CN117987444A - Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production - Google Patents
Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production Download PDFInfo
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- CN117987444A CN117987444A CN202211380895.7A CN202211380895A CN117987444A CN 117987444 A CN117987444 A CN 117987444A CN 202211380895 A CN202211380895 A CN 202211380895A CN 117987444 A CN117987444 A CN 117987444A
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- lysine
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Abstract
The invention discloses an expression cassette, genetically engineered bacteria containing the same and application of the genetically engineered bacteria in L-lysine production. The gene expression cassette comprises an NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene, and the nucleotide sequences of the NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene are respectively shown in SEQ ID NO. 1-5. The genetic engineering bacteria which are constructed by the invention and take the corynebacterium glutamicum as a starting strain and comprise the NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene can effectively utilize agricultural wastes such as straws and the like for fermentation, obviously improves the yield of L-lysine, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an expression cassette, genetically engineered bacteria containing the expression cassette and application of the genetically engineered bacteria in L-lysine production.
Background
Lysine (lysine), also known as 2, 6-diaminocaproic acid, belongs to the basic amino acid. Lysine has important nutrition physiological functions and is widely applied in the industries of medicine, food and feed. At the same time, it can also be used as a precursor substance for synthesizing nylon polymer materials. The production path of lysine mainly comprises three methods of a protein hydrolysis method, a chemical synthesis method and a fermentation method, wherein the microbial fermentation method has the characteristics of low production cost, high production strength, high specificity, small environmental pollution and the like, and becomes the method with the widest application of industrial production of lysine.
The prokaryotic microorganism for producing lysine mainly includes coryneform bacteria, brevibacterium, nocardia, pseudomonas, escherichia, bacillus, and the like. Corynebacterium glutamicum (c.glutamicum) is the most important and safe strain for fermentative production of amino acids, and improving its ability to excessively synthesize various amino acids by metabolic engineering has been a research hotspot. For example, overexpression of a gene involved in lysine synthesis pathway and a gene involved in desensitization of feedback inhibition, or enhancement of energy supply pathway from glucose metabolism and optimization of lysine transporter on cell membrane, etc. are effective in improving productivity of lysine.
In addition to constantly enhancing metabolic pathways for lysine production, fermentation methods that open other bypasses to non-lysine metabolism are also worth the deep excavation. Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is the most important redox carrier in cellular metabolism, and is considered as an important cofactor, which not only serves as an electron acceptor catalyzing substrate catabolism, but also provides reducing power for energy-dependent redox reactions, and is a main driving force in the synthesis of various metabolites including lysine. At present, some researches on improving lysine yield by regulating NADPH through glyceraldehyde 3-phosphate dehydrogenase exist, but no report on efficient production of lysine by optimally breeding the enzyme exists.
Disclosure of Invention
In order to solve the problem of low yield of L-amino acid in the prior art, the invention provides an expression cassette for preparing L-lysine, genetically engineered bacteria and application thereof. Specifically, the invention provides an expression cassette containing NADP dependent glyceraldehyde 3-phosphate dehydrogenase and a genetic engineering bacterium containing the expression cassette, wherein the genetic engineering bacterium can effectively utilize glucose in straws in straw hydrolysate. The invention screens the multifunctional glyceraldehyde 3-phosphate dehydrogenase and obviously improves the yield of L-lysine by regulating and controlling the supply of intracellular cofactor and the metabolism angle of acyl-CoA.
To solve the above technical problem, a first aspect of the present invention provides an expression cassette, which comprises the following components: the nucleotide sequences of the NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding genes are shown as SEQ ID NO. 1-5 respectively.
In a second aspect the present invention provides a recombinant expression vector comprising an expression cassette according to the first aspect of the invention or comprising a nucleotide sequence as defined in the first aspect of the invention.
In a preferred embodiment, the backbone plasmid of the recombinant expression vector is pK18mob.
The specific information of the pK18mob can be referred to as the following web page:
http://www.biovector.net/product/1089.html。
In a third aspect, the invention provides a genetically engineered bacterium transformed into an expression cassette according to the first aspect of the invention, or a nucleotide sequence as defined in the first aspect of the invention, or a recombinant expression vector according to the second aspect of the invention.
In the genetically engineered bacterium, the starting bacterium is corynebacterium glutamicum (Corynebacterium glutamicum), for example, c.glutamicum B253 or c.glutamicum CathS.
In the genetically engineered bacterium, the expression cassette is preferably integrated in the genome of the starting bacterium by homologous recombination or is present in the starting bacterium in non-integrated form.
In the genetically engineered bacterium, it is preferable that the glyceraldehyde 3-phosphate dehydrogenase (gapdh) of the original strain is not expressed, and for example, the gapdh gene (NCBI GeneID of CP 025533.1) is knocked out.
In a preferred embodiment, the expression cassette is integrated into the gapdh gene at a site.
The starting strain can be a wild strain or a modified strain.
In a fourth aspect, the present invention provides a process for producing L-lysine, which comprises fermentatively culturing the genetically engineered bacterium according to the third aspect of the present invention, and recovering L-lysine from the culture broth of the fermentative culture.
In a preferred embodiment, the fermentation culture is performed in a fermentation medium suitable for the growth of the genetically engineered bacterium.
In a preferred embodiment, the fermentation medium contains not less than 25g/L glucose, for example 80-150g/L glucose. In addition to glucose, the fermentation medium contains other components for the growth, propagation or synthesis of L-lysine by the genetically engineered bacteria, such as potassium dihydrogen phosphate, and/or urea, and/or magnesium sulfate, and/or methionine, and/or corn steep liquor, and/or yeast extract.
In a preferred embodiment, the fermentation temperature is 28 to 37 ℃, for example 35 ℃.
In a preferred embodiment, the pH of the fermentation is from 6 to 8, for example 7.
In a preferred embodiment, the fermentation medium comprises a hydrolysate of lignocellulosic material.
In a preferred embodiment, the carbon source in the fermentation medium is derived from a hydrolysate of lignocellulosic material. The carbon source is preferably glucose.
The hydrolysate of the lignocellulose material is formed by enzymolysis and saccharification of the lignocellulose material. The enzymolysis uses cellulase.
The lignocellulose material comprises at least one of, but not limited to, straw, rice husk, bagasse, wood chips. The straw preferably comprises corn straw, wheat straw and cotton straw.
In a preferred embodiment, the lignocellulosic material is subjected to a pretreatment prior to the enzymatic saccharification, the pretreatment comprising screening, impurity removal, dry acid pretreatment and/or detoxification treatment of the lignocellulosic material.
In some embodiments, the lignocellulosic material has a particle size of 2 to 20mm, e.g., 5mm, 10mm.
In a fifth aspect, the present invention provides the use of an expression cassette according to the first aspect of the invention, a nucleotide sequence as defined in the first aspect of the invention, a recombinant expression vector according to the second aspect of the invention, or a genetically engineered bacterium according to the third aspect of the invention for the preparation of L-lysine.
The sixth aspect of the invention provides the use of the expression cassette according to the first aspect of the invention and the recombinant expression vector according to the second aspect of the invention in the preparation of corynebacterium glutamicum engineering bacteria.
In a seventh aspect, the present invention provides a method for preparing a genetically engineered bacterium obtained by introducing an expression cassette according to the present invention into a starting bacterium as defined in the present invention.
The preparation method preferably further comprises: blocking the expression of lactate dehydrogenase in the genetically engineered bacterium, e.g., knocking out gapdh gene.
In the preparation method, preferably, the expression cassette is integrated into the gapdh gene at a site.
In a preferred embodiment, the starting bacterium is C.glutamicum CathS141,141, and the preservation number is CCTCC NO: M20211495.
In a preferred embodiment, the starting bacterium of the present invention is C.glutamicum B253, purchased from Shanghai Industrial microorganism.
The NADP dependent glyceraldehyde 3-phosphate dehydrogenase is respectively from Clostridium sp.NJ4,Chryseobacterium joostei,Streptococcus mutans serotype c,Chryseobacterium piscicola and Pedobacter alluvionis strains, and the nucleotide sequences of the NADP dependent glyceraldehyde 3-phosphate dehydrogenase are respectively shown in SEQ ID NO. 1-5.
The starting strain can be a wild strain or a modified strain.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
After the expression cassette provided by the invention is introduced into the starting bacteria, the electron supply and the like in the fermentation process are effectively improved, so that the glucose utilization efficiency is improved, and the yield of L-lysine is further improved. When the lignocellulose material hydrolysate is used, the lysine yield of the genetically engineered bacterium is obviously improved compared with that of the starting bacterium. The genetically engineered bacterium provided by the invention can effectively utilize agricultural wastes such as straw and the like for fermentation, has good application prospect, and is also beneficial to the efficient production of lysine derivatives such as pentanediamine.
Biological material preservation information
The corynebacterium glutamicum CathS141,141 of the present invention has been preserved in China Center for Type Culture Collection (CCTCC) at 11/29 of 2021, and the preservation address is: chinese university of Wuhan, post code 430072, deposit number: CCTCC NO: M20211495, culture designation CathS, classification designation Corynebacterium glutamicum (Corynebacterium glutamicum).
Paecilomyces variotii CATHTD891 of the invention is preserved in China Center for Type Culture Collection (CCTCC) at the year 2021, 11 and 4, and the preservation address is as follows: chinese university of Wuhan, post code 430072, deposit number: CCTCC NO: M20211366, culture name CATHTD891, classification name Paecilomyces variotii (Paecilomyces variotii Bainier).
Detailed Description
1. Strains used in the present invention
Coli E.coli DH 5. Alpha. Was used for construction of expression plasmid and knock-out plasmid, C.glutamicum CathS141 was a strain for lysine production, and C.glutamicum CathS141 was used mainly as a starting strain in this experiment.
2. Reagent and culture medium
Cellulase CTec 2.0.0 was used to hydrolyze cellulose and hemicellulose in lignocellulose and was purchased from novelin (china) corporation of beijing, china. The cellulase enzyme activity was 203.2FPU/mL according to the method in the NREL LAP-006 guide, the cellobiase activity was 4900.0CBU/mL, and the protein concentration was 87.3mg/mL according to the Bradford method. Restriction enzymes are used to cleave plasmids or gene fragments to produce cohesive ends, available from Thermo Scientific (Wilmington, DE, USA). DNA polymerase is used to amplify the gene fragment, DNA ligase is used to ligate the digested gene fragment and plasmid vector, both of which are available from Takara (Otsu, japan). The seamless cloning kit was used to ligate gene fragments containing homologous fragments to plasmid vectors available from hantao biotechnology company (Nanjing, china). Plasmid extraction kits, PCR product purification recovery kits and gel recovery kits were all purchased from Shanghai swirley biotechnology company (Shanghai, china). Other reagents were purchased from local suppliers.
The culture medium used for culturing the escherichia coli is Luria-Bertani (LB) culture medium, and the specific components are as follows: 10.0g/L sodium chloride, 10.0g/L peptone and 5.0g/L yeast extract.
The specific components of the culture medium used for culturing the corynebacterium glutamicum are as follows:
(1) Seed culture medium: 25g/L glucose, 1.5g/L potassium dihydrogen phosphate, 2.5g/L urea and 0.6g/L magnesium sulfate, 25g/L corn steep liquor.
(2) Fermentation medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and optionally glucose as a carbon source.
Example 1: acquisition of Corynebacterium glutamicum CathS141 as a starting bacterium
Soil samples of Sinkiang urso were collected, added to 10mL of sterile water per 1g of soil sample, vigorously mixed for 1min, allowed to stand for precipitation for a period of time, and the samples were diluted to 10 -3、10-4、10-5 respectively, spread on LB agar plates containing 100mg/L nystatin, and incubated at 30 ℃. And obtaining a purified single colony through repeated streak separation culture. The strain was identified as Corynebacterium glutamicum by 16S rDNA sequence alignment (Corynebacterium glutamicum).
The wheat straw is subjected to acid pretreatment, detoxification and enzymatic hydrolysis to obtain wheat straw hydrolysate, the corynebacterium glutamicum obtained by separation and purification is used as an initial strain, and ultraviolet rays, nitrosoguanidine, 5-fluorouracil, ARTP and the like are used for multiple independent mutagenesis and composite mutagenesis to obtain a stable strain capable of resisting normal growth and lysine production of toxicity inhibitors (acetic acid, furfural, 5-hydroxybenzaldehyde and the like) in the straw hydrolysate. The strain is named CathS141,141 and is now deposited in China center for type culture Collection, address: chinese university of Wuhan, post code 430072, preservation number CCTCC NO: M20211495, preservation date 2021, 11 months and 29 days.
Example 2: construction of the starting Strain gapdh knockout vector
Taking the genome of C.glutamicum CathS141,141 as a template, and knocking out the glyceraldehyde 3-phosphate dehydrogenase gapdh gene (NCBI GeneID of CP 025533.1) of the original strain by designing primers, PCR amplification and other modes by the conventional technical means in the field to obtain a delta-gapdh fragment. The pK18mob plasmid was treated with EcoRI and XbaI endonucleases and then ligated with the delta-gapdh fragment by Gibson and transferred into E.coli by conventional chemical transformation methods. During this time, successfully ligated plasmids, designated pcg0, were selected using seed culture plates containing kanamycin resistance.
Example 3: construction of engineering bacteria containing NADP-dependent glyceraldehyde 3-phosphate dehydrogenase
The glyceraldehyde 3-phosphate dehydrogenase editing genes in reference Clostridium sp.NJ4,Chryseobacterium joostei,Streptococcus mutans serotype c,Chryseobacterium piscicola and Pedobacter alluvionis are synthesized in Huada genes respectively to obtain gapdh1, gapdh2, gapdh3, gapdh4 and gapdh5, and the nucleotide sequences of the gapdh1 to the gapdh5 are shown in SEQ ID NOs. Then, the gene is used as a template, a primer is designed, PCR amplification and other modes are carried out to obtain fragments, and then the fragments are connected into a pcg0 vector which is digested with BamHI by a Gibson method, and the obtained plasmids are named pcg1, pcg2, pcg3, pcg4 and pcg5 in sequence.
The plasmids pcg1, pcg2, pcg3, pcg4 and pcg5 were then transferred into C.glutamicum CathS141, respectively, by electrotransformation. Strains with correct homologous recombination are selected by a PCR method to obtain recombinant corynebacterium glutamicum, and the recombinant corynebacterium glutamicum is named cg1, cg2, cg3, cg4 and cg5 in sequence.
EXAMPLE 4 fermentation of different recombinant Corynebacterium glutamicum
The genetically engineered strain and the original strain Corynebacterium glutamicum CathS were inoculated into a seed medium for two rounds of activation and then transferred into a fermentation medium. 80g/L glucose was added as a carbon source to the fermentation medium. The fermentation was carried out in 250mL shake flasks at an inoculum size of 10% (v/v), at 35℃and 200rpm, and at a pH of 6.5, with glucose consumption as the fermentation termination time. The fermentation results are shown in Table 1: by replacing gapdh of the starting strain with NADP dependent glyceraldehyde 3-phosphate dehydrogenase, the glucose-lysine conversion rate is improved, which shows that the regulation of cofactor angle is an effective means for improving lysine production. Meanwhile, compared with 5 engineering bacteria, the cg4 lifting effect can be found to be most remarkable. In the course of the actual study, it was found that the insertion of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase without destroying gapdh of the starting strain also had an increasing effect, but was not as remarkable as the knockout effect.
TABLE 1 fermentation of different recombinant Corynebacterium glutamicum
| Strain | Glucose-lysine conversion (%) |
| CathS141 | 23.45±0.32 |
| Cg1 | 24.89±0.23 |
| Cg2 | 26.32±0.21 |
| Cg3 | 25.28±0.47 |
| Cg4 | 29.81±0.34 |
| Cg5 | 27.29±0.15 |
As can be seen from example 4, the genetically engineered bacterium of the present invention produced L-lysine with better effect than the starting strain.
Example 5: fermentation of recombinant corynebacterium glutamicum in lignocellulose material hydrolysate
The wheat straw is crushed and sieved, the grain diameter is 10mm, then the impurities such as soil, stones and metal are removed by washing, and the wheat straw is dried to constant weight in a drying oven at 105 ℃. Adding 8wt% sulfuric acid solution into the dried wheat straw, controlling the solid-to-liquid ratio (g/g) to be 7:3, heating for 10min by saturated steam at 180 ℃, and then carrying out enzymolysis at 50 ℃ by using cellulase to obtain straw hydrolysate. The straw hydrolysate is inoculated with a detoxified bacterial strain Paecilomyces variotii (Paecilomyces variotii Bainier) CATHTD891 (with the preservation number of CCTCC NO: M20211366), biological detoxication is carried out for 36 hours at the temperature of 33 ℃, and then centrifugation is carried out, and the supernatant is collected, so that the detoxified straw hydrolysate which contains 95.4g/L glucose is obtained to provide a carbon source. And (3) adding a fermentation medium into the straw hydrolysate, inoculating the transformed strain cg4 to the original strain CathS, performing fermentation comparison, and performing fermentation in a 250mL shaking flask, wherein the inoculum size is 10% (v/v), the culture condition is 35 ℃, the rpm is 200, the pH value is controlled to be 6.5, and the glucose consumption is taken as the fermentation termination time.
The results show that when the fermentation medium carbon source is provided by the wheat straw hydrolysate, the two strains have a significant difference in lysine yield, wherein the final lysine yield of the recombinant strain is 26.11g/L, and the yield of the original strain is 19.85g/L. Meanwhile, the fermentation time of the engineering bacteria cg4 is 61h, which is shortened by 11h compared with that of the original strain. Thus, the two strains exhibited a significant difference in production intensity, with the production intensity (lysine production per unit time) of the recombinant strain being significantly higher than that of the control strain. In addition, when Corynebacterium glutamicum C.glutamicum B253 was used as a starting strain, the strain was obtained by modification in the same modification method as the strain cg4 and designated as Bcg4, and the fermentation effect of the fermentation under the same fermentation conditions described above was confirmed, it was found that the final lysine yield of Bcg4 was increased by 12.21% as compared with the control strain B253. And strain Bcg4 ended fermentation 10h earlier than strain B253.
Therefore, the recombinant strain obtained by the invention has high-efficiency lysine production capacity in real lignocellulose material hydrolysate, and thus has good application prospect.
The foregoing specifically describes an operation example of the technical solution of the present invention, and is not to be construed as limiting the application of the present invention. All equivalent substitutions of operating conditions are within the scope of the present invention.
Claims (10)
1. The gene expression cassette is characterized by comprising an NADP-dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene, wherein the nucleotide sequences of the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase encoding genes are shown as SEQ ID NO. 1-5 respectively.
2. A recombinant expression vector comprising the gene expression cassette of claim 1.
3. The recombinant expression vector of claim 2, wherein the backbone plasmid of the recombinant expression vector is pK18mob.
4. A genetically engineered bacterium, comprising the gene expression cassette of claim 1 or the recombinant expression vector of claim 2 or 3; the starting strain of the genetically engineered bacterium is corynebacterium glutamicum (Corynebacterium glutamicum);
preferably, the gene expression cassette is integrated in the genome of the corynebacterium glutamicum by homologous recombination or is present in non-integrated form in the starting organism.
5. The genetically engineered bacterium of claim 4, wherein the corynebacterium glutamicum is c.glutamicum B253 or c.glutamicum CathS141 with a preservation number of cctccc No. M20211495;
Preferably, the genetically engineered bacterium weakly expresses or does not express glyceraldehyde 3-phosphate dehydrogenase of the original strain;
more preferably, the gene expression cassette is integrated into the site of the gene encoding the glyceraldehyde 3-phosphate dehydrogenase.
6. A method for producing L-lysine, comprising fermentatively culturing the corynebacterium glutamicum engineering bacterium according to claim 4 or 5, and recovering L-lysine from the culture broth of the fermentative culture.
7. The method according to claim 6, wherein the fermentation culture is at a temperature of 28 to 37℃and/or the fermentation culture has a pH of 6 to 8.
8. The method according to claim 6, wherein the carbon source in the fermentation medium used in the fermentation culture is derived from a hydrolysate of lignocellulosic material; the lignocellulose material comprises at least one of straw, rice husk, bagasse, wood and wood chips.
9. Use of the gene expression cassette of claim 1 or the recombinant expression vector of claim 2 or 3 in the preparation of genetically engineered bacteria; preferably, the starting strain of the genetically engineered bacterium is corynebacterium glutamicum (Corynebacterium glutamicum); more preferably C.glutamicum B253 or C.glutamicum CathS with the preservation number of CCTCC NO: M20211495.
10. Use of the gene expression cassette of claim 1, the recombinant expression vector of claim 2 or 3 or the genetically engineered bacterium of claim 4 or 5 in the production of L-lysine.
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