterium glutamicum that produces L-isoleucine, which is classified and named as Corynebacterium glutamicum GIL-018, and was deposited at the China Center for Type Culture Collection on October 16, 2023. The deposit address is Wuhan, China, Wuhan University, and the deposit number is CCTCC NO: M20231914.

[0007] According to the technical solution of the present invention, the Corynebacterium glutamicum that produces L-isoleucine is tolerant to L-isoleucine and its derivatives.

[0008] According to the technical solution of the present invention, the L-isoleucine derivative is isoleucine hydroxamic acid (IleHx).

[0009] In one embodiment of the present invention, the L-isoleucine fermentation yield of the Corynebacterium glutamicum is 51.69 g / L, and the conversion rate is 28.57%.

[0010] In a second aspect, the present invention provides the use of the above-mentioned Corynebacterium glutamicum GIL-018 in the production of isoleucine.

[0011] According to the technical solution of the present invention, the method for producing isoleucine includes: fermentatively culturing Corynebacterium glutamicum GIL-018.

[0012] According to the technical solution of the present invention, the fermentative culture is carried out under conditions that can enable Corynebacterium glutamicum GIL-018 to grow and synthesize L-isoleucine. As long as it can enable Corynebacterium glutamicum GIL-018 to grow and synthesize L-isoleucine, it belongs to the protection scope of the present invention.

[0013] In one embodiment of the present invention, the Corynebacterium glutamicum GIL-018 uses glucose as a carbon source and produces L-isoleucine through fermentation.

[0014] In one embodiment of the present invention, the method for fermentatively producing L-isoleucine includes: inoculating Corynebacterium glutamicum GIL-018 into a fermentation medium at an inoculation amount of 5-15% (v / v) and performing shaking culture under aeration conditions.

[0015] In one embodiment of the present invention, the fermentation medium used is: glucose 100-200 g / L, peptone 2-7 g / L, sodium chloride 1-5 g / L, beef extract powder 1-3 g / L, disodium hydrogen phosphate 0.3-1 g / L, VB1 5-20 mg / L, ammonium sulfate 1-6 g / L, magnesium sulfate heptahydrate 2-4 g / L, ferrous sulfate 1-2 g / L, and the pH of the medium is 6.0-7.5.

[0016] In one embodiment of the present invention, ammonia water is used to adjust the pH of the fermentation medium or the fermentation system.

[0017] In a preferred embodiment of the present invention, the fermentation medium used is: glucose 182 g / L, peptone 2 g / L, sodium chloride 1 g / L, beef extract powder 1 g / L, disodium hydrogen phosphate 0.5 g / L, VB1 5 mg / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 3 g / L, ferrous sulfate 1.5 g / L, and the pH of the medium is 6.5.

[0018] In one embodiment of the present invention, the pH is controlled at 6.9 throughout the fermentation process.

[0019] In one embodiment of the present invention, the fermentation culture is shake flask culture or stirred ventilation culture in a fermenter.

[0020] In one embodiment of the present invention, the shaking speed of the shake flask culture can be 250 - 400 rpm.

[0021] In one embodiment of the present invention, the ventilation rate of the stirred ventilation culture in the fermenter is 2 - 4 vvm, and the stirring speed can be 300 - 500 rpm.

[0022] The purpose of the fermentation culture is to obtain L-isoleucine; specifically, the culture temperature can be 28 - 32 °C, and the culture time is 40 - 60 h.

[0023] In one embodiment of the present invention, the culture temperature is 30 °C and the culture time is 48 h.

[0024] In one embodiment of the present invention, the method for fermentatively producing L-isoleucine further includes: strain activation, and the activation method is: inoculating the well-cultured Corynebacterium glutamicum GIL-018 on the slant into the seed medium, and culturing at 28 - 32 °C, ventilation rate 2 - 4 vvm, stirring speed 300 - 500 rpm for 12 - 16 h to obtain the seed liquid;

[0025] In one specific embodiment of the present invention, the composition of the seed medium is: glucose 20 g·L -1 、yeast powder 5 g·L -1 、peptone 10 g·L -1 , and the pH is 6.5 - 7.0.

[0026] In one specific embodiment of the present invention, the seed liquid is inoculated into the fermentation medium at an inoculation amount of 5 - 15% (v / v).

[0027] In one embodiment of the present invention, the method for fermentatively producing L-isoleucine further includes: separating and extracting L-isoleucine from the fermentation broth, and the specific separation and extraction method is not limited as long as L-isoleucine can be separated from the fermentation broth.

[0028] Advantages of the present invention:

[0029] 1. Using Corynebacterium glutamicum ATCC13869 as the parental strain, Corynebacterium glutamicum GIL-018 producing L-isoleucine was obtained through ultraviolet mutagenesis combined with resistance screening. This strain uses glucose as the sole carbon source for direct fermentation to produce L-isoleucine, and the yield of L-isoleucine can reach 51.69 g / L, and the conversion rate can reach 28.57%, realizing de novo synthesis and high-yield production of L-isoleucine.

[0030] 2. The Corynebacterium glutamicum GIL-018 provided by the present invention has a food safety-type host of Corynebacterium glutamicum, expanding the product application market.

[0031] 3. The screened Corynebacterium glutamicum GIL-018 of the present invention can achieve high yield without genetic modification, alleviating the impact of genetically engineered strains on production instability. Moreover, the strain has strong robustness and a strong response ability to external adverse factors, which is more conducive to the industrial production of L-isoleucine.

[0032] 4. The screened Corynebacterium glutamicum GIL-018 of the present invention uses glucose as the sole carbon source and does not require additional addition of amino acid precursors, reducing the production cost of L-isoleucine. Glucose is inexpensive and easily available, meeting the concept of green production. Description of the drawings

[0033] Figure 1 It is the HPLC detection result chart of the L-isoleucine standard product in the example.

[0034] Figure 2 It is the tank fermentation result of Corynebacterium glutamicum GIL-018 in Example 2. Detailed implementation manners

[0035] The present invention will be further described in detail below in combination with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0036] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0037] The detection methods for L-isoleucine and glucose involved in the following examples are:

[0038] (1) The L-isoleucine and glucose contents in the fermentation broth were analyzed by HPLC. The detection method was as follows: the fermentation broth was diluted 200 times with sterile water, filtered with a 0.22 μm filter membrane, and then injected into a high performance liquid chromatograph.

[0039] Among them, the HPLC detection conditions are as follows:

[0040] Detection instrument: Agilent HPLC 1260-VWD; Chromatographic column: Yuexu Ultimate HILIC Amphion II;

[0041] Mobile phase: organic phase pure acetonitrile; aqueous phase: 0.05M potassium dihydrogen phosphate pH 3.0;

[0042] The preparation method of 0.05M potassium dihydrogen phosphate is as follows: 0.05M potassium dihydrogen phosphate of corresponding concentration is weighed and dissolved in ultrapure water, and the pH is adjusted to 3.0 with phosphoric acid. After filtration, the corresponding volume of acetonitrile is added in a ratio of acetonitrile: 0.05M potassium dihydrogen phosphate = 75:25, and the bubbles are removed by ultrasonication after mixing.

[0043] Flow rate: 1 mL / min; detection wavelength: 206 nm; injection volume: 10 μL; detection time: 20 min.

[0044] Among them, the L-isoleucine standard is a product of Mo, product number I2752; the glucose standard is a product of Aladdin, CAS number: 50-99-7; the peak time of L-isoleucine is about 5.7min, see Figure 1 .

[0045] (2) The glucose content was detected using SBA biosensor analyzer.

[0046] Example 1: Screening of Corynebacterium glutamicum producing L-isoleucine

[0047] S1. Inoculate Corynebacterium glutamicum ATCC13869 (purchased from Beijing Bio-Bowei, catalog number bio-53104) into LBG medium and activate for 12 hours to obtain a bacterial suspension;

[0048] LBG medium: glucose 10 g L -1 , Yeast powder 5g·L -1 , Peptone 10 g·L -1 、NaCl 5g·L -1 .

[0049] S2, placing the bacterial suspension in a culture dish and subjecting it to mutagenesis treatment under ultraviolet irradiation, the mutagenesis conditions are: ultraviolet lamp 20W, mutagenesis distance 20cm, mutagenesis time 90s;

[0050] S3. Dilute the mutagenized bacterial solution 100 times, take 100 μL of the diluted bacterial solution and spread it on the resistant medium for primary screening.

[0051] Among them, in order to screen for mutant strains tolerant to isoleucine hydroxamate, the present invention adds an isoleucine analog, namely isoleucine hydroxamate, to the resistant medium. After a large number of screening verifications, the resistant medium can be: glucose 5 - 10 g / L, peptone 1 - 5 g / L, sodium chloride 1 - 5 g / L, beef extract powder 1 - 3 g / L, disodium hydrogen phosphate 0.03 - 1 g / L, agar 20 g / L, isoleucine hydroxamate 30 - 45 g / L, and adjust the pH to 7.0 with 4M sodium hydroxide.

[0052] In this example, the resistant medium used is: glucose 10 g / L, peptone 1.5 g / L, sodium chloride 1 g / L, beef extract powder 1 g / L, disodium hydrogen phosphate 1 g / L, agar 20 g, isoleucine hydroxamate 40 g / L, pH 7.0.

[0053] S4. Select the strains with yellow, smooth, well - growing colonies and relatively excellent biological shapes on the primary screening culture for flask rescreening, and detect the OD 600 value of the fermentation broth and the L - isoleucine content in the fermentation broth, and screen out the strains with good growth and high L - isoleucine production.

[0054] Among them, the rescreening culture conditions are: culture temperature 28 - 32 °C, flask rotation speed 150 - 200 rpm, culture time 12 - 36 h;

[0055] The flask rescreening medium can be: glucose 50 - 100 g / L, peptone 2 - 7 g / L, sodium chloride 1 - 5 g / L, beef extract powder 1 - 3 g / L, disodium hydrogen phosphate 0.3 - 1 g / L, yeast extract powder 1 - 5 g / L, ammonium sulfate 1 - 6 g / L, magnesium sulfate heptahydrate 2 - 4 g / L, ferrous sulfate 1 - 2 g / L.

[0056] In this example, the rescreening culture conditions are: culture temperature 30 °C, flask rotation speed 200 rpm, culture for 48 h;

[0057] The flask rescreening medium used is: glucose 50 g / L, peptone 2 g / L, sodium chloride 1 g / L, beef extract powder 3 g / L, disodium hydrogen phosphate 0.5 g / L, yeast extract powder 2 g / L, ammonium sulfate 3 g / L, magnesium sulfate heptahydrate 3 g / L, ferrous sulfate heptahydrate 1 g / L.

[0058] Detect the biomass and L - isoleucine fermentation yield of 40 Corynebacterium glutamicum mutant strains rescreened, and the results are shown in Table 1. Screen out 1 strain with high L - isoleucine production and high biomass, namely strain No. 37.

[0059] Table 1: OD values and L-isoleucine yields of different strains cultured for 48 h 600 Value and L-isoleucine production

[0060]

[0061]

[0062] The screened strain No. 37 was classified and named Corynebacterium glutamicum GIL-018, which was deposited at the China Center for Type Culture Collection (CCTCC) on October 16, 2023, with the deposit number CCTCC NO: M20231914. The deposit address is Wuhan University, Wuhan, China.

[0063] Example 2: Fermentation of Corynebacterium glutamicum GIL-018 to produce L-isoleucine

[0064] Using Corynebacterium glutamicum GIL-018 screened in Example 1 to ferment and produce L-isoleucine, including:

[0065] S1. Inoculate a loop of the strain cultured on the slant into the seed medium, and culture it at 30 °C, with an air flow rate of 2 vvm and a rotation speed of 400 rpm for 16 h to obtain a seed solution;

[0066] Among them, the composition of the seed medium is as follows: glucose 20 g·L -1 、yeast powder 5 g·L -1 、peptone 10 g·L -1 , pH 6.5.

[0067] S2. Transfer the seed solution to a 5 L fermenter containing 3 L of fermentation medium at a volume ratio of 10%, and ferment and culture it at 30 °C, with an air flow rate of 4 vvm and a stirring speed of 400 rpm for 48 h; during the fermentation process, the pH is controlled at 6.9 throughout.

[0068] Among them, the composition of the fermentation medium is as follows: glucose 182 g·L -1 、peptone 2 g·L -1 、sodium chloride 1 g·L -1 、beef extract powder 1 g·L -1 、disodium hydrogen phosphate 0.5 g / L, VB1 5 mg / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 3 g / L, ferrous sulfate 1.5 g / L, and adjust the pH to 6.5 with ammonia water.

[0069] Use HPLC to detect the yield of L-isoleucine (ILE) and the remaining amount of glucose (i.e., residual sugar content) in the fermentation broth, and calculate the sugar-acid conversion rate. The results are as followsFigure 2 as shown

[0070] To understand the change in the fermentation ability of Corynebacterium glutamicum GIL-018, under the same above fermentation conditions, fermentation verification of the parental strain - Corynebacterium glutamicum ATCC13869 was carried out, and the results are shown in Table 2.

[0071] Sugar-acid conversion rate = (L-isoleucine yield × fermentation volume) / [glucose dosage - (residual sugar content × fermentation volume)] × 100%.

[0072] Table 2: Comparison of L-isoleucine production capabilities between the parental strain and the mutant strain

[0073] Strain <![CDATA[OD 600 > L-isoleucine production / g / L Sugar-acid conversion rate / % Corynebacterium glutamicum ATCC13869 (parent strain) 40.75 3.12 12％ Corynebacterium glutamicum GIL-018 (mutant strain) 25.4 51.69 28％

[0074] As can be seen from Table 2, compared with Corynebacterium glutamicum ATCC13869, the acid production rate of Corynebacterium glutamicum GIL-018 screened in the present invention increased by 15.57 times, and the sugar-acid conversion rate increased by 133%, indicating that the mutant strain tolerant to L-isoleucine derivatives can be free from the feedback inhibition of L-isoleucine, and Corynebacterium glutamicum GIL-018 is more conducive to improving the production capacity and reducing the cost of industrial production of L-isoleucine.

[0075] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A Corynebacterium glutamicum strain that produces L-isoleucine, classified and named as Corynebacterium glutamicum GIL-018, with a deposit number of CCTCC NO: M20231914.

2. The Corynebacterium glutamicum according to claim 1, characterized in that The Corynebacterium glutamicum is tolerant to L-isoleucine and its derivatives.

3. The Corynebacterium glutamicum according to claim 1, characterized in that The L-isoleucine derivative is isoleucine hydroxamic acid.

4. Use of the Corynebacterium glutamicum according to any one of claims 1 - 3 in the production of isoleucine.

5. The use according to claim 4, characterized in that The method for producing isoleucine includes: fermentatively culturing the Corynebacterium glutamicum according to any one of claims 1-3.

6. The use according to claim 5, characterized in that The method for fermentative culture includes: inoculating the Corynebacterium glutamicum into a fermentation medium at an inoculum size of 5-15% (v / v) and performing shake culture under aeration conditions.

7. The use according to claim 6, characterized in that The fermentation medium is: 100-200 g / L of glucose, 2-7 g / L of peptone, 1-5 g / L of sodium chloride, 1-3 g / L of beef extract powder, 0.3-1 g / L of disodium hydrogen phosphate, 5-20 mg / L of VB1, 1-6 g / L of ammonium sulfate, 2-4 g / L of magnesium sulfate heptahydrate, 1-2 g / L of ferrous sulfate, with a pH of 6.0-7.

5.

8. The use according to claim 6, characterized in that Under aeration conditions, shake flask culture or stirred ventilation culture in a fermenter is performed.

9. The use according to claim 8, characterized in that The shaking speed of the shake flask culture is 250-400 rpm; the ventilation rate of the stirred ventilation culture in the fermenter is 2-4 vvm, and the stirring speed is 300-500 rpm.

10. The use according to claim 6, characterized in that The culture temperature is 28-32 °C, and the culture time is 40-60 h.

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