Genetic engineering strain for producing L-homoserine as well as preparation method and application of genetic engineering strain

Abstract

The invention belongs to the field of metabolic engineering, and particularly relates to a genetic engineering strain for producing L-homoserine as well as a preparation method and application of the genetic engineering strain. According to the invention, directed evolution is utilized, and the strain SHD275 for efficiently fermenting and producing L-homoserine is screened through continuous enrichment of spontaneous mutation of the homoserine producing strain SH017. Compared with the original strain SH017, the fermentation yield and the sugar conversion rate of the production strain are improved, and the production cost is greatly saved.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering, and specifically relates to a genetic engineering strain for producing L-homoserine, a preparation method and application thereof. Background Art

[0002] L-homoserine (2-amino-4-hydroxybutyric acid, HS) is a naturally occurring non-essential amino acid and an important precursor for the biosynthesis of aspartic acid family derived amino acids. It can not only be used to produce many important compounds, such as methionine, tetrahydrofuran, isobutanol, threonine and γ-butyrolactone, but can also be widely used in food, feed, biomedicine and other fields. At present, there are mainly the following methods for producing L-homoserine at home and abroad: chemical method, chemical chiral resolution method and biological method. The biological method mainly includes two methods: bioenzymatic method and fermentation method. The commonly reported bioenzymatic process is to use pyruvate and formaldehyde to generate L-homoserine under the joint action of aldolase and L-amino acid dehydrogenase. This process is costly and requires the use of toxic raw materials formaldehyde and formic acid, as well as expensive coenzymes. Microbial fermentation has many advantages such as low cost, mild conditions and less environmental pollution. In recent years, it has become the preferred process for producing various amino acids. Prior art CN112375726B discloses a genetically engineered bacterium with high yield of L homoserine, wherein the expression of the L homoserine kinase encoding gene thrB in the bacterium is weakened or eliminated, and the expression of the aspartate kinase feedback inhibition mutant gene lysC, the homoserine dehydrogenase feedback inhibition mutant gene hom, the overexpression aspartate kinase feedback inhibition mutant gene thrA and the transporter gene are further enhanced. The L homoserine high-yielding strain constructed by this invention can produce a higher level of L homoserine. In the best embodiment, the genetically engineered bacterium of the present invention is fermented for 72h, and the L homoserine content can reach 63.2±5.4g / L. Existing patent CN113151127A discloses the use of genetic engineering means to transform Escherichia coli, knock out or weaken the branch metabolic genes, and one or more synthetic pathway genes are expressed in a strengthened manner, thereby obtaining an engineered bacterium with high yield of homoserine.

[0003] However, since homoserine has a strong inhibitory effect on bacterial growth, large-scale production of L-homoserine has problems such as low yield and the need for expensive culture medium additives (such as amino acids, inducers, antibiotics), making direct fermentation production of L-homoserine challenging. Summary of the invention

[0004] The present invention aims at the deficiencies of the prior art and provides a genetic engineering strain for producing L-homoserine and a method for producing homoserine.

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

[0006] On the one hand, the present invention provides an L-homoserine production strain SHD275, wherein the production strain SHD275 is derived from Escherichia coli and is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and its deposit number is CGMCC No.32038.

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

[0008]

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

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

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

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

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

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

[0015] In another aspect, the present invention provides a method for producing homoserine by producing strain SHD275, comprising the following steps:

[0016] (1) Activation of SHD275: Inoculate SHD275 with a certain inoculum into a shake flask containing 30 mL of LB medium and culture at 37°C for 16 h until the OD600 reaches 3-4;

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

[0018] (3) inoculating a certain amount of the bacterial solution in step (2) into a 5 L fermenter containing 2 L of semi-synthetic medium, culturing at 37° C., and adjusting the pH to 6.9 with aqueous ammonia;

[0019] (4) maintaining the dissolved oxygen at 30%-40%. When the dissolved oxygen is higher than 40%, starting to feed the culture medium, homoserine is obtained after 19 hours of fermentation.

[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) comprises: 10-20 g glucose, 4-6 g ammonium sulfate, 2 g sodium chloride, 4 g potassium dihydrogen phosphate, 2 g magnesium sulfate heptahydrate, 0.105 g calcium chloride, 0.01 g zinc chloride, 1 mL TM3, 94 mg ferric citrate, 5 g corn steep liquor, 2.5 mg VB1, and 1 g dapoxetine per 1 L.

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

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This patent provides a strain that can efficiently ferment and produce L-homoserine. After ALE evolution, the strain can tolerate 80g / L homoserine. Compared with the non-evolved bacteria, the L-homoserine yield and glucose conversion rate are significantly improved, which is of great significance for reducing production costs.

[0025] Collection Instructions

[0026] Bacteria species: Escherichia coli;

[0027] Latin name: Escherichia coli;

[0028] Strain ID: SHD275;

[0029] Deposit number: CGMCC No.32038;

[0030] Deposit date: September 23, 2024;

[0031] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0032] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 2 This is a graph showing the tolerance of SH017 to homoserine.

[0035] Figure 3 This is the tolerance OD of the evolved strain after 3 months of subculture.

[0036] Figure 4 Screening of evolved strains for shake flasks.

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

[0038] Figure 6 Comparison of fermentation OD, homoserine production and sugar conversion rate of SH017 and SHD275 in 5L fermenter. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually selected according to conventional methods and conditions or according to the product specifications.

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

[0041] 1. Experimental reagents

[0042] (1) LB medium: Each liter of the medium contains 5 g of yeast powder, 10 g of sodium chloride, and 10 g of peptone. The volume is adjusted to 1 L with deionized water. Sterilize with high-pressure steam at 121°C for 20-30 min.

[0043] (2) Fermentation medium: 40 g glucose, 2 g yeast powder FM408, 2 g potassium dihydrogen phosphate, 15 g ammonium sulfate, 0.5 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.01 g MnSO4·7H2O, 100 mg / L Met, 100 mg / L Thre, dilute to 1 L with deionized water. Sterilize at 121°C for 20-30 min. Meanwhile, prepare empty shake flasks (250 mL) and weigh 0.4 g calcium carbonate into each flask to make the final concentration of calcium carbonate 20 g / L.

[0044] 2. Experimental equipment: constant temperature shaking 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 at 250 rpm; (2) Take 700 μL of the seeds after 16 h of culture and transfer them to 2 mL of fermentation medium containing antibiotics and culture them in a shaker at 37°C at 250 rpm for 4 h; (3) Transfer all 2 mL of secondary seeds into a shake flask containing 18 mL of fermentation medium and culture it in a shaker at 37°C at 250 rpm for about 20 h. Take 80 μL of the fermentation liquid and add 1520 μL of deionized water for dilution. After shaking evenly, centrifuge at 12,000 rpm for 1 min. Take 100 μL of the supernatant for derivatization treatment and sample preparation. For details of the detection method, see Basic Experiment 3.

[0047] Derivatization method: Take 100ul of the supernatant, add 100uL of 0.5M boric acid solution (pH7.7), mix well, incubate in a 30°C water bath for 3min, add 200uL of Fmoc-Cl solution, mix well, incubate in a 30°C water bath for 5min, add 800uL of ADAM solution, mix well for 2min, filter the organic phase and detect by HPLC.

[0048] Basic experiment 2: Method for producing L-homoserine by genetically engineered strains (fermentation tank)

[0049] The fermentation medium is 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 VB1, 1g dapoxetine per liter of medium, and the volume is made up with deionized water. The feed medium contains 500g glucose per liter, and the pH is adjusted to 6.9 with ammonia water; the TM3 solution is 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 the volume is made up to 1L with deionized water.

[0050] Experimental method: (1) Activate seeds, inoculate 0.25% of the seed glycerol tube into a 250mL shake flask containing 30mL LB, and culture at 37℃ for 16 hours until OD600 reaches 3-4; (2) Inoculate 1% into a 500mL seed shake flask containing 100mL LB medium, and culture at 37℃ for 4 hours until OD600 reaches 1-2; (3) Inoculate 10% into a 5L fermenter containing 2L semi-synthetic medium, and culture at 37℃. Adjust the pH to 6.9 with ammonia water, couple the dissolved oxygen speed, and maintain the dissolved oxygen at 30%. When the dissolved oxygen is higher than 40%, start coupling feeding to maintain the dissolved oxygen at 30%-45%. After 19 hours of fermentation, start sampling for HPLC detection. The detection method is shown in Basic Experiment 3.

[0051] Basic Experiment 3: Determination of L-homoserine in fermentation broth by HPLC

[0052] Accurately pipette the derivatized supernatant through a 0.22um organic filter membrane and detect by HPLC. The HPLC parameters are as follows: Agilent ZORBAX SB-C18, 4.6*150*5um; 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 1.0mL / min, column temperature 30°C; wavelength 254nm, injection volume 5ul; detection time 12min. Use an ultraviolet detector to detect at a wavelength of 254nm. The peak time of L-homoserine is 5.492 minutes. The HPLC spectrum is as follows Figure 1 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 experiment of homoserine on the growth of original production bacteria SH017

[0056] Referring to the fermentation medium formula in basic experiment 1, 0g / L, 40g / L, 60g / L, 80g / L, 100g / L, 120g / L and 140g / L homoserine solutions were prepared respectively. After high-temperature sterilization, they were divided into 20mL / 250mL Erlenmeyer flasks. 800uL of the overnight cultured SH017 seed solution was transferred to a shake flask and cultured at 37℃ and 250rpm for 24h. 100uL of the bacterial solution was taken out the next day, diluted 30 times with 50mM HCl, and the bacterial concentration was detected at a wavelength of 600nm. The results are as follows: Figure 2 SH017 strain began to become toxic to strain growth at a homoserine concentration higher than 60g / L, thus affecting metabolic synthesis.

[0057] Example 2 Production method of evolved strains

[0058] The homoserine-producing bacteria SH017 was streaked on a plate, and a single clone was obtained after culture at 37°C. Eight clones were randomly picked to activate the liquid shake tube. The next day, 500uL of the bacterial solution was transferred to a fermentation medium containing homoserine (20mL was dispensed into a 250mL Erlenmeyer flask). After culture at 37°C for 24h, 500uL of the bacterial solution was transferred to a fresh fermentation medium containing homoserine. 100uL of the remaining bacterial solution was taken out, diluted 30 times with 50mM HCl, and the bacterial concentration was measured (absorption was detected at a wavelength of 600nm). At the same time, the bacterial solution in each bottle was streaked on a plate and stored in a glycerol tube to ensure that each generation of strains was not contaminated; the control bacteria was transferred to a conventional fermentation medium.

[0059] The initial concentration of homoserine in the fermentation medium was set at 60 g / L. After obtaining the evolved bacteria that tolerated 60 g / L homoserine by daily transfer culture, the external pressure was continued to be increased to 80 g / L. After 3 months of continuous subculture, 3 stable evolved strains were obtained. The results are as follows Figure 3 As shown, the growth OD of the evolved strain was equal to or even slightly higher than that of the control strain.

[0060] Example 3 Shake flask screening of evolved strain SHD275

[0061] The three stable evolved strains in Example 2 were streaked on LB plates and cultured at 37°C to obtain single clones. 60 clones were randomly selected and fermented according to the shake flask fermentation method provided in Basic Experiment 1. The original production strain SH017 was used as the control strain, and clones with initial screening yield and OD better than SH017 were selected for rescreening. The rescreening results showed that Figure 4 The homoserine production of strains 1#-3, 5#-3, and 5#-10 was significantly higher than that of SH017. At the same time, the fermentation OD of 1#-3 and 5#-10 was also higher than that of the control strain. The strains obtained by repeated screening were tested again according to Example 1 to test the tolerance OD at a homoserine concentration of 80 g / L. The results are as follows Figure 5 As shown, 5#-10 was superior to SH017 in terms of fermentation yield, fermentation OD and tolerance OD, and the evolved bacteria 5#-10 was named SHD275.

[0062] Example 4 Fermentation yield, tolerance OD, and glucose conversion rate detection of the evolved strain SHD275

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

[0064] Table 2 Comparison of fermentation tank data of SHD275 and SH017

[0065] SH017 SHD275 Lower tank concentration g / L 132.8 144.5 Total volume / L 3.4 3.8 Total yield / 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 output / 60% glucose dosage / 0.6

[0067] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An L-homoserine producing strain SHD275, characterized in that: The production strain SHD275 is derived from Escherichia coli and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC No.32038.

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

3. The culture according to claim 2, characterized in that The culture medium comprises: 35-45 g of glucose, 1-3 g of yeast powder FM408, 1-3 g of potassium dihydrogen phosphate, 10-20 g of ammonium sulfate, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 0.01 g of MnSO4·7H2O, 100 mg / L of Met and 100 mg / L of Thre.

4. The culture according to claim 2 or 3, characterized in that The culture is culture fluid, culture fluid extract, whole bacteria, whole bacteria extract, fermentation fluid and / or fermentation fluid extract.

5. A bacterial agent, characterized in that The method comprises the production strain SHD275 according to claim 1 or the culture according to any one of claims 2 to 4.

6. The bacterial agent according to claim 5, characterized in that The bacterial agent is a powder or a liquid preparation.

7. Use of the production strain SHD275 according to claim 1, the culture according to any one of claims 2 to 4, or the bacterial agent according to any one of claims 5 to 6 in the preparation of L-homoserine.

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