CN117802171A - Recombinant escherichia coli for producing creatine and method for producing creatine by whole cell transformation - Google Patents
Recombinant escherichia coli for producing creatine and method for producing creatine by whole cell transformation Download PDFInfo
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- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 229960003624 creatine Drugs 0.000 title claims abstract description 26
- 239000006046 creatine Substances 0.000 title claims abstract description 26
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 25
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- BPMFZUMJYQTVII-UHFFFAOYSA-N guanidinoacetic acid Chemical compound NC(=N)NCC(O)=O BPMFZUMJYQTVII-UHFFFAOYSA-N 0.000 claims abstract description 51
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- 239000013612 plasmid Substances 0.000 claims abstract description 28
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- 238000000034 method Methods 0.000 claims abstract description 18
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Abstract
The invention discloses recombinant escherichia coli for producing creatine and a method for producing creatine by whole cell transformation, and belongs to the technical field of bioengineering. The invention takes escherichia coli as an initial strain, and introduces exogenous arginine on a plasmid pET28 a: glycine amidino transferase AGAT and guanidinoacetic acid N-methyltransferase GAMT are obtained by screening 4 different sources of guanidinoacetic acid N-methyltransferase GAMT and three double-enzyme co-expression strategies and using different types of fusion Linker to fuse key genes agaT and gamT in a Cr biosynthesis pathway, and the yield of the recombinant strain with high creatine yield reaches 5.6g/L, so that the two-step synthesis of creatine from glycine in escherichia coli is realized for the first time. The method lays a foundation for the high-efficiency production of creatine in escherichia coli metabolic engineering.
Description
Technical Field
The invention relates to recombinant escherichia coli for producing creatine and a construction method thereof, belonging to the technical fields of metabolic engineering and genetic engineering.
Background
Creatine (Cr) is known by the chemical name N-methylguanidine acetic acid, also known as guanylacetic acid, creatine, etc., an amino acid derivative naturally occurring in vertebrates, and 95% of Creatine is present in skeletal muscle in humans. Cr plays an important role in cell energy homeostasis, and is considered an energy buffer; the biological functions of improving organism energy metabolism, promoting muscle cell development, resisting oxidation and the like are achieved, and the biological functions are applied to the fields of foods, medicines and the like. At present, cr is mainly obtained by a chemical synthesis method, the synthetic route of Cr has the problems of long reaction time, complex operation process, low product purity and the like, and Cr produced by the chemical synthesis method generally has bitter taste, cannot be directly used as a food additive, has complex post-treatment process and has the yield of only 50%, so that the Cr is heterologously expressed by a microbiological method and has important significance. Coli (Escherichia coli) is widely used in industry and has a very important role in the production of proteins and high-value-added compounds. In addition, escherichia coli becomes an excellent industrial mode microorganism due to long research history, clear genetic background, mature gene editing system, wide applicability and the like. Coli is considered a suitable host for the production of Cr.
The whole cell transformation method is a process of constructing a cell factory by metabolic engineering technology and catalyzing a substrate to be transformed into a target product by taking a microbial cell as a catalyst. Compared with the traditional enzyme catalysis method, the whole cell transformation method has certain advantages: (1) The method omits complex cell lysis and enzyme purification processes, and has the characteristics of simplicity and easiness in operation; (2) The deficiency of cascade catalysis in the reaction process can be reduced to the greatest extent by utilizing a multienzyme system in microbial cells, and the catalysis efficiency of enzymes is improved; (3) Intracellular enzymes are protected by intact cell bodies and are more stable than free enzymes, thus the process of catalyzing the reaction is more stable and efficient. In whole cell transformation, cells are in a high density environment, growth is limited, and metabolic processes are mainly used for synthesis of target products. The whole cell transformation method is widely applied due to the advantages of mild reaction conditions, few byproducts, high product yield, no pollution to the environment and the like.
At present, many studies are being conducted on microbiological synthesis of Cr and its intermediate Guanidinoacetate (GAA). A process for preparing creatine by fermentation of non-pathogenic microorganisms such as C.glutamicum is disclosed for example by Fan Wenchao (CN 106065411A). The microorganism has the following bioconversion functions: converting glucose to L-glutamic acid; conversion of L-glutamic acid to N-acetyl-L-glutamic acid; conversion of N-acetyl-L-glutamic acid to N-acetyl-L-glutamic semialdehyde; conversion of N-acetyl-L-glutamic semialdehyde to N-acetyl-L-ornithine; conversion of N-acetyl-L-ornithine to L-ornithine; conversion of L-ornithine to L-citrulline; conversion of L-citrulline to arginino-succinic acid; conversion of arginino-succinic acid to L-arginine; conversion of L-arginine to guanidinoacetic acid; and finally, the conversion of guanidinoacetic acid to creatine. For the first time, a recombinant ornithine cycle was designed in recombinant E.coli by introducing heterologous arginine as described by YIWEN Zhang et al (YIWEN Zhang et al ACS Synthetic Biology, 2020): glycine Aminotransferase (AGAT), a high efficiency whole cell catalyst was designed for GAA production.
In recent years, more and more researches are carried out on the catalytic conversion of biological enzymes, but most of the biological enzymes are used for enzymatically catalyzing and synthesizing GAA by taking arginine and glycine as substrates, and less researches are carried out on enzymatically catalyzing and synthesizing Cr by taking arginine, glycine and methionine as substrates, and the functional application of Cr is not negligible. Meanwhile, the two-enzyme co-expression is based on extremely unbalanced, so that the research of synthesizing Cr by taking arginine, glycine and methionine as substrates through two steps of an enzyme method is imperative.
Disclosure of Invention
In order to solve the problems, the invention fuses key genes agaT and gamT in a Cr biosynthesis path by screening 4 different sources of guanylacetic acid N-methyl transferase GAMT and three double-enzyme co-expression strategies and using different types of fusion Linker (flexible, rigid and sheatable), and finally realizes a method for producing creatine by whole cell transformation of genetically modified escherichia coli.
The first object of the present invention is to provide a method for producing creatine by whole cell transformation, which comprises the steps of fermenting glycine, arginine and active methionine as substrates by a system containing recombinant Escherichia coli,
the recombinant escherichia coli fusion expresses glycine guanyl transferase and guanidinoacetic acid N-methyltransferase, wherein the fusion expression is that a single promoter is adopted to start the expression of glycine guanyl transferase genes and guanidinoacetic acid N-methyltransferase genes, and the glycine guanyl transferase genes and the guanidinoacetic acid N-methyltransferase genes are connected through connecting peptides with amino acid sequences shown as SEQ ID NO.11 or SEQ ID NO. 13.
Further, the amino acid sequence of the glycine guanyltransferase is shown as SEQ ID NO. 1; the amino acid sequence of the guanidinoacetic acid N-methyltransferase is shown as SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO. 9.
Further, the nucleotide sequence of the glycine guanyltransferase gene is shown as SEQ ID NO. 2; the nucleotide sequence of the guanidinoacetic acid N-methyltransferase gene is shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO. 10.
Further, insertion of RBS having the nucleotide sequence shown in SEQ ID NO.15 downstream of the single promoter regulates the expression of glycine guanyl transferase gene and guanidinoacetic acid N-methyltransferase gene.
Further, pET series plasmid is used as expression vector.
Furthermore, E.Coli BL21 (DE 3) was used as the host strain.
Further, the single promoter is a T7 promoter.
Further, in the reaction system, the concentration of arginine, arginine or active methionine is 5-25 g/L.
Further, the OD600 of the recombinant escherichia coli in the reaction system is 40+/-2, and the recombinant escherichia coli reacts under the condition of pH 7.0-8.0.
Further, the reaction system contains 9-15 g/L tryptone, 21-27 g/L yeast powder, 10-18 g/L dipotassium phosphate trihydrate, 2-5 g/L potassium dihydrogen phosphate and 1-5 mL/L glycerin.
Further, the reaction is carried out for 24 to 48 hours at the temperature of between 25 and 40 ℃. In the examples of the present invention, the reaction was carried out at 30℃for 48 hours.
The second object of the present invention is to provide a recombinant plasmid, wherein a single promoter, glycine guanyltransferase gene, guanidinoacetic acid N-methyltransferase gene and a connecting peptide for connecting glycine guanyltransferase gene and guanidinoacetic acid N-methyltransferase gene are linked to the recombinant plasmid, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO.11 or SEQ ID NO. 13.
Further, RBS sequence with nucleotide sequence shown as SEQ ID NO.15 is inserted between the single promoter and the encoding gene.
A third object of the present invention is to provide a recombinant E.coli containing the recombinant plasmid.
The fourth object of the invention is to provide the application of the recombinant plasmid or recombinant escherichia coli in preparing creatine or creatine-containing products.
The invention has the beneficial effects that:
the invention provides a method for producing creatine by whole cell transformation, which discovers that a single promoter cooperates with a specific connecting peptide to fusion express double enzymes (glycine amidino transferase AGAT from Amycolatopsis kentuckyensis and guanidinoacetic acid N-methyltransferase GAMT from Mus caroli) through screening of fusion expression strategies, and the system can efficiently produce creatine when arginine, glycine and methionine are used as substrates for fermentation by adopting the recombinant bacteria, and the creatine yield reaches 5.6g/L when the fermentation is performed for 48 hours.
Drawings
FIG. 1 is a schematic diagram of the Cr synthesis process.
FIG. 2 shows the construction and expression results of recombinant plasmid pET28 a-agaT.
FIG. 3 shows the construction and expression results of plasmid pET28 a-gamT.
FIG. 4 shows the yield results of the whole cell transformation of E.coli BL21 (DE 3)/pET 28a-gamT to Cr.
FIG. 5 shows plasmids constructed from different agaT and gamT co-expression strategies.
FIG. 6 shows comparison of Cr synthesis yield by coexpression of agaT-gamT.
FIG. 7 shows the Cr yields when two enzymes were co-expressed in different fusion Linker.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The materials and methods involved in the following examples are as follows:
culturing and fermenting recombinant escherichia coli seeds:
seed liquid culture medium: 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast powder.
Fermentation medium: 12g/L tryptone, 24g/L yeast powder, 16.43g/L dipotassium phosphate trihydrate, 2.31g/L potassium dihydrogen phosphate and 4mL/L glycerol.
Whole cell transformation process: single colonies were first picked into 5mL of seed solution medium and incubated at 37℃for 12 hours at 220 rpm. Then, 0.5mL of the seed solution is transferred to 50mL of fermentation medium, and the corresponding antibiotics are added to culture at 37 ℃ and 220rpm until the OD 600 =0.8 to 1.2, IPTG was added at a final concentration of 0.1mM, and cultivation was continued at 30 ℃ at 220rpm for 12 hours. Diluting small amount of bacterial liquid to proper multiple for OD measurement 600 About 40, fermentingAfter centrifugation of the solution at 4000rpm for 10min at 4℃to collect the bacterial pellet, the pellet was washed once with a volume of 0.01M PBS buffer at pH 7.5. All cells were added to 25mL of a catalytic reaction system containing 10g/L arginine, 10g/L glycine, 10g/L methionine and one thousandth of kanamycin, and the cells were homogenized, at which point the OD600 in the system was 40.+ -. 2, and the catalytic reaction was performed in 0.01M PBS buffer at pH 7.5. And 5mL of the reaction system is added into a 50mL centrifuge tube of a reaction container, the whole cell conversion reaction is carried out in a constant temperature shaking table at 30 ℃ and 220rpm, and the concentration change of the sample detection products is respectively collected after 4h,10h,24h and 48h of conversion.
The sample detection method comprises the following steps: creatine and the intermediate guanidinoacetic acid were detected by Agilent high performance liquid chromatography (equipped with UV absorber) with a column of ZORBAX Hilic Plus 3.5.5 μm column (4.6 mm. Times.100 mm). The mobile phase was 75% acetonitrile (chromatographic grade) (ammonia adjusted pH 10), the detection wavelength was 210nm, and the flow rate was 1mL/min. Every 400 mu L of whole cell transformed sample is added with 100 mu L of pure acetonitrile (chromatographic grade), and after being uniformly mixed, the mixture is centrifuged at 14000rpm for 10min, and the supernatant is taken out for detection after passing through a membrane.
EXAMPLE 1 construction and expression of plasmid pET28a-agaT
According to glycine guanyltransferase AGAT (the amino acid sequence of which is shown as SEQ ID NO. 1) from Amycolatopsis kentuckyensis, synthesizing a nucleotide sequence of a coding gene agaT (shown as SEQ ID NO. 2), designing a primer, connecting the fragment to a pET28a plasmid in a Gibson assembly mode, and constructing a recombinant plasmid for expressing agaT. Transferring the recombinant plasmid into E.Coli BL21 (DE 3) to construct recombinant bacteria, fermenting and culturing the recombinant bacteria, performing SDS-PAGE analysis, taking E.Coli BL21 (DE 3) as a blank control, and performing SDS-PAGE on cell disruption supernatant as shown in figure 2, wherein AgaT protein is expressed normally.
Example 2 construction and expression of plasmid pET28a-gamT
According to N-methyltransferase GAMT of different sources of guanidinoacetic acid, including Mus caroli (Mcgamt), paramormyrops kingsleyae (Pkgamt), winogradskyella sp.PG-2 (Wingamt) and Streptomyces koyangensis (Skygamt) (the amino acid sequences are shown in SEQ ID NO.3, 5, 7 and 9 in sequence), nucleotide sequences encoding gene gamT (shown in SEQ ID NO.4, 6, 8 and 10) are synthesized, primers are designed, and the fragments are connected to pET28a plasmid in a Gibson assembly mode to construct 4 recombinant plasmids expressing gamT. The 4 recombinant plasmids are respectively transferred into E.Coli BL21 (DE 3) to construct recombinant bacteria, and after fermentation culture, SDS-PAGE analysis is carried out on the recombinant bacteria, and the E.Coli BL21 (DE 3) is taken as a blank control, and the SDS-PAGE result of cell disruption supernatant is shown in figure 3, so that GamT protein is expressed normally.
Example 3 Effect of different sources of gamT on Cr production in engineering bacteria
First, 4 recombinant bacteria single colonies of example 2 were picked into 5mL of seed liquid medium, and cultured at 37℃and 220rpm for 12 hours. Then 0.5mL of seed solution is transferred into 50mL of fermentation medium, corresponding antibiotics are added, the culture is carried out at 37 ℃ and 220rpm until the OD600 = 0.8-1.2, 0.1mM IPTG is added, the culture is continued at 30 ℃ and 220rpm for 12 hours, bacterial precipitation is collected by centrifugation at 4000rpm and 10min, PBS buffer is washed and resuspended to 5mL, and substrate (guanidinoacetic acid/methionine) is added for whole cell transformation. Under the conditions of 48h whole cell transformation and the same other conditions, the final reaction solution of the sample derived from Mcgamt had a Cr yield of at most about 1.8g/L (FIG. 4).
EXAMPLE 4 construction of double enzyme Co-expression plasmid
Based on the extreme unbalance of double-enzyme co-expression, the co-expression of agaT and gamT is realized by adopting a single plasmid single promoter, a single plasmid double promoter and 3 modes of fusion expression of double enzymes. agaT (amino acid sequence shown in SEQ ID NO. 1) from Amycolatopsis kentuckyensis and gamT from Mus caroli were selected to construct a double enzyme co-expression plasmid as shown in FIG. 5. Designing primers to obtain recombinant fragments, connecting the obtained recombinant fragments to pET28a plasmids through a Gibson assembly mode, and constructing recombinant plasmids pAG1, pAG2 and pAG3 with 3 different co-expression strategies (RBS is a sequence shown as SEQ ID NO.15 in the figure, and GS sequence is 5'-GGTGGTGGCGGATCT-3').
Example 5 Effect of different double enzyme Co-expression strategies on Cr production in engineering bacteria
The three double enzyme co-expressed agaT-gamT recombinant plasmids pAG 1-3 of example 4 were transferred into an expression host E.coli BL21 (DE 3) for whole cell transformation, and the product concentration was measured after 48h fermentation, which showed that the Cr yield was about 2.7g/L at the highest in the final reaction solution of the sample by fusion expression of double enzymes under the same conditions (FIG. 6). Presumably, the reason is that fusion expression reduces substrate transport pressure, and constructing corresponding substrate channels relieves thermodynamic bottlenecks; meanwhile, the N end of the gene agaT is possibly modified due to proper fusion expression, so that the translation of the gene is possibly influenced, and the expression quantity is improved.
EXAMPLE 6 construction of Co-expression plasmids containing different fusion Linker
As an integral part of fusion protein recombination, the Linker plays an important role in constructing a stable and bioactive fusion protein. The principle of constructing fusion protein is to delete the stop codon of the first protein gene and connect with the second protein gene with stop codon to realize the fusion expression of 2 genes. The fusion expression double enzyme plasmid pAG2 obtained in example 5 was selected, primers were designed, and co-expression plasmids containing different fusion Linker were constructed by means of Gibson assembly, and the nucleotide sequences of different fusion Linker types were shown in the following table.
TABLE 1 fusion Linker sequences
Example 7 Effect of different fusion Linker on Cr production in engineering bacteria
The co-expression plasmids containing different fusion markers obtained in example 6 are transferred into expression host escherichia coli BL21 (DE 3) for whole cell transformation, and after 48h of fermentation, the concentration of the final product Cr and the intermediate product GAA is measured, and as a result, the concentration of the flexible short chain L (PKVSPEAVKKEAEL) product encoding 14 amino acids is 5.6g/L at maximum. 42.7mM Cr was obtained from 133.2mM glycine, 57.4mM arginine and 67.1mM methionine, and the Cr conversion rates of arginine and glycine were 74.4mol% and 32.1mol%, respectively.
Substrate conversion = moles of substrate actually reacted/total moles of substrate x 100%.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. A method for producing creatine by whole cell transformation is characterized in that glycine, arginine and active methionine are used as substrates, a system containing recombinant escherichia coli is adopted for fermentation production,
the recombinant escherichia coli fusion expresses glycine guanyl transferase and guanidinoacetic acid N-methyltransferase, wherein the fusion expression is that a single promoter is adopted to start the expression of glycine guanyl transferase genes and guanidinoacetic acid N-methyltransferase genes, and the glycine guanyl transferase genes and the guanidinoacetic acid N-methyltransferase genes are connected through connecting peptides with amino acid sequences shown as SEQ ID NO.11 or SEQ ID NO. 13.
2. The method of claim 1, wherein the glycine amidino transferase has the amino acid sequence shown in SEQ ID No. 1; the amino acid sequence of the guanidinoacetic acid N-methyltransferase is shown as SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO. 9.
3. The method of claim 1, wherein insertion of RBS having the nucleotide sequence shown in SEQ ID No.15 downstream of the single promoter regulates expression of glycine guanyltransferase genes and guanidinoacetic acid N-methyltransferase genes.
4. The method of claim 1, wherein the single promoter is a T7 promoter.
5. The method according to claim 1, wherein the concentration of arginine, arginine or active methionine in the reaction system is 5-25g/L and the OD600 of the recombinant E.coli is 40.+ -.2.
6. The method according to claim 1, wherein the reaction conditions are: 25-40 ℃,200-240rpm, and pH 7.0-8.0.
7. The recombinant plasmid is characterized in that a single promoter, a glycine guanyl transferase gene, a guanidinoacetic acid N-methyltransferase gene and a connecting peptide for connecting the glycine guanyl transferase gene and the guanidinoacetic acid N-methyltransferase gene are connected to the recombinant plasmid, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO.11 or SEQ ID NO. 13.
8. The recombinant plasmid according to claim 7, wherein an RBS sequence having a nucleotide sequence shown in SEQ ID NO.15 is inserted between the single promoter and the coding gene.
9. A recombinant escherichia coli comprising the recombinant plasmid of claim 7 or 8.
10. Use of the recombinant plasmid of claim 7 or 8 or the recombinant escherichia coli of claim 9 for preparing creatine or a creatine-containing product.
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