A genetically engineered strain for producing L-homoserine, a preparation method and application thereof

Abstract

The present application belongs to the field of metabolic engineering, and particularly relates to a genetically engineered strain for producing L-homoserine, a preparation method and application thereof. The present application utilizes directed evolution, and through continuous enrichment of spontaneous mutation of homoserine-producing bacteria SH017, a strain SHD275 for efficiently fermenting and producing L-homoserine is screened. The fermentation yield and sugar conversion rate of the production strain are both higher than those of the original strain SH017, thereby greatly saving the production cost.

Description

Technical Field

[0001] This invention belongs to the field of metabolic engineering, specifically relating to a genetically engineered strain that produces L-homoserine, its preparation method, and its application. Background Technology

[0002] L-homoserine (2-amino-4-hydroxybutyric acid, HS) is a naturally occurring non-essential amino acid and an important precursor in the biosynthesis of aspartic acid family-derived amino acids. It is used not only to produce many important compounds, such as methionine, tetrahydrofuran, isobutanol, threonine, and γ-butyrolactone, but also has wide applications in food, feed, and biomedicine. Currently, the main methods for producing L-homoserine both domestically and internationally include chemical methods, chemical chiral resolution methods, and biological methods. Biological methods mainly include enzymatic methods and fermentation methods. A commonly reported enzymatic process utilizes pyruvate and formaldehyde to produce L-homoserine under the combined action of aldolase and L-amino acid dehydrogenase. This process is costly, requiring the use of toxic raw materials such as formaldehyde and formic acid, as well as expensive coenzymes. In contrast, microbial fermentation has many advantages such as low cost, mild conditions, and less environmental pollution, and has become the preferred process for producing various amino acids in recent years. Existing technology CN112375726B discloses a genetically engineered bacterium that produces high levels of L-homoserine. In this bacterium, the expression of the L-homoserine kinase encoding gene thrB is weakened or eliminated, and the expression of the aspartate kinase defecation feedback inhibition mutant gene lysC, the homoserine dehydrogenase defecation feedback inhibition mutant gene hom, the aspartate kinase defecation feedback inhibition mutant gene thrA, and transport protein genes are further enhanced. The L-homoserine-producing strain constructed by this invention can produce a high level of L-homoserine. In the preferred embodiment, after 72 hours of fermentation, the L-homoserine content of the genetically engineered bacterium can reach 63.2 ± 5.4 g / L. Existing patent CN113151127A discloses the use of genetic engineering to modify *E. coli*, knocking out or weakening branch metabolic genes, and enhancing the expression of one or more synthetic pathway genes to obtain a high-homoserine-producing engineered bacterium.

[0003] However, due to the strong inhibitory effect of homoserine on bacterial growth, large-scale production of L-homoserine faces challenges such as low yield and the need for expensive culture medium additives (such as amino acids, inducers, and antibiotics). Therefore, the direct fermentation production of L-homoserine is challenging. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a genetically engineered strain for producing L-homoserine and a method for producing homoserine.

[0005] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, the present invention provides a production strain of L-homoserine, SHD275, which is derived from Escherichia coli and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32038.

[0007] Specifically, the 16S rRNA sequence of the production strain SHD275 contains the nucleotide sequence shown in SED ID NO.1.

[0008]

[0009] On the other hand, the present invention provides a culture obtained by culturing or fermenting the aforementioned production strain SHD275 in a culture medium.

[0010] Specifically, the culture is a culture medium, a culture medium extract, whole bacteria, a whole bacteria extract, a fermentation broth, and / or a fermentation broth extract.

[0011] Specifically, the culture medium includes: 35-45g glucose, 1-3g yeast powder FM408, 1-3g potassium dihydrogen phosphate, 10-20g ammonium sulfate, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 0.01g MnSO4·7H2O, 100mg / L Met and 100mg / L Thre.

[0012] In some embodiments, the culture medium comprises: 40g glucose, 2g yeast extract FM408, 2g potassium dihydrogen phosphate, 15g ammonium sulfate, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 0.01g MnSO4·7H2O, 100mg / L Met and 100mg / L Thre.

[0013] In another aspect, the present invention provides a microbial agent comprising the aforementioned production strain SHD275 or the aforementioned culture.

[0014] Specifically, the bacterial agent is a powder or liquid preparation.

[0015] Furthermore, this invention provides a method for producing homoserine using strain SHD275, comprising the following steps:

[0016] (1) Activation of SHD275: Inoculate SHD275 into a shake flask containing 30mL LB medium at a certain inoculation amount, and incubate at 37℃ for 16h, OD600 to 3-4;

[0017] (2) Inoculate the bacterial solution from step (1) into a shake flask containing 100 mL of LB medium with a certain inoculation amount, and incubate at 37°C for 4 h until OD600 1-2;

[0018] (3) Inoculate the bacterial solution from step (2) with a certain amount of inoculation into a 5L fermenter containing 2L of semi-synthetic culture medium, and incubate at 37℃. Adjust the pH to 6.9 with ammonia water.

[0019] (4) Maintain dissolved oxygen at 30%-40%. When dissolved oxygen is above 40%, start feeding the culture medium. After fermentation for 19 hours, high serine is obtained.

[0020] Specifically, the inoculation amounts in steps (1) to (3) are 0.25%, 1%, and 10%, respectively.

[0021] Specifically, the semi-synthetic culture medium in step (3) includes: 10-20g glucose, 4-6g ammonium sulfate, 2g sodium chloride, 4g potassium dihydrogen phosphate, 2g magnesium sulfate heptahydrate, 0.105g calcium chloride, 0.01g zinc chloride, 1mL TM3, 94mg ferric citrate, 5g corn steep liquor, 2.5mg VB1, and 1g bubbly antagonist per 1L.

[0022] In another aspect, the present invention discloses the application of the above-mentioned production strain SHD275 in the preparation of L-homoserine.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This patent provides a strain capable of efficiently fermenting and producing L-homoserine. After ALE evolution, this strain can tolerate 80g / L of homoserine. Compared with non-evolved strains, it significantly improves the yield of L-homoserine and glucose conversion rate, which is of great significance for reducing production costs.

[0025] Preservation Instructions

[0026] Bacterial species name: Escherichia coli;

[0027] Latin name: Escherichia coli;

[0028] Strain number: SHD275;

[0029] Accession number: CGMCC No. 32038;

[0030] Date of deposit: September 23, 2024;

[0031] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0032] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0033] Figure 1 This is a chromatogram of HPLC peaks.

[0034] Figure 2 The graph shows the tolerance results of SH017 to homoserine.

[0035] Figure 3 The tolerance OD of the evolved strain after 3 months of subculturing.

[0036] Figure 4 To screen for evolutionary strains in shake flasks.

[0037] Figure 5 To retest the tolerance of the evolved dominant strain.

[0038] Figure 6 Comparison of fermentation OD, homoserine yield and sugar conversion rate of SH017 and SHD275 in 5L fermenters. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional methods and conditions or as selected in the product instructions.

[0040] Basic Experiment 1: Method for producing L-homoserine using genetically engineered strains (shake flask fermentation)

[0041] 1. Experimental reagents

[0042] (1) LB medium: Each liter of medium contains 5g yeast extract, 10g sodium chloride, 10g peptone, and deionized water to a final volume of 1L. Sterilize by high-pressure steam at 121℃ for 20-30 minutes.

[0043] (2) Fermentation medium: 40g glucose, 2g yeast powder FM408, 2g potassium dihydrogen phosphate, 15g ammonium sulfate, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 0.01g MnSO4·7H2O, 100mg / L Met, 100mg / L Thre, and deionized water to a final volume of 1L. Sterilize by autoclaving at 121℃ for 20-30 minutes. Simultaneously prepare empty shake flasks (250mL), weighing 0.4g of calcium carbonate into each flask to achieve a final calcium carbonate concentration of 20g / L.

[0044] 2. Experimental apparatus: constant temperature shaker incubator.

[0045] 3. Experimental methods:

[0046] (1) Inoculate the genetically engineered strain into 3 mL of LB medium containing antibiotics and culture it in a shaker at 37°C and 250 rpm. (2) Take 700 μL of the seed after 16 h of culture and transfer it to 2 mL of fermentation medium containing antibiotics. Culture it in a shaker at 37°C and 250 rpm for 4 h. (3) Transfer all 2 mL of secondary seed into a shake flask containing 18 mL of fermentation medium. Culture it in a shaker at 37°C and 250 rpm for about 20 h. Take 80 μL of fermentation broth and dilute it with 1520 μL of deionized water. After shaking evenly, centrifuge at 12000 rpm for 1 min. Take 100 μL of supernatant for derivatization treatment to prepare samples. For the detection method, please refer to Basic Experiment 3.

[0047] Derivatization method: Take 100 μL of supernatant, add 100 μL of 0.5 M boric acid solution (pH 7.7), mix well, incubate at 30 °C for 3 min, add 200 μL of Fmoc-Cl solution, mix well, incubate at 30 °C for 5 min, add 800 μL of ADAM solution, mix well and react for 2 min, filter with organic phase and detect by HPLC.

[0048] Basic Experiment 2: Method for producing L-homoserine using genetically engineered strains (fermenter)

[0049] The fermentation medium was a semi-synthetic medium, containing 5g ammonium sulfate, 2g sodium chloride, 4g potassium dihydrogen phosphate, 2g magnesium sulfate heptahydrate, 15g glucose, 0.105g calcium chloride, 0.01g zinc chloride, 1mL TM3, 94mg ferric citrate, 5g corn steep liquor, 2.5mg vitamin B1, and 1g bufotoxin per liter, diluted to volume with deionized water. The feed medium contained 500g glucose per liter, with the pH adjusted to 6.9 with ammonia. The TM3 solution consisted of 2.0g zinc chloride tetrahydrate, 2.0g calcium chloride hexahydrate, 2.0g sodium molybdate dihydrate, 1.9g copper sulfate pentahydrate, 0.5g boric acid, 100ml hydrochloric acid, and diluted to 1L with deionized water.

[0050] Experimental methods: (1) Activate the seeds by inoculating them into a 250mL shake flask containing 30mL LB medium at a seed glycerol tube at an inoculation rate of 0.25%, and incubate at 37℃ for 16 hours until the OD600 reaches 3-4; (2) Inoculate the seeds into a 500mL shake flask containing 100mL LB medium at a seed inoculation rate of 1%, and incubate at 37℃ for 4 hours until the OD600 reaches 1-2; (3) Inoculate the seeds into a 5L fermenter containing 2L semi-synthetic medium at a seed inoculation rate of 10%, and incubate at 37℃. Adjust the pH to 6.9 with ammonia water, couple the dissolved oxygen pump to maintain dissolved oxygen at 30%, and start feeding when the dissolved oxygen is higher than 40% to maintain dissolved oxygen at 30%-45%. After 19 hours of fermentation, samples are taken for HPLC detection. The detection method is described in Basic Experiment 3.

[0051] Basic Experiment 3: HPLC Determination of L-homoserine in Fermentation Broth

[0052] The derivatized supernatant was precisely pipetted through a 0.22 μm organic filter membrane and analyzed by HPLC. The HPLC parameters were as follows: Agilent ZORBAX SB-C18, 4.6*150*5 μm; mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile, initial ratio A:B = 60:40; gradient elution program (see Table 1); column flow rate: 1.0 mL / min; column temperature: 30℃; wavelength: 254 nm; injection volume: 5 μL; detection time: 12 min. A UV detector at 254 nm was used for detection. The peak time for L-homoserine was 5.492 min. The HPLC chromatogram is shown below. Figure 1 As shown.

[0053] Table 1 Gradient elution program

[0054] Time (min) Phase A (%) Phase B (%) 0.0 60 40 4.0 60 40 5.0 0 100 8.5 0 100 9.0 60 40 12.0 60 40

[0055] Example 1: Toxicity test of homoserine on the growth of the original production strain SH017

[0056] Following the fermentation medium formula in Basic Experiment 1, homoserine solutions of 0 g / L, 40 g / L, 60 g / L, 80 g / L, 100 g / L, 120 g / L, and 140 g / L were prepared. After high-temperature sterilization, the solutions were dispensed into 20 mL / 250 mL Erlenmeyer flasks. 800 μL of the overnight cultured SH017 seed culture was transferred to a shake flask and incubated at 37°C and 250 rpm for 24 h. The next day, 100 μL of the bacterial culture was taken out and diluted 30 times with 50 mM HCl. The bacterial concentration was detected at a wavelength of 600 nm. The results are as follows: Figure 2 The SH017 strain began to exhibit toxicity at concentrations above 60 g / L of high serine, thereby affecting metabolic synthesis.

[0057] Example 2: Method for producing the evolved strain

[0058] The homoserine-producing strain SH017 was streaked on a plate and cultured at 37°C to obtain single clones. Eight clones were randomly selected for activation and liquid culture in shake tubes. The next day, 500 μL of the bacterial culture was transferred to fermentation medium containing homoserine (20 mL aliquots in 250 mL Erlenmeyer flasks). After culturing at 37°C for 24 h, another 500 μL of the bacterial culture was transferred to fresh fermentation medium containing homoserine. 100 μL of the remaining bacterial culture was taken out, diluted 30 times with 50 mM HCl, and the bacterial concentration was measured (absorption was detected at 600 nm). At the same time, the bacterial culture in each flask was streaked on a plate and preserved in glycerol tubes to ensure that there was no contamination in each generation of the strain. The control strain was transferred to conventional fermentation medium.

[0059] The initial concentration of homoserine in the fermentation medium was set at 60 g / L. After obtaining evolved strains tolerant to 60 g / L homoserine through daily subculturing, the external pressure was further increased to 80 g / L. Through three months of continuous subculturing, three stable evolved strains were obtained. The results are as follows: Figure 3 As shown, the growth OD of the evolved strain was the same as or even slightly higher than that of the control strain.

[0060] Example 3: Shake-flask screening of evolutionary strain SHD275

[0061] The three stable evolutionary strains from Example 2 were streaked on LB agar plates and cultured at 37°C to obtain single clones. Sixty clones were randomly selected and fermented using the shake-flask fermentation method provided in Basic Experiment 1. The original production strain SH017 was used as a control strain. Clones with higher yields and OD values ​​than SH017 in the initial screening were selected for further screening. The results of the secondary screening showed... Figure 4 The homoserine production of strains 1#-3, 5#-3, and 5#-10 was significantly higher than that of SH017, and the fermentation OD of strains 1#-3 and 5#-10 was also higher than that of the control strain. The strains obtained from the secondary screening were then tested again according to Example 1 for tolerance OD at a homoserine concentration of 80 g / L, and the results are as follows... Figure 5 As shown, 5#-10 is superior to SH017 in terms of fermentation yield, fermentation OD, and tolerance OD. The evolved strain 5#-10 is named SHD275.

[0062] Example 4: Detection of fermentation yield, OD tolerance, and glucose conversion rate of the evolved strain SHD275

[0063] The evolved strain SHD275, screened in shake flasks, was scaled up and verified according to the operating procedures for a 5L fermenter provided in Basic Experiment 2, with SH017 used as a control. The fermentation results are as follows: Figure 6 As shown, the evolved strain SHD275 performed significantly better than the unevolved SH017 in a 5L fermenter. The OD was maintained at a relatively stable level after 43 hours. The fermentation yield after 48 hours was significantly higher than that of SH017. The yield reached 144 g / L after 67 hours, which was 12 g / L higher than that before evolution. The glucose conversion rate was increased by 5% (Table 2), which greatly saved production costs.

[0064] Table 2 Comparison of data from fermenters SHD275 and SH017

[0065] SH017 SHD275 Lower tank concentration g / L 132.8 144.5 Total volume / L 3.4 3.8 Total output / g 451.5 548.9 60% glucose dosage / g 1447.1 1605.0 Glucose conversion rate % 52.40 57.20

[0066] Note: Glucose conversion rate % = Total yield / 60% glucose usage / 0.6

[0067] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A L-homoserine production strain SHD275 of L-homoserine, which is significantly improved in L-homoserine tolerance and yield, characterized in that, The production strain SHD275 is derived from Escherichia coli (E. coli) Escherichia coli , and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 32038.

2. A culture, characterized in that, The culture is obtained by culturing or fermenting the production strain SHD275 of claim 1 in a medium.

3. The culture of claim 2, wherein, The culture is a culture solution, whole bacteria and / or a fermentation solution.

4. An inoculant characterized in that, The bacterial agent is a powder or a liquid preparation.

5. The bacterial agent of claim 4, wherein 6. Use of the production strain SHD275 of claim 1 or the culture of any one of claims 2-3 or the bacterial agent of any one of claims 4-5 in the preparation of L-homoserine. ​

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