CN107760736B - Method for promoting indigo biosynthesis conversion yield - Google Patents
Method for promoting indigo biosynthesis conversion yield Download PDFInfo
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- CN107760736B CN107760736B CN201610705447.8A CN201610705447A CN107760736B CN 107760736 B CN107760736 B CN 107760736B CN 201610705447 A CN201610705447 A CN 201610705447A CN 107760736 B CN107760736 B CN 107760736B
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Abstract
The invention relates to a method for promoting the indigo biosynthesis and conversion yield. The invention discloses that the Prefoldin protein and cytochrome P450enzyme are co-expressed in cells for the first time, the Prefoldin protein can remarkably promote the biosynthesis of indigo catalyzed by cytochrome P450 by taking indole as a substrate, and a more efficient method is provided for the biosynthesis of the indigo.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for promoting the conversion yield of indigo biosynthesis.
Background
The indigo pigment is one of the oldest pigments, and the application of indigo as a textile dye dates back to 2500 b.c. before the year of. Indigo is currently mainly used for dyeing of blue jeans, overalls and the like and remains one of the most important industrial compounds. The indigo pigment is also widely applied to the industries of food, daily cosmetics and the like and is mainly used as a coloring agent. There is increasing evidence that indigo and its derivatives are widely used in the pharmaceutical industry for the treatment of human diseases such as inflammation, bacterial infections, epilepsy, anti-tumor therapy and chronic myelogenous leukemia.
Traditional indigo synthesis is mainly extracted from some plants such as sophora japonica, isatis tinctoria, polygonum tinctoria, India tinctoria and the like, and the yield of indigo obtained by the method is extremely low. The chemical synthesis of indigo has emerged in 1897 and gradually replaced the traditional extraction of indigo. However, the raw materials and catalysts used in the chemical synthesis of indigo pose serious health risks, and the resulting waste liquid can cause serious environmental pollution. Therefore, researchers have been working on developing a method for synthesizing indigo that is environmentally friendly and harmless to human health. The method for synthesizing the indigo by catalyzing the substrate by an enzyme catalysis method is a green synthesis method.
Cytochrome P450enzymes are a superfamily of proteins that act extensively on endogenous and exogenous substances and are present in almost all organisms. It mainly catalyzes the terminal hydroxylation of long-chain saturated fatty acids and the epoxidation of medium-and long-chain unsaturated fatty acids. Researchers have found that P450enzymes can be modified by directed evolution technology to synthesize indigo and derivatives using indole as a substrate (Glieder A et al, Laboratory evolution of a soluble, self-sufficient, high activity alkane Hydroxylase. Nat Biotechnol.2002,20(11): 1135) 1139). For example, indigo can be synthesized by hydroxylation of indole catalyzed by the directed evolution of several human-derived P450enzymes CYP2A6, CYP2C and CYP2C (Zhang Z G et al, inactivation of amino acids inactivated in 4-chloroindole 3-hydroxylation by cytochrome P4502A 6using discovery of random ligands. journal of Biotechnology.2009,139(1): 12-18; Huang W et al, A-short CYP2C library with a high degree of specificity. archives of Biochemistry and biochemistry.2007, 467(2): 205; Gilam E M et al, Biochemistry of Biochemistry and Biochemistry 13817. Biophys.13817: 45017). However, the industrial use of human P450enzymes has been limited by factors such as low expression levels, low stability, low activity on indoles and the need for redox partners (Meijer L et al, Indirubin, the red shade of indigo, Life in Progress Editions, Illinois 2006).
CYP102A1, also known as P450BM3, is a cytochrome P450 from Bacillus megaterium. The enzyme can be expressed in a soluble form in Escherichia coli (facilitating large-scale preparation) and has high enzymatic activity, and functions only with NADPH and oxygen molecules (Miura Y and Fulco A J. (Omega-2) hydrolysis of fatty acids by a soluble system from Bacillus megaterium.J.biol.chem.1974,249(6): 0. biochem.1978; Hare S and Fulco A J.Carbon monooxide and hydroxymerry sensitivity of a fatty acid (Omega-2) hydrolase from Bacillus megaterium.M.Biophys.Res.1975, 65. 665), thus the enzyme becomes one of the most industrially applicable and engineered enzymes. Li Qi-Shan et al obtained mutants capable of catalyzing indole synthesis of indigo by mutating three sites A47G, F87V, L188Q of P450BM3 by site-Directed mutagenesis (Li Q S et al, Directed evolution of the fatty acid hydrolase P450BM-3in an indole-hydrogenating catalyst. chem Eur J.2000,6(9): 1531-1536). Because the mutant has very low yield of indigo synthesis and other byproducts are produced by the catalytic reaction, researchers have further mutated the mutant as a parent in an attempt to increase yield and reduce byproduct formation. In the research of saturated directed mutation of P450BM3, the D168 site can significantly improve the catalytic activity of parents (A74, F87V and L188Q) on indole. The mutant (A74, F87V, L188Q, D168H) can enhance the affinity of P450 and coenzyme NADPH so as to increase the synthetic amount of indigo blue, and the mutant can reduce the synthetic ratio of byproducts (Li H M, etc., Cytochrome P450BM3evolved by random and synthesis of a major evolution as an effective index-hydrogenation catalyst. applied Biochem Biotechnology.2008, 144(1): 27-36).
Although there is currently a great deal of research into the synthesis of indigo, there is still a need in the art for further optimization to improve the efficiency of indigo synthesis to meet the needs of the industry.
Disclosure of Invention
The invention aims to provide a method for promoting the indigo biosynthesis and conversion yield.
In a first aspect of the present invention, there is provided a method for biosynthesis of indigo, comprising:
(1) providing an indigo biosynthetic cell that co-expresses a cytochrome P450enzyme and a Prefoldin protein;
(2) and (3) culturing the indigo biosynthesis cells in the step (1), and adding indole as a substrate to biosynthesize indigo.
In a preferred embodiment, the molecular chaperone protein Prefoldin is derived from archaea thermophila p.
In another preferred embodiment, the cytochrome P450enzyme and the Prefoldin protein are foreign proteins.
In another preferred embodiment, the cytochrome P450enzyme is a cytochrome P450enzyme or a variant thereof that catalyzes the synthesis of indigo with indole as a substrate.
In another preferred embodiment, the cytochrome P450enzyme is CYP102a 1.
In another preferred embodiment, the CYP102a1 is a mutant CYP102a 1; the amino acid sequence has the mutation from A to G at the 74 th site, F to V at the 87 th site, L to Q at the 188 th site, and D to H at the 168 th site.
In another preferred embodiment, the cell is selected from the group consisting of: prokaryotic cells, eukaryotic cells.
In another preferred embodiment, the prokaryotic cells include, but are not limited to, E.coli cells, Bacillus subtilis cells, Bacillus megaterium cells; such eukaryotic cells include, but are not limited to, yeast cells. Preferably, the expression host is an E.coli cell.
In another preferred embodiment, in step (1), the method for coexpressing cytochrome P450enzyme and Prefoldin protein in cells comprises:
(a) providing an expression vector, wherein the expression vector contains a cytochrome P450enzyme expression cassette and a Prefoldin expression cassette;
(b) transforming the host cell with the expression vector of (a) to co-express cytochrome P450enzyme and Prefoldin protein in the cell.
In another preferred embodiment, the cytochrome P450enzyme expression cassette and the Prefoldin expression cassette are respectively positioned in different expression vectors; or in the same expression vector.
In another preferred example, in step (1), the method further includes: co-expressing a chaperone protein selected from the group consisting of: heat shock protein 60(hsp60), small heat shock protein (shsp), or a combination thereof.
In another aspect of the present invention, there is provided a method for increasing intracellular NADPH/NADP+A method of ratio, comprising: expressing the exogenous Prefoldin protein in cells.
In another aspect of the present invention, there is provided the use of a Prefoldin protein for promoting the synthesis of indigo by a cell expressing a cytochrome P450 enzyme; or for increasing intracellular NADPH/NADP+A ratio.
In a preferred embodiment, the cytochrome P450enzyme is CYP102a 1.
In another preferred embodiment, the CYP102a1 is a mutant CYP102a 1; the amino acid sequence has the mutation from A to G at the 74 th site, F to V at the 87 th site, L to Q at the 188 th site, and D to H at the 168 th site.
In another preferred embodiment, the cell is selected from the group consisting of: the prokaryotic cells include but are not limited to escherichia coli cells, Bacillus subtilis cells, Bacillus megaterium cells; such eukaryotic cells include, but are not limited to, yeast cells.
In another aspect of the present invention, there is provided an indigo-biosynthesis cell that includes a cytochrome P450enzyme expression cassette and a Prefoldin expression cassette and co-expresses a cytochrome P450enzyme and a Prefoldin protein.
In a preferred embodiment, the indigo biosynthesis cell further comprises an expression cassette for a chaperone protein selected from the group consisting of: heat shock protein 60(hsp60), small heat shock protein (shsp), or a combination thereof.
In another aspect of the present invention, there is provided a kit for indigo biosynthesis, the kit comprising: contains cytochrome P450enzyme expression box and Prefoldin expression box expression vector; wherein the two expression vectors are located in the same expression vector or in different expression vectors; or
In another aspect of the present invention, there is provided a kit for indigo biosynthesis, the kit comprising: co-expressing cytochrome P450enzyme and Prefoldin protein minor indigo biosynthesis cell.
Other aspects of the invention will be apparent to those skilled in the art in view of this disclosure.
Drawings
FIG. 1 shows the change of the amount of indigo biosynthesis of each recombinant bacterium with the lapse of culture time.
A: the intracellular indigo synthesis amount of the strains (pACYC-Prefoldin + P450, pACYC-Prefoldin-hsp60+ P450 and pACYC-Prefoldin-shp + P450) which excessively express the Prefoldin protein.
B: the amount of indigo synthesized in the P450 species (P450, pACYC-Prefoldin + P450) varied over a period of 0-18h with indole added.
FIG. 2 shows the enzyme amount, enzyme activity and expression of cytochrome P450enzyme in each bacterial strain.
A: the enzyme amount and the enzyme activity of the P450enzyme in the two strains;
b: expression of P450enzymes in both strains; m represents protein marker, T represents whole bacteria, S represents supernatant, and P represents precipitate.
Detailed Description
The present inventors have conducted extensive studies and found for the first time that the Prefoldin protein co-expresses with cytochrome P450enzyme in a cell, and that the Prefoldin protein can significantly promote the biosynthesis of indigo catalyzed by cytochrome P450 using indole as a substrate. The method is simple and easy to implement, and provides a new idea and a new method for biotransformation by taking the P450 as an enzyme catalyst and improving the conversion amount of the target product.
Term(s) for
As used herein, the term "expression cassette" refers to a gene expression system comprising all the necessary elements for expression of a polypeptide of interest (in the present invention, a cytochrome P450enzyme or a Prefoldin polypeptide), and typically includes the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator; in addition, a signal peptide coding sequence and the like can be optionally included. These elements are operatively connected.
As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.
As used herein, the term "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.
Prefoldin
Prefoldin (pfd), also known as GimC/Gim protein, is a hexameric chaperone protein complex composed of two subunits, present in all eukaryotes and archaea. Prefoldin from Thermus thermophilus consists of two alpha subunits and four beta subunits to form protein oligomer (alpha 2 beta 4) similar to jellyfish in shape, and its structure consists of two beta folding barrels, six long curled tentacles extending to the outside, and the tip of the tentacle exposing hydrophobic region.
The α subunits of the Prefoldin protein are: (a) 1, or a protein having an amino acid sequence shown in SEQ ID NO; or (b) a protein which is formed by substituting, deleting or adding one or more (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acid residues in the amino acid sequence shown in SEQ ID NO. 1, and is derived from (a) after the complete Prefoldin is formed, so that the Prefoldin has the function of promoting the enzyme catalysis of the P450enzyme to synthesize the indigo; or (c) a biologically active fragment of a protein having the amino acid sequence shown in SEQ ID NO: 1. Alternatively, the β subunit of the Prefoldin protein is: (a) 2, a protein of an amino acid sequence shown in SEQ ID NO; or (b) a protein which is formed by substituting, deleting or adding one or more (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acid residues in the amino acid sequence shown in SEQ ID NO. 2, is formed into complete Prefoldin and enables the Prefoldin to have the function of promoting the enzyme catalysis of P450enzyme to synthesize indigo, and is derived from the protein (a); or (c) a biologically active fragment of a protein having the amino acid sequence shown in SEQ ID NO. 2.
The amino acid sequence of the α subunit of the Prefoldin protein can also be found in Genbank accession numbers: NP _ 578104. The amino acid sequence of the β subunit of the Prefoldin protein can also be found in Genbank accession numbers: NP _ 578111.1.
The gene encoding Prefoldin protein is readily available, and in the case of known proteins, those skilled in the art will understand how to obtain the gene encoding Prefoldin, for example, by PCR amplification from the genome or by chemical synthesis. For example, the genes encoding the subunits of Prefoldin protein α can be found in Genbank accession numbers: PF 0375. The coding gene of the beta subunit of the Prefoldin protein can be found in Genbank accession number: PF 0382. In addition, a sequence degenerate to the gene sequence obtained by the above amplification is also usable.
Cytochrome P450enzymes
The cytochrome P450enzyme of the present invention is a cytochrome P450enzyme or a variant thereof that catalyzes the synthesis of indigo with indole as a substrate. Whereas the use of cytochrome P450enzymes to catalyze indoles purporting to be indigo is well known in the art, those skilled in the art are well known for cytochrome P450enzymes suitable for use in the present invention. There have been numerous studies in the art that indicate suitable cytochrome P450enzymes. Including some cytochrome P450enzymes engineered by directed evolution techniques. For example, Glieder A et al, Laboratory evaluation of a soluble, self-sufficient, high activity aldehyde Hydroxylase. Nat Biotechnol.2002,20(11): 1135) 1139 indicate suitable cytochrome P450 enzymes; zhang Z G et al, dentistry of amino acids inactivated in 4-chloro 3-hydroxylation by cytochromes P4502A 6using screening of random libraries. journal of Biotechnology.2009,139(1): 12-18; huang W et al, A shuffled CYP2C library with a high degree of specificity of structural integrity and functional university, apparatuses of Biochemistry and Biophysics, 2007,467(2): 193-; a number of suitable cytochrome P450enzymes are also described in Gillam E M et al, Oxidation of enzyme by cytochromes P450enzymes, biochemistry 2000,39:13817-13824 and others. These cytochrome P450enzymes and other cytochrome P450enzymes reported in other related documents that catalyze the synthesis of indigo with indole as a substrate can be used in the present invention.
In a preferred embodiment of the present invention, the cytochrome P450enzyme is selected from (but not limited to): CYP102a 1.
In a preferred embodiment of the present invention, the cytochrome P450enzyme is: (a) 3, amino acid sequence protein of SEQ ID NO; (b) 3 and amino acid sequence protein formed by mutating the 74 th site from A to G, the 87 th site from F to V, the 188 th site from L to Q and the 168 th site from D to H; or (c) a protein derived from (a) or (b) and having a function of promoting the synthesis of indigo from indole, wherein the protein is formed by substituting, deleting or adding one or more (preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5) amino acid residues in the amino acid sequence of the protein (a) or (b); or (d) a biologically active fragment of the protein of (a) or (b).
The gene encoding the cytochrome P450enzyme is readily available, for example, from the genome by PCR amplification or by chemical synthesis.
Applications of
The inventor is long dedicated to the development and research of the application aspect of Prefoldin, and through extensive research, the invention finds that the Prefoldin protein can obviously improve the method for synthesizing indigo by catalyzing indole with cytochrome P450 enzyme. The recombinant P450 expression bacteria co-express Prefoldin, so that the indigo biosynthesis amount is improved by 15 times. Based on the new finding, the invention provides a new application of Prefoldin protein, which is used for promoting cells to synthesize indigo.
The present inventors also found that Prefoldin modulates intracellular NADPH/NADP+The ratio is such as to increase the bioconversion of indigo. Based on the new discovery, the invention provides a new application of Prefoldin protein, which is used for increasing intracellular NADPH/NADP+A ratio. More deeply, the Prefoldin protein can be applied to the need of increasing the intracellular NADPH/NADP+In many cases of ratios.
Recombinant expression
Based on the new findings of the present inventors, there is provided a method for biosynthesis of indigo, comprising: (1) providing an indigo biosynthetic cell that co-expresses a cytochrome P450enzyme and a Prefoldin protein; (2) and (3) culturing the indigo biosynthesis cell in the step (1), and biosynthesizing indigo by using indole as a substrate.
In a preferred embodiment of the present invention, chaperone proteins selected from the group consisting of: heat shock protein 60(hsp60), small heat shock protein (shsp), or a combination thereof.
Based on the new discovery of the inventor, the invention also provides a method for improving the intracellular NADPH/NADP+A method of ratio, comprising: expressing the exogenous Prefoldin protein in cells.
The present invention also relates to an expression vector comprising the gene encoding the cytochrome P450enzyme and/or the gene encoding Prefoldin, and a host cell produced by genetic engineering transformation using the vector of the present invention.
The expression vector of the present invention is an expression vector suitable for expression in a host cell (expression host), and contains an expression cassette for cytochrome P450enzyme and/or Prefoldin. As a preferred mode of the present invention, the expression vector includes, but is not limited to pT7473 or pACYC.
The cytochrome P450enzyme can be expressed by transforming the expression vector into an expression host and culturing the recombinant expression host transformed with the expression vector. Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
The host cell may be prokaryotic cell, eukaryotic cell, etc. A specific example of such a prokaryotic cell is E.coli.
In a specific example of the present invention, the present inventors performed site-directed mutagenesis of four sites (a74, F87V, L188Q, D168H) of the P450BM3 gene derived from bacillus megaterium and overexpressed it in escherichia coli, and as a result, found that the bioconversion amount of indigo was low. In order to investigate whether Prefoldin can increase the amount of indigo synthesized in escherichia coli, the present inventors co-expressed a recombinant vector containing Prefoldin with P450BM 3in escherichia coli and observed the functions of molecular chaperones by measuring the amount of indigo synthesized. As a result, it was found that the yield of indigo synthesis could be greatly improved by coexpressing Prefoldin in E.coli. Furthermore, the inventor analyzes the relevant factors influencing the indigo synthesis, and finds that Prefoldin can obviously improve the intracellular NADPH/NADP+The ratio, in view of NADPH being an important cofactor for indigo synthesis, suggests that an increase in the ratio is an important factor in increasing the amount of indigo synthesis.
The invention has important reference significance for improving the biotransformation capacity of target products synthesized by different substrates catalyzed by cytochrome P450enzyme (such as P450BM3 mutant).
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
1. Materials and methods
(1) Primary reagents and solutions
LB culture medium: 10g NaCl, 5g yeast powder, 10g peptone were dissolved in 1000ml deionized water.
The Agilent QuikChange Lighting site-directed mutagenesis kit was purchased from Agilent technologies (China) Inc.
Enzychrom NADP+the/NADPH assay kit was purchased from Shanghai Xinui Biotech Ltd.
(2) Extraction of Bacillus megaterium genomic DNA
Bacillus megaterium is available from the institute for agricultural resources and agricultural division, Beijing institute for agricultural sciences (ATCC 14581).
Taking 30ml of overnight cultured Bacillus megaterium bacterial liquid, centrifuging at 5,000rpm for 10min, and removing supernatant; 5ml of SET (75mM NaCl, 25mM EDTA, 20mM Tris & HCl pH 7.5) was added and mixed well; adding appropriate amount of lysozyme, mixing well, and standing in water bath at 37 deg.C for 40 min; adding 600ul of 10% SDS and 1.7ml of proteinase K, and carrying out water bath at 55 ℃ for 2 h; adding 2.8ml NaCl (5M) and mixing; adding phenol: chloroform: extracting the isoamyl alcohol (24:24:1, v/v) twice; adding isopropanol according to the volume of 1:1, and standing for 30min at room temperature; centrifuging at 2,000g for 15min, and discarding the supernatant; then, 70% ethanol was added to wash once, and the mixture was centrifuged at 12,000g for 15min, and the supernatant was discarded, dried, and stored at-20 ℃.
(3) Cloning, site-directed mutagenesis, and recombinant expression of the P450BM3 Gene
The amino acid sequence of the wild type P450BM3 is shown in SEQ ID NO 3.
(4) Preparation of "P450" Strain
P450BM3 gene primers are designed, and Bacillus megaterium genome DNA is used as a template to amplify the P450BM3 gene. The pT7473 vector (see patent 200810039429.6) and the P450BM3 gene were digested with the same endonuclease based on the cleavage sites in the primers, and the desired fragment was ligated to the vector by enzymatic ligation to obtain the pT7473-P450BM3 plasmid. This plasmid was transformed into DH 5. alpha. competence and plated (with ampicillin resistance). Positive clones were picked and then sequenced. Storing the correctly sequenced strain, extracting the plasmid, transferring the plasmid into Ecoli BL21 (DE)3) The recombinant expression strain carrying pT7473-P450BM3 expression vector is obtained and can express P450BM 3.
Furthermore, the inventor also mutates four sites of the P450BM3 gene, which correspond to positions 74, 87, 168 and 188 of the amino acid sequence, and the corresponding sites are transformed into: a74G, F87V, D168H, L188Q. Designing a mutation primer according to the Protocol of the Agilent multi-site mutation kit and carrying out multi-site mutation by taking pT7473-P450BM3 plasmid as a template. The mutation method refers to the protocol of the kit. Since the primer of A74G overlaps with F87V, F87V, D168H and L188Q are mutated, and then the A74G site is subjected to single-site mutation by a conventional method. The mutated plasmid was transformed into DH 5. alpha. competence and plated (with ampicillin resistance). Selecting colony for sequencing identification, storing strain with correct sequencing, extracting plasmid, transferring the plasmid into Ecoli BL21 (DE)3) Thus, Ecoli BL21 (DE) into which pT7473-P450BM3 (with four mutations) had been transferred was obtained3) The strain, the recombinant strain is called as P450 for short.
The primers used in the present invention are shown in Table 1.
TABLE 1 primers
(5) Preparation of the "pACYC + P450" Strain
The pACYCDuet-1 plasmid was transferred into the "P450" strain prepared previously to obtain a "pACYC + P450" strain.
(6) Preparation of the "pACYC-prefoldin + P450" Strain
Construction of pACYC-Prefoldin expression vector: the pPFD plasmid was extracted (see Hui Chen et al, Biotechnol Lett.2010 (32): 429) -434), and the gene expression fragment encoding Prefoldin was obtained by double digestion with restriction enzymes NcoI and SalI, and recovered and purified by agarose gel electrophoresis. pACYCDuet-1 (Novagen) was digested simultaneously with NcoI and SalI, and then recovered by agarose gel electrophoresis. Mixing the double-digested Prefoldin coding gene with the double-digested pACYCDuet-1 plasmid, adding T4DNA ligase, and reacting at 16 ℃ for 16 hours to obtain a pACYC-Prefoldin vector.
The pACYC-Prefoldin expression vector was transformed into the "P450" strain prepared previously to obtain a "pACYC-Prefoldin + P450" strain.
(7) Preparation of the "pACYC-hsp 60+ P450" Strain
Construction of pACYC-hsp60 vector: extracting pET21-PfCPN plasmid (detailed construction of pET21-PfCPN plasmid is described in Hua-you Chen et al, Journal of Basic Microbiology 2007 (47):132-137) containing HSP60 encoding gene, double-digesting with Nde I and Xho I, and recovering and purifying HSP60 encoding gene DNA fragment by agarose gel electrophoresis for later use. The pACYCDuet-1 plasmid is subjected to double enzyme digestion by Nde I and Xhol I and then is subjected to a connection reaction with a recovered HSP60 coding gene DNA fragment to obtain a pACYC-HSP60 vector.
The pACYC-hsp60 expression vector was transformed into the "P450" strain prepared previously, to obtain the "pACYC-hsp 60+ P450" strain.
(8) Preparation of the "pACYC-shsp + P450" Strain
Construction of pACYC-shsp expression vector: extracting pET21-Pfshsp plasmid containing the shsp coding gene (the construction of pET 21-Pfsp plasmid is described in Hua-you Chen et al, Biotechnol lett 2006.28(14): 1089-. The pACYCDuet-1 plasmid is subjected to Nde I and Xho I double enzyme digestion and then is subjected to a connection reaction with the recovered shsp coding gene DNA fragment to obtain a pACYC-shsp vector.
The pACYC-shsp expression vector was transformed into the "P450" strain prepared above to obtain a "pACYC-shsp + P450" strain.
(9) Preparation of the "pACYC-prefoldin-hsp 60+ P450" strain
Construction of pACYC-prefoldin-hsp60 expression vector: the constructed pACYC-prefoldin plasmid (see (6)) was extracted, and the plasmid was digested with Nde I and Xho I, followed by ligation with the recovered DNA fragment of HSP 60-encoding gene digested with Nde I and Xho I to obtain pACYC-prefoldin-HSP60 vector.
The "pACYC-prefoldin-hsp 60+ P450" strain was obtained by transforming the "P450" strain prepared as described above with the pACYC-prefoldin-hsp60 expression vector.
(10) Preparation of the "pACYC-prefoldin-shsp + P450" strain
Construction of pACYC-prefoldin-shsp expression vector: the constructed pACYC-prefoldin plasmid (see (6)) was extracted, digested with Nde I and Xho I, and ligated with the recovered shsp-encoding gene DNA fragment digested with Nde I and Xho I to obtain pACYC-prefoldin-shsp vector.
The pACYC-prefoldin-shsp expression vector was transformed into the "P450" strain prepared as described above to obtain a "pACYC-prefoldin-shsp + P450" strain.
(11) Preparation of the "pACYC-hsp 60-shsp + P450" Strain
Construction of pACYC-hsp60-shsp expression vector: extracting pET21-Pfshsp plasmid containing the shsp encoding gene (the construction of pET 21-Pfsp plasmid is described in Hua-you Chen et al, Biotechnol lett 2006.28(14): 1089-. And simultaneously extracting the constructed pACYC-hsp60 plasmid (shown in (6)), carrying out double digestion on the plasmid by using Nco I and Sal I, and carrying out a connection reaction on the plasmid and the recovered shsp coding gene DNA fragment to obtain the pACYC-hsp60-shsp vector.
The pACYC-hsp60-shsp expression vector was transformed into the "P450" strain prepared as described above to obtain a "pACYC-hsp 60-shsp + P450" strain.
(12) Strain culture
The strain preserved at-80 ℃ was inoculated from a glycerol tube into 3ml of LB liquid medium containing the corresponding resistance and cultured overnight at 37 ℃ at 250 rpm. The following day, the inoculum was inoculated at 1% (v/v) and cultured in 50/250ml (containing the corresponding resistance) of fresh medium to an OD600 of between 0.8 and 1.0. And subpackaging the bacterial liquid into 20/50ml shake flasks, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM for induction for 3h, adding indole with the final concentration of 0.5mM for 1h, 2h, 3h, 4h and 18h respectively, and sampling to determine the OD600 value and the content of the indigo blue.
(13) Cell disruption
The above-mentioned cells after IPTG induction for 3 hours were centrifuged at 3,000rpm and 4 ℃ for 30min, and the cells were collected. The cells were resuspended in the appropriate amount of disruption solution and sonicated. The specific ultrasonic process is as follows: 200W, 2S start, 2S off, 99 cycles, all steps were performed in an ice bath. The disruption solution was centrifuged at 12,800rpm at 4 ℃ for 30min, and the collected supernatant was the crude enzyme solution. The crude enzyme solution was filtered through a 0.22 μm filtration membrane, and the filtered supernatant was subjected to the subsequent experiment.
(14) Determination of the indigo content
1ml of the above-mentioned cell culture solution at each time point (1H, 2H, 3H, 4H, 18H) was centrifuged at 9,000g 4 ℃ for 2min in a 1.5ml centrifuge tube, the supernatant was discarded, DMSO was added to the centrifuge tube to extract indigo blue, and H was added to the mixture2The mixture was extracted for 15min on an O-type mixer, and the OD610 value was measured.
(15) P450enzyme assay
The cytochrome P450 has a characteristic absorption peak at 450nm after being combined with CO, and the enzyme content of the P450 is measured by utilizing CO-difference spectroscopy. The specific method comprises the following steps: equal amounts of supernatant were added to 2 cuvettes 1cm in diameter, one cuvette labeled R and the other labeled S. Introducing CO into the S cuvette for 1min, not introducing into the R cuvette, and respectively adding Na into the two cuvettes in equal amount2S2O4And reversing and mixing uniformly, scanning the light absorption value of the supernatant in the S cuvette at the 400-plus 500nm waveband by taking the R cuvette as a base line, and calculating the enzyme content in the supernatant according to the following formula.
Molar absorptivity of 0.091L nmol-1·cm-1L is cuvette width: 1cm, f is the dilution factor.
(16) Enzyme Activity assay
An appropriate amount of the diluted cells was disrupted, and the supernatant (P450 crude enzyme solution) was centrifuged and placed in a clean glass tube, to which indole was added at a final concentration of 0.5mM and a solution of 0.2mM NADPH was added, followed by reaction in a water bath at 37 ℃ to determine the OD610 value of the reaction solution.
(17)NADPH/NADP+Measurement experiment
NADP+And NADPH content by Enzychrom NADP+the/NADPH assay kit (Bioassay Systems, Hayward, Calif.). The inventor makes some minor changes to the experimental method with reference to the Protocol of the kit, as follows:
after adding 100. mu.l of Extraction Buffer, the samples were sonicated before heat Extraction as follows: 50W, 10S start, 5S off, 1 cycle (all operations were performed on ice). The rest of the operations are referred to the Protocol.
Examples
Example 1 Effect of molecular chaperone protein Prefoldin on indigo bioconversion amount
The inventors examined whether molecular chaperone proteins Prefoldin, hsp60 and shsp derived from archaea thermophila P. furiosus, when co-expressed in e.coli with P450BM3 mutants in combination of two, respectively, contribute to increasing the content of indigo synthesized by P450 catalytic indole.
The chaperone gene was cloned into pACYCDuet-1 plasmid and the P450BM3 gene was cloned into pT7473 plasmid. The strain containing the pT7473-P450BM3 recombinant plasmid is marked as P450, the strain containing pACYC-Prefoldin and pT7473-P450BM3 plasmids is marked as pACYC-Prefoldin + P450, and the naming of other strains is repeated.
P450 strains were used as a control to observe the synthesis of indigo in the cells of each strain after the addition of indole for 0h, 1h, 2h, 3h, 4h and 18 h. The results are shown in FIG. 1.
The data in FIG. 1A show that the intracellular indigo synthesis amount of strains (pACYC-Prefoldin + P450, pACYC-Prefoldin-hsp60+ P450 and pACYC-Prefoldin-shp + P450) which excessively express the Prefoldin protein is remarkably increased, which indicates that the Prefoldin can remarkably improve the indigo biotransformation amount. Other molecular chaperone proteins have a certain promotion effect on the improvement of the indigo yield, but the effect is far less than that of Prefoldin.
The data in FIG. 1B show that the amount of indigo synthesis in P450 species was low and the yield did not change significantly over the period of 0-4h with indole addition. The strain containing the extreme thermophilic archaea molecular chaperone Prefoldin is 1h after indole is added, and the synthetic amount of indigo is obviously improved and is 15 times of that of a control strain; the synthetic amount of indigo in the pACYC-Prefoldin + P450 strain cells has no obvious change within 0-4h after indole is added. After the indole is added for 18 hours, the synthesis amount of the indigo in the cells of the two strains is improved to a certain extent, but the improvement range is not changed greatly. The experiment is repeated three times, and the trend of the experimental result of each time is consistent.
These experimental results show that Prefoldin can significantly improve the bioconversion amount of indigo.
Example 2 Prefoldin influences intracellular NADPH/NADP+Ratio of
In order to investigate factors affecting the amount of indigo biotransformation, the present inventors studied the amount of enzyme, the enzyme activity, and the intracellular NADPH level of P450. The results are shown in Table 2.
TABLE 2 intracellular NADPH/NADP of the respective species+Ratio of
The experimental results show that the enzyme amount and the enzyme activity are not limiting factors influencing the indigo biosynthesis. In cells expressing P450 protein alone (with low bioconversion of indigo), the content and activity of P450enzyme were higher than those of pACYC-Prefoldin + P450 strain, as shown in FIGS. 2A-B.
NADPH is an important cofactor involved in the reaction of synthesizing indigo by catalyzing indole by P450enzyme, and is obtained by measuring intracellular NADPH/NADP+Ratio, the inventors found that the intracellular NADPH/NADP of the strain expressing the pepoldin protein of Thermus thermophilus (pACYC-Prefoldin strain) after 3h induction with IPTG+The ratio is higher than that of the strain expressing the empty plasmid (pACYC)This result indicates that Prefoldin can regulate intracellular NADPH/NADP+And (4) horizontal. After the strain (P450 strain) which singly expresses P450 is induced by IPTG for 3h, intracellular NADPH/NADP+The intracellular NADPH/NADP of the species coexpressed with pepoldin (pACYC-Prefoldin + P450) at a ratio of 0.0730+The ratio was 0.1956, which is 2.68 times that of the control strain (P450 strain) (Table 1).
These results suggest that intracellular NADPH/NADP+The ratio is related to the bioconversion of indigo and when the ratio is high it favours intracellular indigo synthesis.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (8)
1. A method of biosynthesis of indigo comprising:
(1) providing an indigo biosynthetic cell which is an E.coli cell that co-expresses a cytochrome P450enzyme and a Prefoldin protein; the cytochrome P450enzyme is a protein with an amino acid sequence shown as SEQ ID NO. 3, wherein the 74 th position of the amino acid sequence is mutated from A to G, the 87 th position is mutated from F to V, the 188 th position is mutated from L to Q, and the 168 th position is mutated from D to H; the alpha subunit of the Prefoldin protein is a protein with an amino acid sequence shown as SEQ ID NO. 1, and the beta subunit is a protein with an amino acid sequence shown as SEQ ID NO. 2;
(2) and (3) culturing the indigo biosynthesis cells in the step (1), and adding indole as a substrate to biosynthesize indigo.
2. The method of claim 1, wherein the step (1) of coexpressing the cytochrome P450enzyme and the Prefoldin protein in the cell comprises:
(a) providing an expression vector, wherein the expression vector contains a cytochrome P450enzyme expression cassette and a Prefoldin expression cassette;
(b) transforming the host cell with the expression vector of (a) to co-express cytochrome P450enzyme and Prefoldin protein in the cell.
3. The method of claim 1, wherein step (1) further comprises: co-expressing a chaperone protein selected from the group consisting of: heat shock protein 60, small heat shock protein or a combination thereof.
4. Method for increasing intracellular NADPH/NADP+A method of ratio, comprising: in the cellExpressing exogenous Prefoldin protein, wherein the cell is an Escherichia coli cell; the alpha subunit of the Prefoldin protein is protein with an amino acid sequence shown as SEQ ID NO. 1, and the beta subunit is protein with an amino acid sequence shown as SEQ ID NO. 2.
5. Use of Prefoldin protein for promoting the synthesis of indigo by e.coli cells that express cytochrome P450 enzymes; or for increasing intracellular NADPH/NADP in Escherichia coli+A ratio; the cytochrome P450enzyme is a protein with an amino acid sequence shown as SEQ ID NO. 3, wherein the 74 th position of the amino acid sequence is mutated from A to G, the 87 th position is mutated from F to V, the 188 th position is mutated from L to Q, and the 168 th position is mutated from D to H; the alpha subunit of the Prefoldin protein is protein with an amino acid sequence shown as SEQ ID NO. 1, and the beta subunit is protein with an amino acid sequence shown as SEQ ID NO. 2.
6. An indigo biosynthesis cell which is an Escherichia coli cell and contains a cytochrome P450enzyme expression cassette and a Prefoldin expression cassette and can co-express cytochrome P450enzyme and Prefoldin protein; the cytochrome P450enzyme is a protein with an amino acid sequence shown as SEQ ID NO. 3, wherein the 74 th position of the amino acid sequence is mutated from A to G, the 87 th position is mutated from F to V, the 188 th position is mutated from L to Q, and the 168 th position is mutated from D to H; the alpha subunit of the Prefoldin protein is protein with an amino acid sequence shown as SEQ ID NO. 1, and the beta subunit is protein with an amino acid sequence shown as SEQ ID NO. 2.
7. An indigo biosynthetic cell according to claim 6, further comprising an expression cassette for a chaperone protein selected from the group consisting of: heat shock protein 60, small heat shock protein, or a combination thereof.
8. A kit for indigo biosynthesis comprising: contains cytochrome P450enzyme expression box and Prefoldin expression box expression vector; or
The kit comprises: coexpressing a cytochrome P450enzyme and a Prefoldin protein together with an indigo biosynthesis cell, wherein the cell is an Escherichia coli cell;
wherein, the cytochrome P450enzyme is protein with amino acid sequence as SEQ ID NO. 3, and the 74 th site of the amino acid sequence is mutated from A to G, the 87 th site is mutated from F to V, the 188 th site is mutated from L to Q, and the 168 th site is mutated from D to H; the alpha subunit of the Prefoldin protein is protein with an amino acid sequence shown as SEQ ID NO. 1, and the beta subunit is protein with an amino acid sequence shown as SEQ ID NO. 2.
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