CN109468254B - Method for improving production efficiency of L-alanine and application thereof - Google Patents
Method for improving production efficiency of L-alanine and application thereof Download PDFInfo
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Abstract
The invention discloses a method for improving the production efficiency of L-alanine and application thereof, belonging to the technical field of biological engineering. The invention utilizes genetic engineering means to modify screened mutant strain FMME-A232 growing to produce acid under high-concentration glucose, strengthens the transfer path of glucose, and uses pEtac plasmid to express glucose transporter EI protein and EIIA protein in seriesClcThe recombinant Escherichia coli is obtained from the protein, and can improve the consumption rate of glucose, so that the fermentation period of the strain is reduced from 55h to 45h, the yield of L-alanine reaches 160.8g/L, and the production efficiency of L-alanine is improved.
Description
Technical Field
The invention relates to a method for improving the production efficiency of L-alanine and application thereof, belonging to the technical field of biological engineering.
Background
L-alanine is a non-essential amino acid for human body, and has wide application in the fields of medicine, food and the like. In the field of medicine, L-alanine is used for synthesizing amino acid infusion, and is also a main raw material for synthesizing vitamin B6 and aminopropanol; in the field of food, L-alanine has special fragrance and sweet taste, and is applied to beverages, cakes, dairy products and the like as a flavor seasoning, and the nutritional value of the food can be improved by adding the L-alanine.
The production process of L-alanine mainly comprises an enzyme catalysis method and a fermentation method. The main raw material L-aspartic acid of the enzyme catalysis method has high price, the fumaric acid is produced by refining petroleum, and the petroleum resource belongs to non-renewable resources. The fermentation method generally realizes the high yield of L-alanine by genetically engineering to transform Escherichia coli with clear genetic background. The applicant carries out metabolic engineering transformation on Escherichia coli K12 in a patent application with the application number of 201811234785.3, and further carries out adaptive evolution on the strain to obtain Escherichia coli FMME-232A capable of highly producing L-alanine under the condition of high-concentration glucose, wherein the L-alanine yield can reach 171.0g/L after fermentation in a 30L small-scale fermentation tank for 55 hours. The fermentation of the grapes is utilized to produce the L-alanine, the production cost is reduced, and the method is a sustainable renewable biomass resource,
however, the high concentration of glucose results in a prolonged fermentation period of L-alanine and a reduced production efficiency.
Disclosure of Invention
On the basis of the patent application with the application number of 201811234785.3, the invention constructs a novel strain capable of efficiently producing L-alanine by taking Escherichia coli FMME-232A with high yield of L-alanine as an initial strain and strengthening a glucose transport path, and provides a method for improving the production efficiency of L-alanine and application thereof.
The first purpose of the invention is to provide an Escherichia coli recombinant bacterium, which expresses EI protein coded by ptsI gene and EIIA coded by crr gene in seriesClcA protein.
Optionally, the amino acid sequence of EI protein of ptsI gene is shown in SEQ ID NO.1, and EIIA of crr geneClcThe amino acid sequence of the protein is shown as SEQ ID NO. 2.
Optionally, the escherichia coli recombinant strain is obtained by modifying a glucose transport pathway in escherichia coli FMME-A232, wherein the escherichia coli FMME-A232 is preserved in a China Center for Type Culture Collection (CCTCC) in 2018, 9 and 17 months, the preservation number is CCTCC NO: M2018628, and the preservation address is university of Wuhan, China.
Alternatively, the pEtac plasmid is used for tandem expression of the glucose transporter EI protein and EIIAClcA protein.
Alternatively, the pEtac plasmid is used for tandem expression of the glucose transporter EI protein and the EIIAClcThe protein comprises:
tandem expression of EI transporter encoded by ptsI Gene and EIIA encoded by crr Gene using pEtac plasmidClcTransporter, construction of recombinant plasmid pEtac-EI-EIIAClc(ii) a The recombinant plasmid pEtac-EI-EIIAClcAnd introducing into Escherichia coli FMME-A232 to obtain Escherichia coli recombinant bacteria.
The second purpose of the invention is to provide the application of the escherichia coli recombinant bacteria in food and/or medicines.
Alternatively, the application is the production of L-alanine by using the Escherichia coli recombinant strain.
The third purpose of the invention is to provide a method for producing L-alanine by fermentation, wherein the method is to use the recombinant bacterium of the large intestine bacillus to produce L-alanine by fermentation.
Optionally, the method includes: inoculating the recombinant Escherichia coli to an LB culture medium containing 100mg/L kanamycin sulfate, performing shake culture at 37 ℃ overnight, inoculating the recombinant Escherichia coli to a fermentation culture medium according to the inoculum size of 10-15%, performing early aerobic fermentation at the temperature of 26-30 ℃, with the air amount of 1.0-2.0 vvm, stirring at the rotating speed of 300-400 rpm, culturing for 6-8 h, adding 4-8 g/L lactose to induce EI protein and EIIAClcAnd (3) expressing the protein, continuously culturing for 15-25 h, starting oxygen-limited stage fermentation, reducing the ventilation volume to 0.01-0.1 vvm, reducing the stirring rotation speed to 50-100 rpm, raising the temperature to 40-43 ℃, and immediately finishing the fermentation after the glucose is consumed.
Optionally, the fermentation medium comprises: 180-220 g/L glucose, 3-6 g/L corn steep liquor, 2-6 g/L yeast powder and K2HPO4·12H2O 2～6g/L，KH2PO4 4～6g/L，MgSO4·7H20.4-0.6 g/L of O and 0.2-0.4 g/L of citric acid monohydrate.
The invention has the beneficial effects that:
modifying the screened mutant strain FMME-A232 growing to produce acid under high concentration glucose by using genetic engineering means, strengthening the transport path of glucose, and using pEtac plasmid to express glucose transporter EI protein and EIIA protein in seriesClcThe protein is obtained into an escherichia coli recombinant strain, the consumption rate of glucose can be improved by the escherichia coli recombinant strain, the fermentation period of the strain is reduced from 55 hours to 45 hours, and the yield of L-alanine is reduced160.8g/L is achieved.
Biological material preservation information:
escherichia coli FMME-A232 is preserved in a China center for type culture preservation in 2018, 9 and 17 months, with the preservation number of CCTCC NO: M2018628 and the preservation address of China, Wuhan university.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows pEtac-EI-EIIAClcSchematic representation of the construction of the co-expression plasmid.
Detailed Description
In the following examples, the materials used are all commercially available using routine experimentation.
In the following examples, Escherichia coli FMME-232 was deposited in the China center for type culture Collection in 2018, 9, 17, M2018628 with the deposition number of CCTCC NO, and the deposition address of university of Wuhan, China.
The amino acid detection adopts conventional high performance liquid chromatography.
The glucose determination method comprises the following steps: the analysis was performed using an SBA-40 biosensing analyzer.
Calculation of production strength: production intensity (g/L/h) ═ L-alanine yield (g/L)/fermentation time (h).
Calculation of glucose yield: glucose yield (%) ═ L-alanine maximum yield (g/L)/total glucose addition (g/L) x 100.
Example 1: construction of recombinant Escherichia coli expressed by single gene
The PTS transport pathway is composed of 4 proteins EI (ptsI), HPr (ptsH), EIIAGlc(err)、EIIBCGlc(ptsG) and constructing a single gene expression strain and screening genes capable of shortening the fermentation period of L-alanine.
(1) Extracting the genome DNA of the Escherichia coli K12 by using a bacterial genome extraction kit;
(2) the pstI (SEQ ID NO.1), ptsH (SEQ ID NO.3), crr (SEQ ID NO.2), ptsG (SEQ ID NO.4) genes were cloned from genomic DNA using the primers ptsI-up and ptsI-down, ptsH-up and ptsH-down, err-up and err-down, ptsG-up and ptsG-down in Table 1;
(3) using SalI and HindIII to cut target expression vector pEtac and target fragment;
(4) connecting the cloned double enzyme digestion vector with a target fragment by using a DNA connection kit to obtain a recombinant plasmid, and thermally shocking and transforming the plasmid into FMME-A232 to obtain recombinant strains FMME-A232EI, FMME-A232HPr, FMME-A232EIIA and FMME-A232 EIIBC;
TABLE 1 construction primers for single expression plasmids
Example 2: fermentation characteristics of recombinant Escherichia coli
Fermentation medium components: 180g/L glucose, 3g/L corn steep liquor, 2g/L yeast powder and K2HPO4·12H2O 2g/L，KH2PO45g/L，MgSO4·7H20.5g/L of O and 0.3g/L of citric acid monohydrate.
Examining the fermentation characteristics of the recombinant strain through flask fermentation: the original strain FMME-A232 and the recombinant strains FMME-A232EI, FMME-A232HPr, FMME-A232EIIA and FMME-A232EIIBC constructed in example 1 were inoculated into LB medium (100 mg/L kanamycin sulfate was added to the culture medium of the recombinant bacteria) respectively for overnight culture. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 10 percent, and performing aerobic-oxygen-limited two-stage fermentation culture. And (3) performing shake culture on the aerobic bacteria at 30 ℃ and 150rpm until the OD600 of the cells reaches 10, adding 2g/L lactose to induce the expression of the target protein, continuing to perform culture until the OD600 reaches 25, and performing shake culture at 25rpm and 42 ℃ in an oxygen-limited fermentation stage for 85 hours. The experimental results are shown in Table 2, and the fermentation periods of the recombinant strain FMME-A232EI and FMME-A232EIIA are compared with that of the original strain FMME-A232 are all shortened by 10h, the production intensity limit is enhanced, which shows that EI and EIIA are over-expressedClcThe protein can increase the glucose transport rate, shorten the fermentation period and further improve the production efficiency of the L-alanine.
TABLE 2 fermentation characteristics of different recombinant E.coli
Example 3: construction of recombinant Escherichia coli with double gene expression
(1) The pstI gene was cloned from the genomic DNA of E.coli K12 using the primers ptsI-up 1 and ptsI-down1 in Table 2, and the crr gene was cloned from the genomic DNA of E.coli K12 using crr-up1 and crr-down 1;
(2) the expression vector pEtac is cut by HindIII and EcoRI;
(3) connecting the two target fragments obtained by cloning with a double enzyme digestion vector by using a one-step recombinant cloning kit to obtain a recombinant plasmid pEtac-EI-EIIAClc(see FIG. 1), the plasmid was transformed into FMME-A232 competent cells by heat shock to obtain recombinant strain FMME-A232 EE.
TABLE 3 construction primers for tandem co-expression plasmids
Example 4: l-alanine produced by fermenting original strain and recombinant strain
Fermentation medium components: glucose 180g/L, corn steep liquor 4g/L, yeast powder 4g/L, K2HPO4·12H2O 5g/L， KH2PO45g/L，MgSO4·7H20.5g/L of O and 0.3g/L of citric acid monohydrate.
FMME-A232 fermentation culture: inoculating FMME-A232 strain in LB culture medium, shaking for overnight culture at 37 ℃, inoculating in fermentation culture medium according to 10% inoculum concentration, performing early aerobic fermentation at 26 ℃, air amount of 1.0vvm, stirring at 350rpm for 15h, starting oxygen-limited stage fermentation, reducing ventilation to 0.1vvm, stirring at 75rpm, raising temperature to 42 ℃, and immediately ending fermentation after glucose is consumed.
FMME-A232EE fermentation culture: inoculating FMME-A232EE strain in LB medium containing 100mg/L kanamycin sulfate, shake culturing at 37 deg.C overnight, inoculating 10% of strain in fermentation medium, performing aerobic fermentation at 26 deg.C with air amount of 1.0vvm, stirring at 350rpm, culturing for 6 hr, adding 4g/L lactose to induce EI protein and EIIAClcProtein expression, continuing culturing for 15h, starting oxygen-limited stage fermentation, reducing ventilation to 0.1vvm, reducing stirring speed to 75rpm, raising temperature to 42 ℃, and ending fermentation immediately after glucose is consumed.
The results of fermentation experiments are shown in Table 4, and under the same culture conditions, when the glucose concentration is 180g/L, the fermentation period of FMME-A232EE is shortened by 15h compared with that of FMME-A232, and the yields of the two strains are basically consistent.
TABLE 4 fermentation characteristics of the original and recombinant strains
Example 5: influence of glucose concentration on L-alanine production by fermentation of recombinant strains
The prepared glucose concentrations in the fermentation medium were 180g/L, 200g/L and 220g/L, respectively, and the effect of different glucose concentrations on the fermentation production of L-alanine from FMME-A232EE was examined. The rest of the fermentation medium was the same as in example 4, and the fermentation was carried out in the same manner as in example 4. The results are shown in Table 5: when the concentration of glucose in the fermentation medium is 200g/L, the concentration of L-alanine reaches 160.8g/L when the fermentation is carried out for 45 hours, and the yield of glucose and the production intensity of L-alanine reach 80.4 percent and 3.57 g/L/h; when the glucose concentration was further increased to 220g/L, a significant decrease in the glucose yield occurred.
TABLE 5 Effect of different glucose concentrations on L-alanine production by recombinant bacteria
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for improving production efficiency of L-alanine and application thereof
<160> 16
<170> PatentIn version 3.3
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Claims (7)
1. An Escherichia coli recombinant bacterium, which is characterized in that the Escherichia coli recombinant bacterium expresses EI protein coded by ptsI gene and EIIA coded by crr gene in seriesClcA protein; the escherichia coli recombinant strain is obtained by modifying a glucose transport path in escherichia coli FMME-A232, wherein the escherichia coli FMME-A232 is preserved in a China Center for Type Culture Collection (CCTCC) in 2018, 9 and 17 months, the preservation number is CCTCC NO: M2018628, and the preservation address is China, Wuhan university and Wuhan university;
wherein EI transporter encoded by ptsI gene and EIIA encoded by crr gene are expressed in tandem using pEtac plasmidClcTransporter, construction of recombinant plasmid pEtac-EI-EIIAClc(ii) a The recombinant plasmid pEtac-EI-EIIAClcAnd introducing into Escherichia coli FMME-A232 to obtain Escherichia coli recombinant bacteria.
2. The recombinant Escherichia coli as claimed in claim 1, wherein the ptsI gene encoding the EI transporter has a nucleotide sequence shown in SEQ ID No.1, and the nucleotide sequence encodes the EIIAClcThe nucleotide sequence of the crr gene of the transporter is shown in SEQ ID NO. 2.
3. Use of the recombinant Escherichia coli according to claim 1 or 2 for producing a food or pharmaceutical product containing L-alanine.
4. The use of claim 3, wherein the use is for the production of L-alanine using the recombinant Escherichia coli.
5. A method for producing L-alanine by fermentation, which is characterized in that the method is used for producing L-alanine by fermentation of the Escherichia coli recombinant strain as claimed in claim 1 or 2.
6. The method of claim 5, wherein the method comprises: inoculating the recombinant Escherichia coli to an LB culture medium containing 100mg/L kanamycin sulfate, performing shake culture at 37 ℃ overnight, inoculating the recombinant Escherichia coli to a fermentation culture medium according to the inoculum size of 10-15%, performing early aerobic fermentation at the temperature of 26-30 ℃, with the air amount of 1.0-2.0 vvm, stirring at the rotating speed of 300-400 rpm, culturing for 6-8 h, and adding 4-8 g/L lactose to induce EI protein and EIIAClcAnd (3) expressing the protein, continuously culturing for 15-25 h, starting oxygen-limited stage fermentation, reducing the ventilation volume to 0.01-0.1 vvm, reducing the stirring rotation speed to 50-100 rpm, raising the temperature to 40-43 ℃, and immediately finishing the fermentation after the glucose is consumed.
7. The method of claim 6, wherein the fermentation medium comprises: 180-220 g/L glucose, 3-6 g/L corn steep liquor, 2-6 g/L yeast powder and K2HPO4·12H2O 2～6g/L，KH2PO4 4～6g/L，MgSO4·7H20.4-0.6 g/L of O and 0.2-0.4 g/L of citric acid monohydrate.
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