Application of L-amino acid transporter or its encoding gene in improving the ability of Escherichia coli to produce L-amino acids

Abstract

The present invention discloses an application of an L-amino acid transporter or a gene encoded thereof in improving the ability of Escherichia coli to produce L-amino acids, and relates to the field of genetic engineering. aaeA , adeQ , yijE , ydfJ and acrE The mutant strain has an improved L-amino acid production capacity by comprising one or more genes in the Escherichia coli ). The mutant strain can exert an improved L-amino acid production capacity compared to the parent strain and can produce high-concentration L-amino acids.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to the application of an L-amino acid transport protein or a gene encoded by the L-amino acid transport protein in improving the ability of Escherichia coli to produce L-amino acids. Background Art

[0002] With the rapid development of biosynthesis technology, the production and application of industrial amino acids have become a key force in promoting innovation and upgrading in many industries such as medicine, food and feed. In recent years, with the in-depth exploration of microbial resources and the continuous advancement of gene editing technology, people have developed a series of engineered strains that can efficiently produce amino acids, which has significantly reduced the production cost of amino acids and broadened their application range. Among them, aromatic amino acids - tryptophan, phenylalanine and tyrosine, with their unique molecular structure and wide range of physiological activities, have shown great application potential in the above fields.

[0003] Microbial fermentation, as the mainstream technology for the production of aromatic amino acids, has become the preferred process in the industry due to its advantages such as environmental friendliness, wide range of raw materials and large-scale production. However, the biosynthetic pathway of aromatic amino acids is complex and lengthy, involving multi-step enzymatic reactions and sophisticated metabolic regulation mechanisms, which greatly limits the improvement of production efficiency. Therefore, how to accurately transform the production strain through metabolic engineering technology has become the key to improving the yield and quality of aromatic amino acids.

[0004] In response to this challenge, researchers continue to explore and apply advanced strategies such as system metabolic engineering and synthetic biology to optimize the biosynthetic pathway of aromatic amino acids. In particular, strategies that enhance product efflux capacity can reduce feedback inhibition and cytotoxicity caused by intracellular product accumulation.

[0005] In terms of product efflux, although studies have reported the application of the transporter YddG in increasing the production of aromatic amino acids, the improvement effect is limited, suggesting that there may be other unexplored aromatic amino acid transporters in microbial cells. These potential transporters may have higher transport efficiency and broader substrate specificity, which is crucial for further improving the production efficiency of aromatic amino acids.

[0006] In the prior art, for example, a patent application with publication number CN116515918A discloses an L-tryptophan (L-Trp) transport application of MdtL and a production method and strain, which relates to the field of microbial technology. The invention also provides the application of MdtL in the production of tryptophan. The production of tryptophan is to ferment the recombinant strain overexpressing the MdtL transport protein to produce tryptophan. The invention found that the yield of L-tryptophan fermented in a shake flask by the recombinant strain overexpressing the MdtL transport protein was higher than that of the control group, and the content of the byproduct shikimic acid was reduced. And the patent application with publication number CN118006520A discloses a mutant strain of Escherichia coli or a mutant strain of Corynebacterium glutamicum that produces L-amino acids and a method for producing L-amino acids using the same. According to the invention, the mutant strain of Escherichia coli and the mutant strain of Corynebacterium glutamicum that produces L-amino acids have the ability to improve the production of L-amino acids compared with the parent strain, and can efficiently produce high-concentration L-amino acids.

[0007] Based on such prior studies, the present invention has produced various L-amino acid-producing mutants having improved L-amino acid production capabilities by regulating the expression of L-amino acid excretion genes in Escherichia coli strains. Summary of the invention

[0008] Based on the deficiencies in the prior art, the present invention provides an application of an L-amino acid transporter or a gene encoding thereof in improving the ability of Escherichia coli to produce L-amino acids. By overexpressing one or more L-amino acid transporters mentioned in the present invention in an Escherichia coli mutant strain, its production capacity of producing a variety of aromatic amino acids can be effectively improved.

[0009] The present invention aims to construct a series of efficient and stable engineered strains, which can not only effectively increase the yield of aromatic amino acids, but also reduce production costs, and provide more economical and environmentally friendly amino acid raw materials for industries such as medicine, food and feed.

[0010] The invention provides application of an L-amino acid transporter or a gene encoded thereby in improving the ability of Escherichia coli to produce L-amino acids. The L-amino acid transporter is AaeA, AdeQ, YijE, YdfJ or AcrE.

[0011] Preferably, the amino acid sequences of AaeA, AdeQ, YijE, YdfJ and AcrE are shown in SEQ ID NO.1-5, respectively; the L-amino acid is selected from one of L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamine, L-lysine, L-arginine, L-valine, L-leucine, L-isoleucine, L-threonine, L-histidine, L-serine and L-citrulline.

[0012] Further preferably, the base sequences of the genes encoding the AaeA, AdeQ, YijE, YdfJ and AcrE are shown as SEQ ID NO.6-10 respectively; and the L-amino acid is L-tryptophan and / or L-phenylalanine.

[0013] The present invention also provides a mutant strain, which is a mutant strain having improved L-amino acid production capacity by overexpressing one or more genes selected from AaeA, AdeQ, YijE, YdfJ and AcrE, wherein the mutant strain is Escherichia coli ( Escherichia coli ).

[0014] Preferably, the base sequences of AaeA, AdeQ, YijE, YdfJ and AcrE are shown in SEQ ID NO.6-10 respectively.

[0015] Further, the L-amino acid is selected from one of L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamine, L-lysine, L-arginine, L-valine, L-leucine, L-isoleucine, L-threonine, L-histidine, L-serine and L-citrulline.

[0016] Preferably, the L-amino acid is L-tryptophan and / or L-phenylalanine.

[0017] The present invention also provides a method for producing L-amino acids, comprising the following steps:

[0018] The mutant strain is fermented and cultured, and L-amino acids are extracted and obtained.

[0019] Furthermore, the L-amino acid is selected from one of L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamine, L-lysine, L-arginine, L-valine, L-leucine, L-isoleucine, L-threonine, L-histidine, L-serine and L-citrulline.

[0020] Preferably, the L-amino acid is L-tryptophan and / or L-phenylalanine.

[0021] The present invention provides a mutant strain with improved L-amino acid production capacity by overexpressing genes encoding one or more L-amino acid transporters selected from AaeA, AdeQ, YijE, YdfJ and AcrE. The L-amino acid-producing Escherichia coli mutant strain has improved L-amino acid production capacity compared with the parent strain and can efficiently (the yield can be increased by up to 71.4% compared with the parent strain) produce high-concentration L-amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the plasmid map of pACYC184-aaeA.

[0023] Figure 2 This is the plasmid map of pACYC184-adeQ.

[0024] Figure 3 This is the plasmid map of pACYC184-yijE.

[0025] Figure 4 This is the plasmid map of pACYC184-ydfJ.

[0026] Figure 5 This is the plasmid map of pACYC184-acrE.

[0027] Figure 6 This is the plasmid map of pACYC184-eGFP.

[0028] Figure 7 This is a schematic diagram of the L-amino acid transporter of the present invention improving the production capacity of L-amino acids. DETAILED DESCRIPTION

[0029] The schematic diagram of the L-amino acid transporter of the present invention improving the production capacity of L-amino acids is as follows Figure 7 As shown, the present invention is further described below in conjunction with specific examples, but is not intended to limit the present invention.

[0030] Example 1

[0031] An L-tryptophan-producing Escherichia coli mutant strain overexpressing AaeA, AdeQ, YijE, YdfJ and AcrE was constructed.

[0032] First, Escherichia coli The genomic DNA of str. K-12 substr. MG1655 was used as a template and amplified using primer pairs aaeA-F&aaeA-R, adeQ-F&adeQ-R, yijE-F&yijE-R, ydfJ-F&ydfJ-R, and acrE-F&acrE-R (Table 1) aae , Q , Yj , Yj Y and acrE Gene fragments (the gene sequences are shown in SEQ ID NOs.6-10, and the amino acid sequences of the proteins encoded by the genes are shown in SEQ ID NOs.1-5);

[0033] Using pEGFP-C1 as a template, eGFP-F & eGFP-R primers were used to amplify the eGFP fragment (gene sequence as shown in SEQ ID NO.11) as an overexpression control group;

[0034] The pACYC184 plasmid was used as a template, and the linear vector fragment was amplified with pACYC184-F & pACYC184-R primers; the connection was performed according to the conditions in Table 2. Then 10 μL of the connection system was transferred into DH5α competent cells and cultured at 37°C overnight. The positive clones were selected and sent for sequencing for verification. The successfully constructed plasmids were named pACYC184-aaeA, pACYC184-adeQ, pACYC184-yijE, pACYC184-ydfJ, pACYC184-acrE and pACYC184-eGFP (see the plasmid map). Figure 1-6 ).

[0035] In the previous work, our laboratory used Escherichia coli K-12 as the starting strain by knocking out TnaA (Gene ID: 948221) and overexpressing AroG S180F (Gene ID: 945605), SerA (Gene ID: 945258) and TrpE S40F A tryptophan high-producing strain was obtained after DCBA (GeneID: 945846), named LTrp. The above six plasmids were transformed into the tryptophan strain LTrp, respectively, and spread on solid LB medium (SangonBiotech, A507003-0250) containing tetracycline (50 mg / L) and ampicillin (50 mg / L) and cultured at 37°C overnight. The next day, the grown strains were tested by PCR, and the correct strains were named LTrp-aaeA, LTrp-adeQ, LTrp-yijE, LTrp-ydfJ, LTrp-acrE and LTrp-eGFP, respectively.

[0036] Table 1. Primers used in Example 1

[0037]

[0038] Table 2. Construction of plasmid ligation reaction system

[0039]

[0040] Example 2

[0041] Fermentation of the tryptophan mutant strain constructed in Example 1 in a micro-bioreactor and detection of L-tryptophan production.

[0042] Biolector fermentation process;

[0043] The LTrp-aaeA, LTrp-adeQ, LTrp-yijE, LTrp-ydfJ, LTrp-acrE and LTrp-eGFP glycerol strains obtained in Example 1 were rewarmed to room temperature, coated on antibiotic solid LB medium containing tetracycline (50 mg / L) and ampicillin (50 mg / L) by the partitioning streak method, and cultured overnight at 37°C, a single colony with good growth status was picked and inoculated into a 48-well plate, LB medium containing tetracycline (50 mg / L) and ampicillin (50 mg / L) was added, and cultured overnight in a Microscreen (Jie Ling Instrument Manufacturing (Tianjin) Co., Ltd.) incubator at 37°C 800 rpm to the logarithmic phase, the logarithmic phase bacterial solution was inoculated into a new 48-well plate at a ratio of 1:100, 900 μL of primary fermentation medium was added to each well, and the plate was incubated in a Microscreen incubator at 37°C 800 rpm. rpm overnight culture to the logarithmic phase; the logarithmic phase bacterial solution was inoculated into the Biolector (BECKMAN COULTER) well plate at a ratio of 1:10, 800 μL of secondary fermentation medium was added to each well, and the fermentation culture was carried out in the Biolector for about 40 hours and then the plates were taken out for detection.

[0044] Table 3. Primary fermentation medium for tryptophan strains

[0045]

[0046] Table 4. Secondary fermentation medium for tryptophan strains

[0047]

[0048] OD 600 And L-tryptophan detection method:

[0049] Take out the sample after fermentation, dilute the bacterial solution 100 times, and measure the OD at 600 nm using a spectrophotometer. 600 and record; L-tryptophan was detected by the p-dimethylaminobenzaldehyde colorimetric method, the fermentation supernatant was diluted 100 times, 100 μL of the diluted supernatant was added to 900 μL of p-dimethylaminobenzaldehyde, mixed thoroughly and boiled in a water bath for 2 minutes, taken out and added a drop of 2% sodium nitrite, inverted and mixed, and boiled in a water bath for 3 minutes, quickly transferred to ice for cooling, and the absorbance value at 600 nm of a spectrophotometer was recorded using a colorimetric dish, a standard curve was drawn according to the detection results of the L-tryptophan standard, and the L-tryptophan concentration was calculated according to the absorbance values ​​of different samples.

[0050] The results are shown in Table 5. Compared with the LTrp-eGFP control strain, the gene aae , Q , Yj , Yj Y , acrE Overexpression of aae The effect was the most significant, with L-tryptophan production increasing by 71.3%.

[0051] Table 5. L-tryptophan (L-Trp) yields of different strains in Biolector fermentation

[0052]

[0053] Example 3

[0054] Constructing overexpressed genes aae , Q , Yj , Yj Y and acrE A mutant strain of Escherichia coli producing L-phenylalanine.

[0055] In the previous work, our laboratory used Escherichia coli K-12 as the starting strain to overexpress AroG S180F (Gene ID: 945605), SerA (Gene ID: 945258), PheA T326P (Gene ID:947081) and TyrB(Gene ID:948563), a phenylalanine high-yielding strain was obtained, named LPhe. The six plasmids pACYC184-aaeA, pACYC184-adeQ, pACYC184-yijE, pACYC184-ydfJ, pACYC184-acrE and pACYC184-eGFP constructed in Example 1 were respectively transferred into LPhe, and coated on antibiotic solid LB medium containing tetracycline (50 mg / L) and ampicillin (50 mg / L) and cultured at 37°C overnight. The next day, the grown strains were tested by PCR, and the correct strains were named LPhe-aaeA, LPhe-adeQ, LPhe-yijE, LPhe-ydfJ, LPhe-acrE and LPhe-eGFP, respectively. Example

[0056] Example 3 Shake flask fermentation of the constructed phenylalanine mutant strain and detection of L-phenylalanine production.

[0057] The phenylalanine mutant strain constructed in Example 3 was taken out of the -80°C refrigerator and revived overnight on an LB plate, eluted with 40 mL of sterile water, inoculated into a seed culture medium (Table 6) at a ratio of 150 mL / 1000 mL, cultured at 32.5°C for 7-8 hours, and then inoculated into a fermentation culture medium (Table 7) at a 10% inoculum amount, with a shake flask volume of 50 mL / 500 mL, cultured at 37.5°C for about 43 hours, and sampled for detection. The L-phenylalanine content in the supernatant of the fermentation broth was detected by HPLC.

[0058] The specific steps for HPLC detection of L-phenylalanine are as follows:

[0059] About 1 mL of fermentation broth was collected and centrifuged at 13000rpm for 2 min, and then the supernatant was taken. The supernatant was diluted to an appropriate concentration with deionized water, filtered using a microporous filter membrane with a pore size of 0.22 μm and injected into a liquid phase bottle for detection. The chromatographic column was a Kromasil C18 column (250 mm×460 mm, 5 μm), and the mobile phase was 10% (v / v) acetonitrile. The detection parameters were: UV detection wavelength of 210 nm, detection temperature of 40°C, flow rate of 1 mL / min, injection volume of 20 μL each time, analysis time of 10 min, and the peak time of L-phenylalanine was 2.612 min. The L-phenylalanine standards with concentrations of 0.0625 g / L, 0.125 g / L, 0.25 g / L, 0.5 g / L, and 1 g / L were set to establish a standard curve, and the L-phenylalanine concentration was calculated based on the peak area and the corresponding linear formula.

[0060] Table 6. Seed culture medium of phenylalanine strain

[0061]

[0062] Table 7. Fermentation medium for phenylalanine strains

[0063]

[0064] Table 8. L-phenylalanine (L-Phe) production in shake flask fermentation

[0065]

[0066] The results are shown in Table 8. Compared with the LPhe-eGFP control strain, the gene aae , Q , Yj , Yj Y , acrE Overexpression of aae The effect is the most significant, and the increase in L-phenylalanine production is the largest.

Claims

1. Use of an overexpressed L-amino acid transporter or a gene encoding the same in improving the ability of Escherichia coli to produce L-amino acids, characterized in that: The L-amino acid transporter is YijE; The L-amino acid is selected from one of L-tryptophan, L-phenylalanine and L-tyrosine; The amino acid sequence of YijE is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The base sequence of the gene encoding the YijE is shown in SEQ ID NO.

8.

3. A mutant strain, characterized in that: A mutant strain having improved L-amino acid production capacity by overexpressing a gene encoding YijE, wherein the mutant strain is Escherichia coli ( Escherichia coli ); The L-amino acid is selected from one of L-tryptophan, L-phenylalanine and L-tyrosine; The amino acid sequence of YijE is shown in SEQ ID NO.

3.

4. The mutant strain according to claim 3, characterized in that The base sequence of the gene encoding YijE is shown in SEQ ID NO.

8.

5. A method for producing L-amino acids, characterized in that: The following steps are involved: Fermenting and culturing the mutant strain according to claim 3, and extracting and obtaining L-amino acids; The L-amino acid is selected from one of L-tryptophan, L-phenylalanine and L-tyrosine.

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