CN114621934B - P450 reductase and application thereof - Google Patents

P450 reductase and application thereof Download PDF

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CN114621934B
CN114621934B CN202011455140.XA CN202011455140A CN114621934B CN 114621934 B CN114621934 B CN 114621934B CN 202011455140 A CN202011455140 A CN 202011455140A CN 114621934 B CN114621934 B CN 114621934B
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赵宗保
李青
王雪颖
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Dalian Institute of Chemical Physics of CAS
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Abstract

The application discloses a P450 reductase and application thereof. The amino acid sequence of the P450 reductase is set forth in SEQ ID NO:1, at least one of the following mutations occurs on the basis of the amino acid sequence indicated in 1: R967D, Q977E, Q1005E, W1047S. The P450 reductase is BMR subjected to directed evolution of protein, and takes non-natural coenzyme NCDH as a cofactor and reducing power, and electrons are transferred to a P450 enzyme catalytic center so as to finish the catalytic conversion of a substrate.

Description

P450 reductase and application thereof
Technical Field
The application relates to P450 reductase and application thereof, and belongs to the technical field of biology.
Background
Nicotinamide cofactor (NAD (P)) and its reduced form NAD (P) H are important coenzymes in life processes, participate in redox metabolism and other important biochemical processes in life, and any manipulation that alters NAD concentration and its redox state has a global effect on cells. The NAD analogue and the mutant oxidoreductase which can only recognize the NAD analogue can realize the regulation and control of the target redox process at the coenzyme level, and has great significance for the biological catalysis and synthesis biological research (Ji DB, et al J Am Chem Soc,2011,133,20857-20862;Wang L,et al.ACS Catal,2017,7,1977-1983). Several NAD analogues with good biocompatibility were reported by this study group. Such as Nicotinamide Cytosine Dinucleotide (NCD), nicotinamide 5-fluorocytosine dinucleotide (NFCD), nicotinamide 5-chlorocytosine dinucleotide (NClCD), nicotinamide 5-bromocytosine dinucleotide (NBrCD), and nicotinamide 5-methylcytosine dinucleotide (NMeCD) (Ji DB, et al J Am Chem Soc,2011,133,20857-20862;Ji DB,et al.Sci China Chem,2013,56,296-300). Meanwhile, some enzymes recognizing NAD analogs, such as NADH oxidase (NOX, genbank S45681) derived from enterococcus faecalis Enterococcus faecalis, D-lactate dehydrogenase (DLDH, gnebank CAA 47255) V152R mutant, malic enzyme (ME, genbank P26616) L310R/Q401C mutant, malate dehydrogenase (MDH, genbank CAA 68326) L6R mutant, have been reported. Modulation of intracellular metabolic reactions using Nicotinamide Cytosine Dinucleotide (NCD) has been achieved by intracellular transport of NCD using either NTT4 derived from Chlamydia or AtNDT2 protein derived from Arabidopsis thaliana, and specific biocatalytic modulation has been achieved by reduction of pyruvate to lactate using NCD by DLDH-V152R (Wang L, et al ACS catalyst, 2017,7,1977-1983).
The P450 enzyme is a mercaptide-heme enzyme protein with various functions, and can selectively activate C-H bond, N-H bond, S-H bond and the like, so that more than 20 different types of reactions are catalyzed, and modification reactions such as selective hydroxylation, epoxidation, dealkylation and the like of a large number of substrates with different structures are realized, and the P450 enzyme is known as a universal catalyst in nature. The activity of the P450 enzyme depends on the redox partner to transfer two electrons of NAD (P) H to heme prosthetic groups of the NAD (P) H, so that the catalytic cycle of the NAD (P) H can be completed, and the substrate conversion is realized. P450 enzymes and redox partners can be divided into five classes, based on their composition and cellular localization: (1) Class I is a three-component system that exists in most bacteria and many eukaryotic mitochondria, including three free components of FAD-containing ferredoxin reductase (FdR), fe-S cluster-containing ferredoxin (Fdx) and heme-containing P450 enzymes, where three proteins of Class I P450 are soluble, where Class I P450 is only Fdx soluble, fdR and P450 are anchored to the membrane. During the reaction, fdR transfers electrons from NAD (P) H to Fdx, fdx and then to P450 enzymes. (2) Class II is a two-component system that is present on most eukaryotic microbial endoplasmic reticulum membranes, including NAD (P) HP450 reductase (NAD (P) H cytochrome P450 reductase, CPR) and P450 enzymes containing cofactors FAD and FMN, both of which are anchored on the membrane. During the reaction, CPR transfers electrons from NAD (P) H to the P450 enzyme. (3) Class III is a single-component system, exists in bacteria, is a difunctional peptide chain formed by fusing a P450 enzyme domain containing heme prosthetic group and a P450 reductase domain containing cofactor FAD and FMN, does not need additional auxiliary electron transfer protein in the reaction process, and transfers electrons in the interior of molecules, thus being an electron self-sufficient catalytic system. (4) Class IV is also a single component system with three components identical to Class I on a fused polypeptide chain, with the P450 domain at the N-terminus linked to the FdR domain containing the cofactor FMN and then to the Fdx domain containing the Fe-S cluster at the C-terminus, and is also an electronic self-sufficient catalytic system. (5) Class V is an unusual NAD (P) H independent P450 enzyme. (Bernhardt R.et al J.Biotechnol,2006,124,128-145)
P450 BM3 is a fatty acid hydroxylase derived from Bacillus megaterium (Bacillus megaterium) and belongs to Class III self-sufficient P450, including heme-containing P450 domains (BMPs) and P450 reductase domains (BMRs), utilizing NADPH and O 2 Catalyzing the hydroxylation of the fatty acid subterminal methylene to generate hydroxy fatty acid. Is the P450 enzyme with highest catalytic efficiency reported in the current literature, and the catalytic efficiency is as high as 17000min -1 The coupling efficiency is as high as 100% (Whitehouse, c.j.c.et al chem Soc Rev,2012,41,1218-1260), and its efficient catalytic and electronic coupling efficiency is thought to be due to the spatial configuration and structural arrangement of its P450 domain and P450 reductase domain. The bioconversion system of other free P450 enzymes has the problems of low overall efficiency, weak host substrate supply capacity, dependence on reduced coenzyme and the like, and severely restricts the industrialized application of the bioconversion system. Fusion of the P450 BM3 reductase domain with different types of free P450 enzymes was constructed self-supporting using the molecular Leachia method (Dodhia, V.R.et al J Biol Inorg Chem 2006,11,903-916)The self-contained bifunctional P450 enzyme can improve the catalysis efficiency and the coupling efficiency of the P450 enzyme.
The construction of the non-natural coenzyme-dependent P450 reductase is expected to realize uncoupling of the non-natural coenzyme-mediated P450 reaction system from the endogenous energy supply. The preference of the cofactor of the non-natural coenzyme-dependent P450 reductase constructed in advance is not strict, and the improvement of the preference of the P450 reductase to the cofactor of the dihydronicotinamide cytosine dinucleotide (NCDH) is still a problem to be solved urgently at present.
Disclosure of Invention
According to one aspect of the present application, there is provided a P450 reductase, wherein the P450 reductase is a protein directed-evolved BMR, and an unnatural coenzyme NCDH is used as a cofactor and a reducing power, and electrons are transferred to a P450 enzyme catalytic center to complete the catalytic conversion of a substrate.
A P450 reductase having an amino acid sequence set forth in SEQ ID NO:1, at least one of the following mutations occurs on the basis of the amino acid sequence indicated in 1: R967D, Q977E, Q1005E, W1047S.
The SEQ ID NO:1 is the amino acid sequence of BMR, which is the P450 reductase domain BMR containing flavin mononucleotide and flavin adenine dinucleotide of P450 BM3 derived from Bacillus megaterium Bacillus megaterium, namely the amino acid sequence from position 472 to position 1049 of P450 BM 3.
The P450 reductase provided by the application is a multi-site mutant obtained by BMR through genetic engineering; the mutation site of the multi-site mutant comprises at least one of R967D, Q977E, Q1005E, W1047S. The mutant recognizes NCDH and relies on NCDH for reduction.
Alternatively, the mutations include R967D, Q977E and W1047S; or (b)
The mutations include R967D, Q977E, Q E and W1047S.
Alternatively, the P450 reductase has the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
Alternatively, the P450 reductase is a non-native coenzyme NCDH-dependent P450 reductase having a structure of formula I:
the non-natural coenzyme NCDH is obtained by reducing non-natural coenzyme NCD by using a regeneration substrate through non-natural coenzyme NCDH regeneration enzyme;
the unnatural coenzyme NCD has a structure represented by formula II:
optionally, the non-native coenzyme NCDH regenerases comprise at least one of the malate enzyme ME-L310R/Q401C, D-lactate dehydrogenase DLDH-V152R/N213E, D-lactate dehydrogenase DLDH-V152R/I177K/N213I, phosphite dehydrogenase PDH-I151R/P176R/M207A, phosphite dehydrogenase PDH-I151R/P176E/M207A, formate dehydrogenase FDH-V198I/C256I/P260S/E261P/S381N/S383F, methanol dehydrogenase MDH-Y171R/I196V/V237T/N240E/K241A.
The regeneration substrate comprises at least one of malic acid compound, phosphorous acid compound, D-lactic acid compound, formic acid compound and methanol.
Optionally, the malic acid compound comprises malic acid and/or a malate salt;
the D-lactic acid compound comprises D-lactic acid and/or D-lactate;
The phosphorous acid compound comprises phosphorous acid and/or phosphorous acid salt;
the formic acid compound comprises formic acid and/or formate.
According to another aspect of the present application, there is provided the P450 reductase as K 3 [Fe(CN) 6 ]The application of any one of reductase, cytochrome c reductase and thiazole blue reductase.
Alternatively, P450 reductase as K 3 [Fe(CN) 6 ]The reaction system of the application of the reductase is as follows: in a buffer system with pH of 5-9, the non-natural coenzyme NCDH is 0.01mM-10mM, K 3 [Fe(CN) 6 ]0.2mM-3mM, unnatural coenzyme NCDH-EpidenThe lisi P450 reductase is 2 nM-100. Mu.M.
Alternatively, the reaction system for the use of P450 reductase as cytochrome c reductase is: in a buffer system with pH of 5-9, the non-natural coenzyme NCDH is 0.01mM-10mM, the cytochrome c is 0.005mM-0.5mM, and the non-natural coenzyme NCDH-dependent P450 reductase is 2 nM-100. Mu.M.
Alternatively, the reaction system for the use of P450 reductase as a thiazole blue reductase is: in a buffer system with pH of 5-9, the non-natural coenzyme NCDH is 0.01mM-10mM, the thiazole blue is 0.01mM-10mM, and the non-natural coenzyme NCDH-dependent P450 reductase is 2nM-100 mu M.
According to another aspect of the application there is provided the use of said P450 reductase in the catalytic substrate conversion of a P450 enzyme.
Optionally, in the application of the P450 reductase in the catalytic substrate conversion of the P450 enzyme, the reaction system is as follows: in a buffer system with pH of 5-9, the non-natural coenzyme NCDH is 0.05mM-50mM, the P450 enzyme is 2 nM-100. Mu.M, the P450 enzyme substrate is 0.05mM-50mM, and the non-natural coenzyme NCDH-dependent P450 reductase is 2 nM-100. Mu.M.
According to another aspect of the present application, there is provided a fusion enzyme, the amino acid sequence of which comprises the amino acid sequence of the P450 reductase as defined in any one of the above and the amino acid sequence of the P450 enzyme or P450 enzyme domain.
Alternatively, the C-terminus of the amino acid sequence of the P450 enzyme or P450 enzyme domain is linked to the N-terminus of the amino acid sequence of the P450 reductase by a linker peptide.
Alternatively, the linker peptide has the amino acid sequence as set forth in SEQ ID NO:12 to 16.
Optionally, the P450 enzyme is selected from any one of a Class I P450 enzyme and a Class II P450 enzyme;
the P450 enzyme domain is selected from any one of Class III P450 enzyme domains.
Optionally, the Class I P450 enzyme is CYP101A1 or CYP152L1.
Alternatively, the Class II P450 enzyme is CYP53a15.
Alternatively, the CYP53A15 does not include an N-terminal 35 amino acid transmembrane sequence.
Alternatively, the Class III P450 enzyme domain is the P450 domain of CYP102 A1.
Optionally, the P450 enzyme or the substrate of the P450 enzyme domain is selected from any one of D-camphor, C12-C18 saturated fatty acid and benzoic acid.
According to another aspect of the present application there is provided an enzyme catalytic system comprising a P450 reductase, a P450 enzyme or a P450 enzyme domain, an unnatural coenzyme NCDH regenerating enzyme as defined in any of the preceding claims; or (b)
Comprising the fusion enzyme of any one of the above-mentioned, non-natural coenzyme NCDH regenerating enzyme.
According to another aspect of the present application, there is provided a nucleic acid encoding any one of the P450 reductase, the fusion enzyme of any one of the above.
According to another aspect of the present application there is provided a vector comprising an expression cassette comprising a nucleic acid as defined in any one of the preceding claims.
Optionally, the vector further comprises an expression cassette comprising a nucleic acid encoding a non-native coenzyme NCDH regenerant.
Optionally, the vector further comprises an expression cassette comprising a nucleic acid encoding a nucleotide transporter.
Alternatively, the nucleotide transporter comprises an NTT4 nucleotide transporter derived from chlamydia and/or an AtNDT2 nucleotide transporter derived from arabidopsis thaliana.
Optionally, the nucleotide transporter in the expression cassette comprising a nucleic acid encoding a nucleotide transporter comprises an NTT4 nucleotide transporter derived from chlamydia and/or an AtNDT2 nucleotide transporter derived from arabidopsis thaliana.
According to another aspect of the application there is provided a host cell comprising a vector as defined in any one of the preceding claims.
Alternatively, the host cell is selected from a prokaryote and/or a eukaryote.
Optionally, the prokaryote is escherichia coli; the eukaryote is Saccharomyces cerevisiae.
According to another aspect of the application there is provided the use of a P450 reductase as defined in any one of the preceding claims, a fusion enzyme as defined in any one of the preceding claims, an enzyme catalytic system as defined in any one of the preceding claims, a nucleic acid as defined in any one of the preceding claims, a vector as defined in any one of the preceding claims, a host cell as defined in any one of the preceding claims, in a biocatalytic reaction mediated by an unnatural coenzyme NCD.
The P450 reductase, the fusion enzyme formed by fusing the P450 reductase and the P450 enzyme, and the fusion enzyme are further co-expressed with nucleotide transport proteins, and have important significance for realizing decoupling of energy consumption of the P450 enzyme and endogenous NADPH supply.
The application has the beneficial effects that:
(1) The P450 reductase provided by the application is a multi-site mutant obtained by BMR through genetic engineering, and has higher K3[ Fe (CN) 6], cytochrome c and thiazole blue reductase activities.
(2) The P450 reductase provided by the application uses the non-natural coenzyme NCDH as a cofactor and reducing power to transfer electrons to the catalytic center of the P450 enzyme, so that the catalytic efficiency and the electron transfer efficiency of the P450 enzyme can be improved.
(3) The enzyme catalysis system provided by the application has mild catalysis conditions and high reaction efficiency; the product selectivity is high; based on a whole-cell catalytic conversion system of non-natural coenzyme NCDH dependent P450 reductase and self-sufficient difunctional P450 enzyme, small molecular chemical energy reducing power is selectively transferred to a substrate molecule through non-natural coenzyme NCD mediation, intracellular natural coenzyme NAD (P) H is not relied on, the problem of endogenous energy dependence is overcome, and the constructed non-natural coenzyme NCD mediated biological orthogonal metabolic pathway realizes decoupling of energy consumption catalyzed by the P450 enzyme and endogenous energy metabolism.
Detailed Description
The present application is described in detail below with reference to examples, but the present application is not limited to these examples.
The NCDH in the specific embodiment of the application is unnatural coenzyme dihydro nicotinamide cytosine dinucleotide; the NCD is non-natural coenzyme nicotinamide cytosine dinucleotide. The NCD was synthesized according to the literature (Ji DB, et al J Am Chem Soc,2011,133,20857-20862).
In the present application, SEQ ID NO:1 represents the amino acid sequence of BMR; for the expression and purification of proteins, the initial amino acid methionine M and the amino acid sequence HHHHH (his tag), i.e.the amino acid sequence MHHHH, are added before the arginine R at the N-terminus of the amino acid sequence, when constructing the expression vector.
SEQ ID NO:2 represents the amino acid sequence of BMR-R967D/Q977E/W1047S; for the expression and purification of proteins, the initial amino acid methionine M and the amino acid sequence HHHHH (his tag), i.e.the amino acid sequence MHHHH, are added before the arginine R at the N-terminus of the amino acid sequence, when constructing the expression vector.
SEQ ID NO:3 represents the amino acid sequence of BMR-R967D/Q977E/Q1005E/W1047S, and in order to express and purify the protein, the initial amino acid methionine M and the amino acid sequence HHHHH (his tag), i.e. the amino acid sequence MHHHH, are added before the arginine R at the N-terminal of the amino acid sequence when constructing the expression vector.
SEQ ID NO:4 represents the amino acid sequence of P450 BM3 (i.e., CYP102A 1), and in order to express and purify the protein, the initial amino acid methionine M and the amino acid sequence HHHHH (his tag) are added before the N-terminal initial amino acid methionine M of the amino acid sequence, i.e., the amino acid sequence MHHHHH is added when constructing the expression vector.
SEQ ID NO:5 shows the nucleic acid sequence of BMR (with amino acid sequence MHHHH).
SEQ ID NO:6 shows the nucleic acid sequence of BMR-R967D/Q977E/W1047S (with amino acid sequence MHHHH).
SEQ ID NO:7 represents the nucleic acid sequence of BMR-R967D/Q977E/Q1005E/W1047S (with amino acid sequence MHHHH).
SEQ ID NO:8 represents the nucleic acid sequence of P450 BM3, i.e.CYP 102A1 (with the amino acid sequence MHHHH).
SEQ ID NO:9 represents the nucleic acid sequence of CYP101A1 (addition of his tag sequence HHHH after the initial amino acid methionine M of CYP10A 1).
SEQ ID NO:10 represents the nucleic acid sequence of CYP152L1 (the his tag sequence HHHH is added after the initial amino acid methionine M of CYP152L 1).
SEQ ID NO:11 shows the nucleic acid sequence of CYP53A15 (the his tag sequence HHHH is added after the initial amino acid methionine M of CYP53A 15).
SEQ ID NO:12 represents the amino acid sequence of Linker 1.
SEQ ID NO:13 represents the amino acid sequence of Linker 2.
SEQ ID NO:14 represents the amino acid sequence of Linker 3.
SEQ ID NO:15 represents the amino acid sequence of Linker 4.
SEQ ID NO:16 represents the amino acid sequence of Linker 5.
The his tag is the amino acid sequence hhhhhhh consisting of 6 histidine residues.
As a specific example, the application provides a non-natural coenzyme dihydro Nicotinamide Cytosine Dinucleotide (NCDH) dependent P450 reductase and application thereof, in particular to BMR subjected to directed evolution of protein, and the NCDH is taken as a cofactor and reducing power to catalyze and reduce the P450 enzyme, so that the P450 enzyme catalyzes substrate conversion. And provides a fusion enzyme, namely: provided is a method for constructing an NCDH-dependent self-sufficient bifunctional P450 enzyme by fusion expression of an NCDH-dependent P450 reductase with a P450 enzyme. The three-element system comprising NCDH dependent P450 reductase, different P450 enzymes and NCDH regenerating enzyme or the two-element system comprising NCDH dependent self-sufficient difunctional P450 enzyme and NCDH regenerating enzyme can be co-expressed with nucleotide transport protein in microbial cells for constructing biological orthogonal metabolic pathways, and the decoupling of the energy consumption catalyzed by the P450 enzyme and the endogenous energy metabolism is realized. Therefore, the method of the application can be applied to the fields of biocatalysis and bioconversion and has important value.
Specifically, an NCDH-dependent P450 reductase is characterized in that:
1) The NCDH-dependent P450 reductase is a P450 reductase with NCDH as cofactor, and can be used for catalyzing and reducing K 3 [Fe(CN) 6 ]Cytochrome c and thiazole blue are K 4 [Fe(CN) 6 ]Reduced cytochrome c and blue formazan; the saidNCDH is obtained by reducing NCD with a regeneration substrate by NCDH regeneration enzyme;
2) The chemical structures of the NCDH and the NCD are respectively shown in the formula I and the formula II:
the NCDH-dependent P450 reductase is further characterized by: the NCDH-dependent P450 reductase is BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S, wherein the BMR-R967D/Q977E/W1047S has the amino acid sequence as shown in SEQ ID NO:2, said BMR-R967D/Q977E/Q1005E/W1047S having the amino acid sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
The NCDH-dependent P450 reductase can utilize NCDH to catalyze and reduce K 3 [Fe(CN) 6 ]Cytochrome c thiazole blue is K 4 [Fe(CN) 6 ]The reaction system of the reduced cytochrome c and blue formazan is as follows: NCDH 0.01mM-10mM, K in a buffer system at pH 5-9 3 [Fe(CN) 6 ]0.2mM-3mM or cytochrome c 0.005mM-0.5mM or thiazole blue 0.01mM-10mM, NCDH-dependent P450 reductase 2 nM-100. Mu.M.
The NCDH-dependent P450 reductase can transfer two electrons of NCDH to the catalytic centers of different types of P450 enzymes to complete the catalytic conversion of substrates, and the reaction system is as follows: in a buffer system with pH of 5-9, NCDH is 0.05mM-50mM, P450 enzyme is 2 nM-100. Mu.M, P450 enzyme substrate is 0.05mM-50mM, NCDH-dependent P450 reductase is 2 nM-100. Mu.M.
The NCDH is obtained by reducing NCD by using a regeneration substrate by NCDH regeneration enzyme; wherein the NCDH regenerates one or more than two of malate enzyme ME-L310R/Q401C, D-lactate dehydrogenase DLDH-V152R/N213E, D-lactate dehydrogenase DLDH-V152R/I177K/N213I, phosphite dehydrogenase PDH-I151R/P176R/M207A, phosphite dehydrogenase PDH-I151R/P176E/M207A, formate dehydrogenase FDH-V198I/C256I/P260S/E261P/S381N/S383F, methanol dehydrogenase MDH-Y171R/I196V/V237T/N240E/K241A, and the corresponding regenerates substrate comprises one or more than two of malate compound, phosphite compound, D-lactate compound, formate compound, methanol; wherein the malic acid compound is one or more than two of malic acid and malate; the D-lactic acid compound is one or more than two of D-lactic acid and D-lactate; the phosphite compound is one or more than two of phosphorous acid and phosphite; the formic acid compound is one or more than two of formic acid and formate.
The NCDH-dependent P450 reductase can be fused with different types of P450 enzymes to express the NCDH-dependent self-sufficient difunctional P450 enzyme through a connecting peptide, wherein the connecting peptide comprises but is not limited to Linker 1:KKIPLGGISPSTQSAKKV or Linker 2-5 (G) 4 S) n Wherein n is an integer of 1 to 4, i.e., the amino acid sequence of Linker 2 is: GGGGS; the amino acid sequence of Linker 3 is: GGGGSGGGGS; the amino acid sequence of Linker 4 is: GGGGSGGGGSGGGGS; the amino acid sequence of Linker 5 is: GGGGSGGGGSGGGGSGGGGS. The self-sufficient double functions are that electrons are transferred inside molecules without adding auxiliary electron transfer proteins in the reaction process.
The different types of P450 enzymes include, but are not limited to, class I P450 enzymes, class II P450 enzymes, class III P450 enzyme domains. Wherein Class I P450 enzymes include, but are not limited to, CYP101A1 or CYP152L1; class II P450 enzymes include, but are not limited to, CYP53a15; class III P450 enzyme domains include, but are not limited to, the P450 domain of CYP102 A1; the P450 enzyme corresponding substrate comprises, but is not limited to, D-camphor, C12-C18 saturated fatty acid and benzoic acid.
The NCDH-dependent P450 reductase, the different types of P450 enzymes, the NCDH regenerating enzyme and the nucleotide transporter are expressed in microbial cells, and the microbial cells catalyze the P450 substrate to be converted into a product; wherein NCDH is transported into the cell by either the NTT4 derived from Chlamydia or the AtNDT2 nucleotide transporter derived from Arabidopsis thaliana; microbial cells include, but are not limited to, prokaryotic E.coli and eukaryotic Saccharomyces cerevisiae.
The NCDH dependent self-sufficient difunctional P450 enzyme and the NCDH regenerated enzyme can construct a double-enzyme conjugate catalytic system, and the reaction system is as follows: in a buffer system with pH of 5-9, NCD is 0.01mM-1mM, NCDH regeneration enzyme is 2U/mL-100U/mL, regeneration substrate is 0.1mM-100mM, NCDH-dependent self-sufficient difunctional P450 enzyme is 1 mu M-100 mu M, and P450 enzyme substrate is 0.05mM-50mM.
The NCDH-dependent self-sufficient difunctional P450 enzyme, the NCDH regenerating enzyme and the nucleotide transporter are coexpressed in microbial cells, and the microbial cells catalyze the P450 substrate to be converted into a product; wherein NCDH is transported into the cell by either the NTT4 derived from Chlamydia or the AtNDT2 nucleotide transporter derived from Arabidopsis thaliana; microbial cells include, but are not limited to, prokaryotic E.coli and eukaryotic Saccharomyces cerevisiae.
The application discloses an enzyme catalysis system constructed by NCDH dependent P450 reductase and NCDH dependent self-sufficient difunctional P450 enzyme, which has mild catalysis conditions and high reaction efficiency; the product selectivity is high; the NCDH-dependent self-sufficient difunctional P450 enzyme improves the catalytic efficiency and the electron transfer efficiency of the P450 enzyme; based on the NCDH-dependent P450 reductase and a whole-cell catalytic conversion system of the NCDH-dependent self-sufficient difunctional P450 enzyme, the chemical energy reducing power of small molecules is selectively transferred to substrate molecules through the mediation of non-natural coenzyme NCD, the problem of endogenous energy dependence is overcome, the constructed non-natural coenzyme NCD-mediated biological orthogonal metabolic pathway is overcome, and the decoupling of the energy consumption catalyzed by the P450 enzyme and the endogenous energy metabolism is realized.
NCD reference methods (Ji DB, et al Sci China Chem,2013,56,296-300) in the examples of the present application were used to prepare an aqueous solution having a concentration of 20 mM.
The method for electric transformation of prokaryotic transformation such as E.coli refers to the method for transformation of eukaryotic organisms such as Saccharomyces cerevisiae in the third edition of molecular cloning guide (Gietz, R.D., et al Nature Protocols 2007,2,31).
The BMR described in the specific example of the present application is a P450 reductase domain BMR containing flavin mononucleotide and flavin adenine dinucleotide derived from P450 BM3 of Bacillus megaterium Bacillus megaterium, i.e., the amino acid sequence from position 472 to position 1049 of P450 BM 3.
In a specific embodiment of the application, the NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S and BMR-R967D/Q977E/Q1005E/W1047S are constructed by genetically engineering BMR into mutants wherein BMR-R967D/Q977E/W1047S has the sequence as set forth in SEQ ID NO:2, and BMR-R967D/Q977E/Q1005E/W1047S has the amino acid sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
In the application, the mutation site expression method of the polypeptide mutant comprises the following steps: amino acid before mutation, amino acid number after mutation. For example, R967D represents the change from an original arginine R to a subsequent aspartic acid D at amino acid 967 in the polypeptide.
The expression method of the polypeptide mutant comprises the following steps: the original polypeptide-amino acid before mutation, amino acid number after mutation/amino acid before mutation, amino acid number after mutation, amino acid after mutation/… …, "/" is used to separate each mutated amino acid. For example, BMR-R967D/Q977E/W1047S represents a mutant of BMR obtained by 3 amino acid mutation, wherein BMR represents a raw polypeptide, R967D represents a change from the original arginine R to the subsequent aspartic acid D at amino acid 967; Q977E represents the change of 977 th amino acid from original glutamine Q to glutamic acid E; W1047S indicates that amino acid 1047 is changed from tryptophan W to serine S. Regarding the numbering of the mutated amino acids, wherein the numbering of the amino acids in BMR-R967D/Q977E/W1047S and BMR-R967D/Q977E/Q1005E/W1047S is identical to that of P450 BM3, i.e., numbering is carried out sequentially backwards with the initial amino acid methionine number of P450 BM3 being 1. The remaining mutants were numbered the same as the number of the polypeptide before mutation, i.e., numbered sequentially backwards with the starting amino acid methionine of the polypeptide before mutation numbered 1, unless otherwise specified. In the artificial construction of expression vectors, neither the initial amino acid methionine M nor histidine H in his tag, which is added for the expression or purification of proteins, is counted.
The construction of the NCDH-dependent self-sufficient bifunctional P450 enzyme used in the specific embodiment of the application is to fuse the C end of the amino acid sequence of the P450 enzyme from different sources with the N end of the amino acid sequence of the NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S) through a connecting peptide, and construct the P450 gene with the connecting peptide sequence between the N end his-tag of the NCDH-dependent P450 reductase expression vector and the NCDH-dependent P450 reductase gene sequence through an RF cloning method (unlimited cloning method), so as to obtain the NCDH-dependent self-sufficient bifunctional P450 enzyme expression vector. Wherein the N-terminal 35 amino acid transmembrane sequence of P450 is deleted when fused to Class II CYP53A 15.
The sequence of the connecting peptide used in the specific embodiment of the application is as follows:
Linker 1：KKIPLGGIPSPSTEQSAKKV；
Linker 3：GGGGSGGGGS；
Linker 5：GGGGSGGGGSGGGGSGGGGS
CYP101A1 used in the embodiment of the application is Pseudomonas putida (UniProt code P00183), CYP152L1 is Jeotogamicacoccus sp.ATCC 8456 (UniProt code E9NSU 2), CYP53A15 is Curvularia lunata (UniProt code B8QM 33), CYP102A1 (P450 BM 3) is Bacillus megaterium (UniProt code P14779), NCDH-dependent P450 BM3 enzymes (CYP 102A1-R967D/Q977E/W1047S or CYP102A 1-R967D/Q977E/Q1005E/W1047S) are obtained by mutating CYP102A1 (UniProt code P14779).
The Malic Enzyme (ME) used in the present application is derived from Escherichia coli K12 (UniProt code P26616), the D-lactate dehydrogenase (DLDH) is derived from Lactobacillus helveticus (UniProt code P30901), the Phosphite Dehydrogenase (PDH) is derived from Ralstonia sp.strain 4506 (UniProt code G4XDR 8), the Formate Dehydrogenase (FDH) is derived from Pseudomonas sp.101 (UniProt code P33160), and the Methanol Dehydrogenase (MDH) is derived from Bacillus stearothermophilus DSM2334 (Unipro code P42327).
The mutant dehydrogenases used in the embodiments of the present application are thoseThe single-site mutation kit is obtained by introducing amino acid mutation, wherein the mutant type malic enzyme (ME-L310R/Q401C) is obtained by mutating Malic Enzyme (ME) (UniProt code P26616), and the mutant type lactic acid dehydrogenase (DLDH-V152R/N213E or DLDH-V152R/I177K)N213I) was obtained by mutating D-lactate dehydrogenase (DLDH) (Uniprot code P30901), mutant phosphite dehydrogenase (PDH-I151R/P176R/M207A or PDH-I151R/P176E/M207A) was obtained by mutating Phosphite Dehydrogenase (PDH) (Uniprot code G4XDR 8), mutant formate dehydrogenase (FDH-V198I/C256I/P260S/E261P/S381N/S383F) was obtained by mutating Formate Dehydrogenase (FDH) (Uniprot code P33160), mutant methanol dehydrogenase (MDH-Y171R/I196V/V237T/N240E/K241A) was obtained by mutating Methanol Dehydrogenase (MDH) (Uniprot code P42327).
Expression and purification of enzyme: NCDH-dependent P450 reductase and self-sufficient bifunctional P450 enzyme were purified by Ni affinity chromatography according to literature methods (Wang JX, et al protein express 2007,53,97) for protein overexpression. Protein content determination: bovine serum albumin ABS was used as a standard protein and was measured using Bradford method.
Detection of regenerated substrate and corresponding product: the content of regenerated substrates such as malic acid, lactic acid, formic acid or phosphorous acid and the corresponding products in the reaction solution is analyzed and measured by using an ICS-2500 ion chromatography system of the Dynamo company in the United states under an ED50 pulse electrochemical detection mode. IonPac AS11-HC anion exchange analytical column (200 mm. Times.4 mm) and IonPac AG11-HC anion exchange guard column (50 mm. Times.4 mm) were used. Analysis conditions: the mobile phase is 1mM-15mM NaOH gradient, the flow rate is 1mL/min, and the column temperature is as follows: the temperature is 30 ℃ and the sample injection amount is 10 mu L. Qualitative analysis was performed by reference to a standard sample, and quantitative analysis was performed by a standard curve method.
C12-C18 fatty acid and hydroxylation products are analyzed by GC-MS with an Angilent7890-5700D equipped with a capillary splitter and FID through N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) silane derivatization products, D-camphor and hydroxylation products, and an analysis column is an HP-5MS capillary column (30 m multiplied by 0.32mm multiplied by 0.4 μm); the temperature of the sample injector is 250 ℃, the initial temperature of the column temperature box is 100 ℃,2min,10 ℃/min is raised to 300 ℃, and the temperature is kept for 3min; detector (FID) temperature 280 ℃; carrier gas He 40mL/min, H 2 30mL/min and 300mL/min of air; the sample injection amount is 1 mu L, and the split ratio is 20:1; MS detector solvent delay 4min; MS was used for characterization, FID was used for quantification, and external standard method was used to determine the relative content.
The benzoic acid, the parahydroxybenzoic acid and the 3, 4-dihydroxybenzoic acid are analyzed by adopting a high performance liquid chromatography-diode array detector, and the analysis column is a C18 reverse silica gel column; solvent a was used: 5% acetonitrile, 0.1% h2so4; solvent B: acetonitrile 90% w/v, H2SO4 0.1% w/v gradient elution, 0-10min 100% A,10-12min down to 60% A,12-24min 100% A; the flow rate is 1mL/min; the sample injection amount is 20 mu L; detecting signals at 215nm and 250 nm; and determining the relative content by adopting an external standard method.
Example 1: NCDH-dependent P450 reductase BMR-R967D/Q977E/1047W1047S catalytic reduction of K using NCDH 3 [Fe(CN) 6 ]For K 4 [Fe(CN) 6 ]
100. Mu.L of 100mM Tris-HCl buffer system pH 5: 0.2mM-3mM K 3 [Fe(CN) 6 ]10mM NCDH, 1. Mu.M NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S,30℃reaction, measurement of change in absorbance at 420nm, determination of K 3 [Fe(CN) 6 ]Is reduced to K 4 [Fe(CN) 6 ]，K 3 [Fe(CN) 6 ]Molar absorptivity at 420nm of 1.020mM - 1 cm -1 . The results are shown in Table 1, where the enzyme activity is expressed in terms of moles per minute per mole of enzyme catalyzed product formation or substrate consumption per unit mol (mol enzyme) -1 min -1 。
TABLE 1 NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S reducing K with NCDH 3 [Fe(CN) 6 ]Is of (2)
Example 2: NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S for catalyzing and reducing cytochrome c to reduced cytochrome c by NCDH
100. Mu.L of 300mM potassium phosphate buffer system pH 7.5: 5 mu M-500 mu M cytochrome C, 200 mu M NCDH, 2nM NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S,30 ℃ reaction, determination of absorbance at 550nM to determine formation of reduced cytochrome C having a molar absorbance of 21.1mM -1 cm -1 . The results are shown in Table 2,the enzyme activity is expressed in moles per minute per mole of enzyme catalyzed product formation or substrate consumption per mol (mol enzyme) -1 min -1 。
TABLE 2 enzyme Activity of NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S for reducing cytochrome c by NCDH
Example 3: NCDH-dependent P450 reductase for catalyzing and reducing thiazole blue into blue formazan by utilizing NCDH
100. Mu.L of 50mM Tris-HCl buffer system pH 9: 0.01mM-10mM thiazole blue, 10mM NCDH, 100. Mu.M NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S), 30℃reaction, determination of absorbance at 570nm, determination of reduced formazan production, molar absorbance of formazan 16.2mM -1 cm -1 . The results are shown in Table 3, where the enzyme activity is expressed in terms of moles per minute per mole of enzyme catalyzed product formation or substrate consumption per unit mol (mol enzyme) -1 min -1 。
TABLE 3 enzyme Activity of NCDH-dependent P450 reductase for reducing thiazole blue Using NCDH
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Example 4: coupling catalytic reduction of K by NCDH-dependent P450 reductase and NCDH regenerating enzyme 3 [Fe(CN) 6 ]
100. Mu.L of 100mM Tris-HCl buffer system pH 6.8: 2.5mM K 3 [Fe(CN) 6 ]0.5mM NCD, 50U/mL ME-L310R/Q401C, 5mM L-malic acid, 5mM MgCl 2 100. Mu.M NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S), 30℃reaction, measurement of decrease in absorbance at 420nm, determination of K 3 [Fe(CN) 6 ]Is reduced, K 3 [Fe(CN) 6 ]Molar absorptivity at 420nm of 1.020mM -1 cm -1 . The results are shown in Table 4, and the enzymeActivity is expressed in moles per minute per mole of enzyme catalyzed product formation or substrate consumption per mol (mol enzyme) -1 min -1 。
TABLE 4 NCDH-dependent P450 reductase coupled with NCDH regeneration enzyme to reduce K 3 [Fe(CN) 6 ]Enzyme activity
Enzymes Enzyme activity (U)
BMR-R967D/Q977E/W1047S 4431±101
BMR-R967D/Q977E/Q1005E/W1047S 4609±72
Example 5: coupling catalytic reduction of cytochrome c by NCDH-dependent P450 reductase and NCDH regenerating enzyme
100. Mu.L of 200mM potassium phosphate buffer system pH 7.5: 40. Mu.M cytochrome c, 200. Mu.M NCD, 100U/mL PDH-I151R/P176R/M207A, 20mM sodium phosphite, 2nM NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S), 30℃reaction, measurement of absorbance at 550nM to determine the production of reduced cytochrome c, the molar absorbance of reduced cytochrome c being 21.1mM -1 cm -1 . The results are shown in Table 5, where the enzyme activity is expressed in terms of moles per minute per mole of enzyme catalyzed product formation or substrate consumption per unit mol (mol enzyme) -1 min -1 。
TABLE 5 enzyme Activity of NCDH-dependent P450 reductase coupled with NCDH regeneration enzyme to reduce cytochrome c
Enzymes Enzyme activity (U)
BMR-R967D/Q977E/W1047S 2891±38
BMR-R967D/Q977E/Q1005E/W1047S 3143±127
Example 6: coupling catalytic reduction of thiazole blue by NCDH dependent P450 reductase and NCDH regenerating enzyme
100. Mu.L of 100mM Tris-HCl buffer system pH 8.1: 0.4mM thiazole blue, 1mM NCD, 2U/mL DLDH-V152R/N213E, 2mM D-lactic acid, 100nM NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S), 30℃reaction, determination of absorbance at 570nM, determination of reduced formazan formation, molar absorbance of formazan of 16.2mM -1 cm -1 . The results are shown in Table 6, where the enzyme activity is expressed in terms of moles per minute per mole of enzyme catalyzed product formation or substrate consumption per unit mol (mol enzyme) -1 min -1 。
TABLE 6 enzyme Activity of NCDH-dependent P450 reductase coupled with NCDH regeneration enzyme to reduce thiazole blue
Enzymes Enzyme activity (U)
BMR-R967D/Q977E/W1047S 657±49
BMR-R967D/Q977E/Q1005E/W1047S 859±76
Example 7: catalytic system of NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S and CYP101A1
100. Mu.L of 50mM Tris-HCl buffer system pH 7.0: 0.1mM NCDH, 0.05mM D-camphor, 2nM CYP101A1, 2nM NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S,30℃for 24h, 5. Mu.L 4M HCl was added to terminate the reaction, D-camphor and hydroxylation products were extracted with an equal volume of ethyl acetate, 10000g were centrifuged for 5min, and the organic phase was detected by GC-MS analysis and the D-camphor conversion was 20.3%.
Example 8: catalytic system of NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S and CYP152L1
100. Mu.L of 50mM Tris-HCl buffer system pH 5.5: 0.05mM NCDH, 1mM stearic acid, 5. Mu.M CYP152L1, 5. Mu.M NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S, reaction at 30℃for 4h, termination of the reaction by adding 5. Mu.L 4M HCl, extraction of stearic acid and decarboxylated product with equal volume of chloroform, centrifugation of 10000g for 5min, derivatization of stearic acid and decarboxylated product with BSTFA followed by GC-MS analysis, stearic acid conversion of 18.8%.
Example 9: catalytic system of NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S and CYP53A15
100. Mu.L of 50mM Tris-HCl buffer system pH 8.8: 50mM NCDH, 50mM benzoic acid, 100. Mu.M CYP53A15 and 100. Mu.M NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S, react for 4 hours at 30 ℃, add 5. Mu.L 4M HCl to terminate the reaction, add an equal volume of acetonitrile to the sample, centrifuge 10000g for 5 minutes, and then analyze by HPLC that the benzoic acid conversion rate is 4.6%.
Example 10: catalytic system of NCDH-dependent P450 reductase and different P450 enzymes
100. Mu.L of 50mM Tris-HCl buffer system pH 7.5: 0.1mM NCDH, 50U/mL MDH-Y171R/I196V/V237T/N240E/K241A, 10mM methanol, 1mM D-camphor (or stearic acid or benzoic acid), 10. Mu.M CYP101A1 (or 5. Mu.M CYP152L1 or 20. Mu.M CYP53A 15) and NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S) equimolar with free P450, the reaction was terminated by adding 5. Mu.L of 4M HCl, the D-camphor and the hydroxylation product were extracted with an equal volume of ethyl acetate (stearic acid and decarboxylation product were extracted with an equal volume of chloroform; benzoic acid sample was directly added with an equal volume of acetonitrile, 10000g was centrifuged for 5min, the organic phase was analyzed by GC-MS (stearic acid and decarboxylation product was analyzed by BSderivatization and GC-MS), the reaction results are shown in Table 7).
TABLE 7 substrate conversion of different P450 enzymes and NCDH-dependent P450 reductase catalytic systems
Example 11: catalytic system for coupling NCDH-dependent self-sufficient difunctional P450 enzyme with NCDH regeneration enzyme
300. Mu.L of 100mM MES buffer system at pH 8.0: 0.1mM NCD, 0.4mg/mL FDH-V198I/C256I/P260S/E261P/S381N/S383F, 20mM sodium formate, 1.1mg/mL NCDH-dependent self-sufficient bifunctional P450 enzyme (CYP 101A1-Linker3-BMR-R967D/Q977E/W1047S or CYP101A1-Linker1-BMR-R967D/Q977E/Q1005E/W1047S or CYP152L1-Linker1-BMR-R967D/Q977E/Q1005E/W1047S or CYP53A15-Linker5-BMR-R967D/Q977E/W1047S or CYP53A15-Linker 1-BMR-R967D/Q977E/W1047S or CYP152L1-Linker1-BMR-R967D/Q977E/W1047S or CYP152L 1-mM 2 or camphor acid or benzoic acid). The reaction solution was treated and analyzed in the same manner as in example 2, and the reaction results are shown in Table 8.
TABLE 8 substrate conversion of NCDH-dependent, self-sufficient bifunctional P450 enzyme-coupled NCDH regenerant System
Example 12: catalytic system for coupling NCDH-dependent CYP102A1 enzyme with NCDH regeneration enzyme
300. Mu.L of 100mM Tris-HCl buffer system pH 8.0: 0.1mM NCD, 0.4mg/mL FDH-V198I/C256I/P260S/E261P/S381N/S383F, 20mM sodium formate, 1.1mg/mL NCDH-dependent P450 BM3 enzyme (CYP 102A1-R967D/Q977E/W1047S or CYP102A 1-R967D/Q977E/Q1005E/W1047S), 2mM fatty acid (C 12:0 Or C 14:0 Or C 16:0 Or C 18:0 ) The reaction was carried out at 30℃for 4 hours. The reaction solution was treated and analyzed in the same manner as in example 2, and the reaction results are shown in Table 9.
TABLE 9 substrate conversion of NCDH-dependent P450 BM3 coupled NCDH regeneration enzyme System
Example 13: NCDH-dependent P450 reductase-mediated prokaryotic cell catalytic conversion system
The NCDH-dependent P450 reductase, the free P450 enzyme, the NCDH regenerating enzyme and the NCD transporter are co-expressed in a host to construct a NCD-mediated biocatalysis system. The biocatalytic system is started after the regeneration substrate and NCD in the medium enter the host cell. Thus, the use of NCDH-dependent P450 reductases will not depend on intracellular NADPH levels, and can selectively transfer to P450 substrates using extracellular regenerative substrate reducing power, enabling decoupling of P450 enzyme-catalyzed energy consumption from endogenous NADPH supply.
The construction of an engineering strain for catalyzing the decarboxylation of fatty acids will be described below by taking E.coli Escherichia coli BL (DE 3) as a host strain.
NAD transporter AtNDT2 (Accession No. NC-003070) has a broad substrate spectrum (Palnieri F, et al J Biol Chem,2009,284,31249-31259) and can transport NCD. The expression of the gene AtNDT2 expressing the transporter was controlled by the gapAP1 promoter (Charpentier B, et al J Bacteriol,1994,176,830-839), the gene encoding BMR-R967D/Q977E/W1047S, the gene encoding CYP152L1, and the gene encoding malic enzyme ME-L310R/Q401C were controlled by lac, trc, and T7 promoters, respectively, and isopropyl thiogalactose (IPTG) was induced, and these four expression cassettes were constructed into the same plasmid pUC18 (bla: kan) by enzyme-cutting links to obtain an engineering plasmid.
The engineering plasmid is introduced into E.coli BL21 (DE 3) to obtain engineering strain E.coli QL001. Inducing engineering strain E.coli QL001 to express the four functional proteins in LB culture medium, adding 50 mug/mL kanamycin, 0.5mM IPTG, 1mM 5-aminolevulinic acid, 1mM vitamin B1 and 0.04mM FeCl into the culture medium 3 Culturing at 25deg.C in a shaking table at 200rpm for 48 hr to reach cell density OD 600 The cells were collected by centrifugation at 2000 Xg for 6min at 4.5.
The cells were resuspended by washing with MOPS medium at pH 7.5, and the cell density OD was determined 600 Adjusted to 9. 2mM lauric acid, 5% DMSO for dissolution assistance, 10mM L-malic acid and 3mM NCD are added into the engineering bacteria suspension, the total volume is 1mL, the reaction is carried out for 4 hours in a shaking table at 30 ℃ and 200rpm, 500 mu L of 4M HCl and 0.5mL chloroform are added, after vortex shaking for 3 minutes, centrifugation is carried out, the lower organic phase is taken out, a 0.22 mu M organic filter membrane is used for filtration, 150 mu L of a silylation reagent N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) is added for derivatization, and the conversion rate of lauric acid is 18.4% by analysis of BSGC-MS. In the control experiment without NCD, the conversion of lauric acid was 4.3%.
Experimental results show that in the whole cell catalysis process of escherichia coli, NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S utilizes malic enzyme ME-L310R/Q401C to regenerate NCDH, and electrons are transferred to CYP152L1 enzyme to catalyze lauric acid decarboxylation reaction.
Example 14: NCDH-dependent P450 reductase-mediated eukaryotic cell catalytic conversion system.
The NCDH-dependent P450 reductase, the free P450 enzyme, the NCDH regenerating enzyme and the NCD transporter are co-expressed in a host to construct a NCD-mediated biocatalysis system. The biocatalytic system is started after the regeneration substrate and NCD in the medium enter the host cell. Thus, the use of NCDH-dependent P450 reductases will not depend on intracellular NADPH levels, and can selectively transfer to P450 substrates using extracellular regenerative substrate reducing power, enabling decoupling of P450 enzyme-catalyzed energy consumption from endogenous NADPH supply.
The construction of an engineering strain for catalyzing benzoic acid hydroxylation using Saccharomyces cerevisiae Saccharomyces cerevisiae BY4741 as a host strain will be described below.
NAD transporter NTT4 (Haferkamp I, et al Nature,2004,432,622-625.) can transport NTD. The gene encoding CYP53A15 is controlled by a TEF1 promoter and a CYC1 terminator, the gene encoding BMR-R967D/Q977E/Q1005E/W1047S is controlled by a TDH3 type promoter and an ADH1 terminator, the gene encoding FDH-V198I/C256I/P260S/E261P/S381N/S383F is controlled by a PGK1 promoter and a PHO5 terminator, the gene encoding NTT4 is controlled by a URA3 promoter and a TDH1 terminator, and the four expression cassettes are integrated into a P416 yeast episomal shuttle expression vector to obtain an engineering plasmid.
The engineering plasmid is introduced into Saccharomyces cerevisiae to obtain engineering strain S.cerevisiae QL002. Inducing engineering bacteria S.cerevisiae QL002 to express the above two functional proteins with YEPD medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone at pH 6.0, culturing in shaking table at 25deg.C and 200rpm for 48 hr to cell density OD 600 The cells were collected by centrifugation at 2000 Xg for 6min at 4.5, and the resuspended cells were washed with Tris-Cl at a concentration of 50mM and pH 7.5 to give a cell density OD 600 Adjusted to 9. Preparation of permeabilized cells: 5mL of frozen cells were thawed in a water bath at room temperature, 5mM EDTA and toluene at a volume ratio of 1% were added, and the mixture was incubated at 30℃for 30 minutes in a shaker at 200rpm, and then left at 4℃for 1 hour. The supernatant containing EDTA and toluene was removed by centrifugation at 2000g for 6min, washed twice with 50mM Tris-Cl at pH 7.5 and resuspended in 5mL Tris-Cl at pH 50mM and pH 7.5 to obtain permeabilized cells.
5mM benzoic acid, 10mM sodium formate and 1mM NCD were added to the above-mentioned suspension of the permeabilized engineering bacteria resuspended in 50mM Tris-Cl at pH 7.5, the total volume was 1mL, the reaction was carried out in a shaker at 200rpm at 30℃for 4 hours, 500. Mu.L was added with 25. Mu.L of 4M HCl and 0.5mL acetonitrile, and after vortexing for 1min, the suspension was filtered with a 0.22 μm organic filter membrane, and the conversion of benzoic acid was 41.3% by HPLC analysis. In the control experiment without NCD, the conversion of benzoic acid was 3.7%.
Experimental results show that in the whole-cell catalysis process of saccharomyces cerevisiae, NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S utilizes formate dehydrogenase FDH-V198I/C256I/P260S/E261P/S381N/S383F to regenerate NCDH, transmits electrons to CYP53A15 enzyme, and catalyzes lauric acid decarboxylation.
Example 15: NCDH-dependent self-sufficient difunctional P450 enzyme-mediated prokaryotic cell catalytic conversion system
The NCDH-dependent self-sufficient difunctional P450 enzyme, the NCDH regeneration enzyme and the NCD transporter are co-expressed in host cells to form a NCD-dependent biocatalysis system. The biocatalytic system is started after the regeneration substrate and NCD in the medium enter the host cell. Thus, the use of an NCDH-dependent self-sufficient bifunctional P450 enzyme will not depend on intracellular NADPH levels, and can be selectively transferred to the P450 enzyme substrate by extracellular regenerative substrate reducing power, enabling decoupling of the energy consumption of the P450 enzyme from the endogenous NADPH supply.
The construction of an engineering strain for catalyzing the hydroxylation of D-camphor is described below by taking Escherichia coli Escherichia coli BW25113 as a host bacterium.
NAD transporter NTT4 (Haferkamp I, et al Nature,2004,432,622-625.) can transport NTD. The expression of the gene for NTT4 expressing the transporter was controlled by the gapAP1 promoter (Charpentier B, et al J Bacteriol,1994,176,830-839). The gene encoding CYP101A1-Linker1-BMR-R967D/Q977E/Q1005E/W1047S and the gene encoding the phosphite dehydrogenase PDH-I151R/P176E/M207A were controlled by lac and trc promoters induced by isopropyl thiogalactose (IPTG), and the above three expression cassettes were cloned into the same plasmid by replacing the LacZ gene of pUC18 (bla:: kan) to obtain an engineering plasmid.
And introducing the engineering plasmid into E.coli BW25113 to obtain an engineering strain E.coli QL 003. Inducing engineering strain E.coll QL003 to express the three functional proteins in LB culture medium, adding 50 μg/mL kanamycin, 0.5mM IPTG, 1mM 5-aminolevulinic acid, 1mM vitamin B1 and 0.04mM FeCl into the culture medium 3 Culturing at 25deg.C in a shaking table at 200rpm for 48 hr to reach cell density OD 600 The cells were collected by centrifugation at 2000 Xg for 6min at 4.5.
Washing and resuspending the cells with MOPS medium at pH 7.5 to obtain cell density OD 600 Adjusted to 9. 10mM D is added into the engineering bacteria suspensionCamphor (5% v/v DMSO-co-solubilised), 20mM sodium phosphite, 5mM NCD, total volume 1mL, in a shaking table at 200rpm at 30℃for 4h, 500. Mu.L of 4M HCl and 0.5mL ethyl acetate were added, after vortexing for 3min, the upper organic phase was centrifuged off, the organic phase was filtered with a 0.22 μm organic filter membrane, 500. Mu.L was analysed by GC-MS and D-camphor conversion was 30.3%. In the control experiment without NCD addition, the D-camphor conversion was 2.6%.
Experimental results show that in the whole cell catalysis process of escherichia coli, NCDH-dependent self-sufficient difunctional P450 enzyme CYP101A1-Linker1-BMR-R967D/Q977E/Q1005E/W1047S utilizes NCDH regenerated by phosphite dehydrogenase PDH-I151R to complete the NCD-mediated D-camphora hydroxylation reaction driven by phosphite.
Example 16: NCDH-dependent self-sufficient difunctional P450 enzyme-mediated eukaryotic cell catalytic conversion system
The NCDH-dependent self-sufficient difunctional P450 enzyme and the NCDH regenerating enzyme are co-expressed in an NCD self-sufficient escherichia coli host to form a NCD-dependent biocatalysis system. After the regeneration substrate in the medium enters the host cell, the biocatalysis system is started. Thus, the use of an NCDH-dependent self-sufficient bifunctional P450 enzyme will not depend on intracellular NADPH levels, and can be selectively transferred to the P450 enzyme substrate by extracellular regenerative substrate reducing power, enabling decoupling of the energy consumption of the P450 enzyme from the endogenous NADPH supply.
The construction of an engineering strain for catalyzing the hydroxylation of fatty acids is described below by taking Saccharomyces cerevisiae Saccharomyces cerevisiae BY4742 as a host strain.
The gene encoding CYP102A1-R967D/Q977E/Q1005E/W1047S is controlled by a TEF1 promoter and a CYC1 terminator, the gene encoding DLDH-V152R/I177K/N213I is controlled by a PGK1 promoter and a PHO5 terminator, the gene encoding NTT4 is controlled by a URA3 promoter and a TDH1 terminator, and the three expression cassettes are integrated into a p416 yeast episomal shuttle expression vector to obtain engineering plasmids.
And introducing the engineering plasmid into saccharomyces cerevisiae to obtain engineering strain S.cerevisiae QL004. Induction of engineering with YEPD medium containing 20g/L glucose, 10g/L Yeast extract, 20g/L peptone at pH 6.0 Bacteria S.cerevisiae QL004 express the two functional proteins, and are cultured for 48 hours in a shaking table at 25 ℃ and 200rpm to reach the bacterial density OD 600 The cells were collected by centrifugation at 2000 Xg for 6min, and the resuspended cells were washed with Tris-Cl at a concentration of 50mM and pH 7.5 to adjust the cell density OD600 to 9. Preparation of permeabilized cells: 5mL of frozen cells were thawed in a water bath at room temperature, 5mM EDTA and toluene at a volume ratio of 1% were added, and the mixture was incubated at 30℃for 30 minutes in a shaker at 200rpm, and then left at 4℃for 1 hour. The supernatant containing EDTA and toluene was removed by centrifugation at 2000g for 6min, washed twice with 50mM Tris-Cl at pH 7.5 and resuspended in 5mL Tris-Cl at pH 50mM and pH 7.5 to obtain permeabilized cells.
1mM lauric acid (5% v/v DMSO-assisted dissolution), 10mM D-sodium lactate, 3mM NCD were added to the above-mentioned suspension of the permeabilized engineering bacteria resuspended in 50mM Tris-Cl at pH 7.5, the total volume was 1mL, reacted for 4 hours at 30℃in a 200rpm shaker, 500. Mu.L of 4M HCl and 0.5mL chloroform were added, after vortexing for 3min, the lower organic phase was removed by centrifugation, the organic phase was filtered with a 0.22 μm organic filter membrane, 500. Mu.L was analyzed by GC-MS, and the conversion of lauric acid hydroxylation was 85.3%. In the control experiment without NCD, the conversion of lauric acid hydroxylation was 5.2%.
Experimental results show that in the whole-cell catalysis process of saccharomyces cerevisiae, NCDH-dependent self-sufficient difunctional P450 enzyme CYP102A1-R967D/Q977E/Q1005E/W1047S utilizes the regenerated NCDH of D-lactate dehydrogenase DLDH-V152R/I177K/N213I to complete the fatty acid hydroxylation reaction mediated by NCD.
The three letter abbreviations and one letter abbreviation correspondence of amino acids in the present application are shown in table 10 below:
TABLE 10 correspondence of one letter abbreviation and three letter abbreviations for amino acids in the present application
Chinese translated name Letter abbreviation Three letter abbreviations
Glycine (Gly) G Gly
Alanine (Ala) A Ala
Valine (valine) V Val
Leucine (leucine) L Leu
Isoleucine (Ile) I Ile
Phenylalanine (Phe) F Phe
Tryptophan W Trp
Tyrosine Y Tyr
Aspartic acid D Asp
Histidine H His
Asparagine derivatives N Asn
Glutamic acid E Glu
Lysine K Lys
Glutamine amide Q Gln
Methionine M Met
Arginine (Arg) R Arg
Serine (serine) S Ser
Threonine (Thr) T Thr
Cysteine (S) C Cys
Proline (proline) P Pro
While the application has been described in terms of preferred embodiments, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the scope of the application, and it is intended that the application is not limited to the specific embodiments disclosed.
Sequence listing
<110> institute of chemical and physical of Dalian of academy of sciences of China
<120> a P450 reductase and its use
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Arg Lys Lys Ala Glu Asn Ala His Asn Thr Pro Leu Leu Val Leu Tyr
1 5 10 15
Gly Ser Asn Met Gly Thr Ala Glu Gly Thr Ala Arg Asp Leu Ala Asp
20 25 30
Ile Ala Met Ser Lys Gly Phe Ala Pro Gln Val Ala Thr Leu Asp Ser
35 40 45
His Ala Gly Asn Leu Pro Arg Glu Gly Ala Val Leu Ile Val Thr Ala
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Ser Tyr Asn Gly His Pro Pro Asp Asn Ala Lys Gln Phe Val Asp Trp
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Leu Asp Gln Ala Ser Ala Asp Glu Val Lys Gly Val Arg Tyr Ser Val
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Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr Thr Tyr Gln Lys Val Pro
100 105 110
Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn Ile Ala
115 120 125
Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu
130 135 140
Glu Trp Arg Glu His Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu
145 150 155 160
Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser Thr Leu Ser Leu Gln Phe
165 170 175
Val Asp Ser Ala Ala Asp Met Pro Leu Ala Lys Met His Gly Ala Phe
180 185 190
Ser Thr Asn Val Val Ala Ser Lys Glu Leu Gln Gln Pro Gly Ser Ala
195 200 205
Arg Ser Thr Arg His Leu Glu Ile Glu Leu Pro Lys Glu Ala Ser Tyr
210 215 220
Gln Glu Gly Asp His Leu Gly Val Ile Pro Arg Asn Tyr Glu Gly Ile
225 230 235 240
Val Asn Arg Val Thr Ala Arg Phe Gly Leu Asp Ala Ser Gln Gln Ile
245 250 255
Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala His Leu Pro Leu Ala Lys
260 265 270
Thr Val Ser Val Glu Glu Leu Leu Gln Tyr Val Glu Leu Gln Asp Pro
275 280 285
Val Thr Arg Thr Gln Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro
290 295 300
Pro His Lys Val Glu Leu Glu Ala Leu Leu Glu Lys Gln Ala Tyr Lys
305 310 315 320
Glu Gln Val Leu Ala Lys Arg Leu Thr Met Leu Glu Leu Leu Glu Lys
325 330 335
Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu Phe Ile Ala Leu Leu Pro
340 345 350
Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Val Asp
355 360 365
Glu Lys Gln Ala Ser Ile Thr Val Ser Val Val Ser Gly Glu Ala Trp
370 375 380
Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala Ser Asn Tyr Leu Ala Glu
385 390 395 400
Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe Ile Ser Thr Pro Gln Ser
405 410 415
Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr Pro Leu Ile Met Val Gly
420 425 430
Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Val Gln Ala Arg Lys
435 440 445
Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe
450 455 460
Gly Cys Arg Ser Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu
465 470 475 480
Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu His Thr Ala Phe Ser Arg
485 490 495
Met Pro Asn Gln Pro Lys Thr Tyr Val Gln His Val Met Glu Gln Asp
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Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile
515 520 525
Cys Gly Asp Gly Ser Gln Met Ala Pro Ala Val Glu Ala Thr Leu Met
530 535 540
Lys Ser Tyr Ala Asp Val His Gln Val Ser Glu Ala Asp Ala Arg Leu
545 550 555 560
Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp
565 570 575
Ala Gly
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Arg Lys Lys Ala Glu Asn Ala His Asn Thr Pro Leu Leu Val Leu Tyr
1 5 10 15
Gly Ser Asn Met Gly Thr Ala Glu Gly Thr Ala Arg Asp Leu Ala Asp
20 25 30
Ile Ala Met Ser Lys Gly Phe Ala Pro Gln Val Ala Thr Leu Asp Ser
35 40 45
His Ala Gly Asn Leu Pro Arg Glu Gly Ala Val Leu Ile Val Thr Ala
50 55 60
Ser Tyr Asn Gly His Pro Pro Asp Asn Ala Lys Gln Phe Val Asp Trp
65 70 75 80
Leu Asp Gln Ala Ser Ala Asp Glu Val Lys Gly Val Arg Tyr Ser Val
85 90 95
Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr Thr Tyr Gln Lys Val Pro
100 105 110
Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn Ile Ala
115 120 125
Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu
130 135 140
Glu Trp Arg Glu His Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu
145 150 155 160
Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser Thr Leu Ser Leu Gln Phe
165 170 175
Val Asp Ser Ala Ala Asp Met Pro Leu Ala Lys Met His Gly Ala Phe
180 185 190
Ser Thr Asn Val Val Ala Ser Lys Glu Leu Gln Gln Pro Gly Ser Ala
195 200 205
Arg Ser Thr Arg His Leu Glu Ile Glu Leu Pro Lys Glu Ala Ser Tyr
210 215 220
Gln Glu Gly Asp His Leu Gly Val Ile Pro Arg Asn Tyr Glu Gly Ile
225 230 235 240
Val Asn Arg Val Thr Ala Arg Phe Gly Leu Asp Ala Ser Gln Gln Ile
245 250 255
Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala His Leu Pro Leu Ala Lys
260 265 270
Thr Val Ser Val Glu Glu Leu Leu Gln Tyr Val Glu Leu Gln Asp Pro
275 280 285
Val Thr Arg Thr Gln Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro
290 295 300
Pro His Lys Val Glu Leu Glu Ala Leu Leu Glu Lys Gln Ala Tyr Lys
305 310 315 320
Glu Gln Val Leu Ala Lys Arg Leu Thr Met Leu Glu Leu Leu Glu Lys
325 330 335
Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu Phe Ile Ala Leu Leu Pro
340 345 350
Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Val Asp
355 360 365
Glu Lys Gln Ala Ser Ile Thr Val Ser Val Val Ser Gly Glu Ala Trp
370 375 380
Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala Ser Asn Tyr Leu Ala Glu
385 390 395 400
Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe Ile Ser Thr Pro Gln Ser
405 410 415
Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr Pro Leu Ile Met Val Gly
420 425 430
Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Val Gln Ala Arg Lys
435 440 445
Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe
450 455 460
Gly Cys Arg Ser Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu
465 470 475 480
Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu His Thr Ala Phe Ser Asp
485 490 495
Met Pro Asn Gln Pro Lys Thr Tyr Val Glu His Val Met Glu Gln Asp
500 505 510
Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile
515 520 525
Cys Gly Asp Gly Ser Gln Met Ala Pro Ala Val Glu Ala Thr Leu Met
530 535 540
Lys Ser Tyr Ala Asp Val His Gln Val Ser Glu Ala Asp Ala Arg Leu
545 550 555 560
Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Ser
565 570 575
Ala Gly
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Arg Lys Lys Ala Glu Asn Ala His Asn Thr Pro Leu Leu Val Leu Tyr
1 5 10 15
Gly Ser Asn Met Gly Thr Ala Glu Gly Thr Ala Arg Asp Leu Ala Asp
20 25 30
Ile Ala Met Ser Lys Gly Phe Ala Pro Gln Val Ala Thr Leu Asp Ser
35 40 45
His Ala Gly Asn Leu Pro Arg Glu Gly Ala Val Leu Ile Val Thr Ala
50 55 60
Ser Tyr Asn Gly His Pro Pro Asp Asn Ala Lys Gln Phe Val Asp Trp
65 70 75 80
Leu Asp Gln Ala Ser Ala Asp Glu Val Lys Gly Val Arg Tyr Ser Val
85 90 95
Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr Thr Tyr Gln Lys Val Pro
100 105 110
Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn Ile Ala
115 120 125
Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu
130 135 140
Glu Trp Arg Glu His Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu
145 150 155 160
Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser Thr Leu Ser Leu Gln Phe
165 170 175
Val Asp Ser Ala Ala Asp Met Pro Leu Ala Lys Met His Gly Ala Phe
180 185 190
Ser Thr Asn Val Val Ala Ser Lys Glu Leu Gln Gln Pro Gly Ser Ala
195 200 205
Arg Ser Thr Arg His Leu Glu Ile Glu Leu Pro Lys Glu Ala Ser Tyr
210 215 220
Gln Glu Gly Asp His Leu Gly Val Ile Pro Arg Asn Tyr Glu Gly Ile
225 230 235 240
Val Asn Arg Val Thr Ala Arg Phe Gly Leu Asp Ala Ser Gln Gln Ile
245 250 255
Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala His Leu Pro Leu Ala Lys
260 265 270
Thr Val Ser Val Glu Glu Leu Leu Gln Tyr Val Glu Leu Gln Asp Pro
275 280 285
Val Thr Arg Thr Gln Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro
290 295 300
Pro His Lys Val Glu Leu Glu Ala Leu Leu Glu Lys Gln Ala Tyr Lys
305 310 315 320
Glu Gln Val Leu Ala Lys Arg Leu Thr Met Leu Glu Leu Leu Glu Lys
325 330 335
Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu Phe Ile Ala Leu Leu Pro
340 345 350
Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Val Asp
355 360 365
Glu Lys Gln Ala Ser Ile Thr Val Ser Val Val Ser Gly Glu Ala Trp
370 375 380
Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala Ser Asn Tyr Leu Ala Glu
385 390 395 400
Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe Ile Ser Thr Pro Gln Ser
405 410 415
Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr Pro Leu Ile Met Val Gly
420 425 430
Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Val Gln Ala Arg Lys
435 440 445
Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe
450 455 460
Gly Cys Arg Ser Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu
465 470 475 480
Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu His Thr Ala Phe Ser Asp
485 490 495
Met Pro Asn Gln Pro Lys Thr Tyr Val Glu His Val Met Glu Gln Asp
500 505 510
Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile
515 520 525
Cys Gly Asp Gly Ser Glu Met Ala Pro Ala Val Glu Ala Thr Leu Met
530 535 540
Lys Ser Tyr Ala Asp Val His Gln Val Ser Glu Ala Asp Ala Arg Leu
545 550 555 560
Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Ser
565 570 575
Ala Gly
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Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys
1 5 10 15
Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys
20 25 30
Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg
35 40 45
Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp
50 55 60
Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg
65 70 75 80
Asp Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn
85 90 95
Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala
100 105 110
Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val
115 120 125
Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu
130 135 140
Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn
145 150 155 160
Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr
165 170 175
Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala
180 185 190
Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu
195 200 205
Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg
210 215 220
Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn
225 230 235 240
Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg
245 250 255
Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly
260 265 270
Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu
275 280 285
Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro
290 295 300
Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn
305 310 315 320
Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala
325 330 335
Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp
340 345 350
Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp
355 360 365
Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser
370 375 380
Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala
385 390 395 400
Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly
405 410 415
Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu
420 425 430
Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys
435 440 445
Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr
450 455 460
Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn
465 470 475 480
Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly
485 490 495
Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro
500 505 510
Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly
515 520 525
Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn
530 535 540
Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val
545 550 555 560
Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala
565 570 575
Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala
580 585 590
Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp
595 600 605
Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp
610 615 620
Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys
625 630 635 640
Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu
645 650 655
Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu
660 665 670
Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu
675 680 685
Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile
690 695 700
Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly
705 710 715 720
Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu
725 730 735
Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln
740 745 750
Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met
755 760 765
Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu
770 775 780
Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr
785 790 795 800
Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser
805 810 815
Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile
820 825 830
Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser
835 840 845
Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile
850 855 860
Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys
865 870 875 880
Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu
885 890 895
Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg
900 905 910
Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu
915 920 925
Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr
930 935 940
Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr
945 950 955 960
Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val
965 970 975
Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp
980 985 990
Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro
995 1000 1005
Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val
1010 1015 1020
Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly
1025 1030 1035 1040
Arg Tyr Ala Lys Asp Val Trp Ala Gly
1045
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<213> Artificial sequence (Artificial Sequence)
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atgcaccatc atcatcatca tcgcaaaaag gcagaaaacg ctcataatac gccgctgctt 60
gtgctatacg gttcaaatat gggaacagct gaaggaacgg cgcgtgattt agcagatatt 120
gcaatgagca aaggatttgc accgcaggtc gcaacgcttg attcacacgc cggaaatctt 180
ccgcgcgaag gagctgtatt aattgtaacg gcgtcttata acggtcatcc gcctgataac 240
gcaaagcaat ttgtcgactg gttagaccaa gcgtctgctg atgaagtaaa aggcgttcgc 300
tactccgtat ttggatgcgg cgataaaaac tgggctacta cgtatcaaaa agtgcctgct 360
tttatcgatg aaacgcttgc cgctaaaggg gcagaaaaca tcgctgaccg cggtgaagca 420
gatgcaagcg acgactttga aggcacatat gaagaatggc gtgaacatat gtggagtgac 480
gtagcagcct actttaacct cgacattgaa aacagtgaag ataataaatc tactctttca 540
cttcaatttg tcgacagcgc cgcggatatg ccgcttgcga aaatgcacgg tgcgttttca 600
acgaacgtcg tagcaagcaa agaacttcaa cagccaggca gtgcacgaag cacgcgacat 660
cttgaaattg aacttccaaa agaagcttct tatcaagaag gagatcattt aggtgttatt 720
cctcgcaact atgaaggaat agtaaaccgt gtaacagcaa ggttcggcct agatgcatca 780
cagcaaatcc gtctggaagc agaagaagaa aaattagctc atttgccact cgctaaaaca 840
gtatccgtag aagagcttct gcaatacgtg gagcttcaag atcctgttac gcgcacgcag 900
cttcgcgcaa tggctgctaa aacggtctgc ccgccgcata aagtagagct tgaagccttg 960
cttgaaaagc aagcctacaa agaacaagtg ctggcaaaac gtttaacaat gcttgaactg 1020
cttgaaaaat acccggcgtg tgaaatgaaa ttcagcgaat ttatcgccct tctgccaagc 1080
atacgcccgc gctattactc gatttcttca tcacctcgtg tcgatgaaaa acaagcaagc 1140
atcacggtca gcgttgtctc aggagaagcg tggagcggat atggagaata taaaggaatt 1200
gcgtcgaact atcttgccga gctgcaagaa ggagatacga ttacgtgctt tatttccaca 1260
ccgcagtcag aatttacgct gccaaaagac cctgaaacgc cgcttatcat ggtcggaccg 1320
ggaacaggcg tcgcgccgtt tagaggcttt gtgcaggcgc gcaaacagct aaaagaacaa 1380
ggacagtcac ttggagaagc acatttatac ttcggctgcc gttcacctca tgaagactat 1440
ctgtatcaag aagagcttga aaacgcccaa agcgaaggca tcattacgct tcataccgct 1500
ttttctcgca tgccaaatca gccgaaaaca tacgttcagc acgtaatgga acaagacggc 1560
aagaaattga ttgaacttct tgatcaagga gcgcacttct atatttgcgg agacggaagc 1620
caaatggcac ctgccgttga agcaacgctt atgaaaagct atgctgacgt tcaccaagtg 1680
agtgaagcag acgctcgctt atggctgcag cagctagaag aaaaaggccg atacgcaaaa 1740
gacgtgtggg ctgggtaa 1758
<210> 6
<211> 1758
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atgcaccatc atcatcatca tcgcaaaaag gcagaaaacg ctcataatac gccgctgctt 60
gtgctatacg gttcaaatat gggaacagct gaaggaacgg cgcgtgattt agcagatatt 120
gcaatgagca aaggatttgc accgcaggtc gcaacgcttg attcacacgc cggaaatctt 180
ccgcgcgaag gagctgtatt aattgtaacg gcgtcttata acggtcatcc gcctgataac 240
gcaaagcaat ttgtcgactg gttagaccaa gcgtctgctg atgaagtaaa aggcgttcgc 300
tactccgtat ttggatgcgg cgataaaaac tgggctacta cgtatcaaaa agtgcctgct 360
tttatcgatg aaacgcttgc cgctaaaggg gcagaaaaca tcgctgaccg cggtgaagca 420
gatgcaagcg acgactttga aggcacatat gaagaatggc gtgaacatat gtggagtgac 480
gtagcagcct actttaacct cgacattgaa aacagtgaag ataataaatc tactctttca 540
cttcaatttg tcgacagcgc cgcggatatg ccgcttgcga aaatgcacgg tgcgttttca 600
acgaacgtcg tagcaagcaa agaacttcaa cagccaggca gtgcacgaag cacgcgacat 660
cttgaaattg aacttccaaa agaagcttct tatcaagaag gagatcattt aggtgttatt 720
cctcgcaact atgaaggaat agtaaaccgt gtaacagcaa ggttcggcct agatgcatca 780
cagcaaatcc gtctggaagc agaagaagaa aaattagctc atttgccact cgctaaaaca 840
gtatccgtag aagagcttct gcaatacgtg gagcttcaag atcctgttac gcgcacgcag 900
cttcgcgcaa tggctgctaa aacggtctgc ccgccgcata aagtagagct tgaagccttg 960
cttgaaaagc aagcctacaa agaacaagtg ctggcaaaac gtttaacaat gcttgaactg 1020
cttgaaaaat acccggcgtg tgaaatgaaa ttcagcgaat ttatcgccct tctgccaagc 1080
atacgcccgc gctattactc gatttcttca tcacctcgtg tcgatgaaaa acaagcaagc 1140
atcacggtca gcgttgtctc aggagaagcg tggagcggat atggagaata taaaggaatt 1200
gcgtcgaact atcttgccga gctgcaagaa ggagatacga ttacgtgctt tatttccaca 1260
ccgcagtcag aatttacgct gccaaaagac cctgaaacgc cgcttatcat ggtcggaccg 1320
ggaacaggcg tcgcgccgtt tagaggcttt gtgcaggcgc gcaaacagct aaaagaacaa 1380
ggacagtcac ttggagaagc acatttatac ttcggctgcc gttcacctca tgaagactat 1440
ctgtatcaag aagagcttga aaacgcccaa agcgaaggca tcattacgct tcataccgct 1500
ttttctgata tgccaaatca gccgaaaaca tacgttgagc acgtaatgga acaagacggc 1560
aagaaattga ttgaacttct tgatcaagga gcgcacttct atatttgcgg agacggaagc 1620
caaatggcac ctgccgttga agcaacgctt atgaaaagct atgctgacgt tcaccaagtg 1680
agtgaagcag acgctcgctt atggctgcag cagctagaag aaaaaggccg atacgcaaaa 1740
gacgtgagcg ctgggtaa 1758
<210> 7
<211> 1758
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgcaccatc atcatcatca tcgcaaaaag gcagaaaacg ctcataatac gccgctgctt 60
gtgctatacg gttcaaatat gggaacagct gaaggaacgg cgcgtgattt agcagatatt 120
gcaatgagca aaggatttgc accgcaggtc gcaacgcttg attcacacgc cggaaatctt 180
ccgcgcgaag gagctgtatt aattgtaacg gcgtcttata acggtcatcc gcctgataac 240
gcaaagcaat ttgtcgactg gttagaccaa gcgtctgctg atgaagtaaa aggcgttcgc 300
tactccgtat ttggatgcgg cgataaaaac tgggctacta cgtatcaaaa agtgcctgct 360
tttatcgatg aaacgcttgc cgctaaaggg gcagaaaaca tcgctgaccg cggtgaagca 420
gatgcaagcg acgactttga aggcacatat gaagaatggc gtgaacatat gtggagtgac 480
gtagcagcct actttaacct cgacattgaa aacagtgaag ataataaatc tactctttca 540
cttcaatttg tcgacagcgc cgcggatatg ccgcttgcga aaatgcacgg tgcgttttca 600
acgaacgtcg tagcaagcaa agaacttcaa cagccaggca gtgcacgaag cacgcgacat 660
cttgaaattg aacttccaaa agaagcttct tatcaagaag gagatcattt aggtgttatt 720
cctcgcaact atgaaggaat agtaaaccgt gtaacagcaa ggttcggcct agatgcatca 780
cagcaaatcc gtctggaagc agaagaagaa aaattagctc atttgccact cgctaaaaca 840
gtatccgtag aagagcttct gcaatacgtg gagcttcaag atcctgttac gcgcacgcag 900
cttcgcgcaa tggctgctaa aacggtctgc ccgccgcata aagtagagct tgaagccttg 960
cttgaaaagc aagcctacaa agaacaagtg ctggcaaaac gtttaacaat gcttgaactg 1020
cttgaaaaat acccggcgtg tgaaatgaaa ttcagcgaat ttatcgccct tctgccaagc 1080
atacgcccgc gctattactc gatttcttca tcacctcgtg tcgatgaaaa acaagcaagc 1140
atcacggtca gcgttgtctc aggagaagcg tggagcggat atggagaata taaaggaatt 1200
gcgtcgaact atcttgccga gctgcaagaa ggagatacga ttacgtgctt tatttccaca 1260
ccgcagtcag aatttacgct gccaaaagac cctgaaacgc cgcttatcat ggtcggaccg 1320
ggaacaggcg tcgcgccgtt tagaggcttt gtgcaggcgc gcaaacagct aaaagaacaa 1380
ggacagtcac ttggagaagc acatttatac ttcggctgcc gttcacctca tgaagactat 1440
ctgtatcaag aagagcttga aaacgcccaa agcgaaggca tcattacgct tcataccgct 1500
ttttctgata tgccaaatca gccgaaaaca tacgttgagc acgtaatgga acaagacggc 1560
aagaaattga ttgaacttct tgatcaagga gcgcacttct atatttgcgg agacggaagc 1620
gagatggcac ctgccgttga agcaacgctt atgaaaagct atgctgacgt tcaccaagtg 1680
agtgaagcag acgctcgctt atggctgcag cagctagaag aaaaaggccg atacgcaaaa 1740
gacgtgagcg ctgggtaa 1758
<210> 8
<211> 3171
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atgcaccatc atcatcatca tatgacaatt aaagaaatgc ctcagccaaa aacgtttgga 60
gagcttaaaa atttaccgtt attaaacaca gataaaccgg ttcaagcttt gatgaaaatt 120
gcggatgaat taggagaaat ctttaaattc gaggcgcctg gtcgtgtaac gcgctactta 180
tcaagtcagc gtctaattaa agaagcatgc gatgaatcac gctttgataa aaacttaagt 240
caagcgctta aatttgtacg tgattttgca ggagacgggt tatttacaag ctggacgcat 300
gaaaaaaatt ggaaaaaagc gcataatatc ttacttccaa gcttcagtca gcaggcaatg 360
aaaggctatc atgcgatgat ggtcgatatc gccgtgcagc ttgttcaaaa gtgggagcgt 420
ctaaatgcag atgagcatat tgaagtaccg gaagacatga cacgtttaac gcttgataca 480
attggtcttt gcggctttaa ctatcgcttt aacagctttt accgagatca gcctcatcca 540
tttattacaa gtatggtccg tgcactggat gaagcaatga acaagctgca gcgagcaaat 600
ccagacgacc cagcttatga tgaaaacaag cgccagtttc aagaagatat caaggtgatg 660
aacgacctag tagataaaat tattgcagat cgcaaagcaa gcggtgaaca aagcgatgat 720
ttattaacgc atatgctaaa cggaaaagat ccagaaacgg gtgagccgct tgatgacgag 780
aacattcgct atcaaattat tacattctta attgcgggac acgaaacaac aagtggtctt 840
ttatcatttg cgctgtattt cttagtgaaa aatccacatg tattacaaaa agcagcagaa 900
gaagcagcac gagttctagt agatcctgtt ccaagctaca aacaagtcaa acagcttaaa 960
tatgtcggca tggtcttaaa cgaagcgctg cgcttatggc caactgctcc tgcgttttcc 1020
ctatatgcaa aagaagatac ggtgcttgga ggagaatatc ctttagaaaa aggcgacgaa 1080
ctaatggttc tgattcctca gcttcaccgt gataaaacaa tttggggaga cgatgtggaa 1140
gagttccgtc cagagcgttt tgaaaatcca agtgcgattc cgcagcatgc gtttaaaccg 1200
tttggaaacg gtcagcgtgc gtgtatcggt cagcagttcg ctcttcatga agcaacgctg 1260
gtacttggta tgatgctaaa acactttgac tttgaagatc atacaaacta cgagctggat 1320
attaaagaaa ctttaacgtt aaaacctgaa ggctttgtgg taaaagcaaa atcgaaaaaa 1380
attccgcttg gcggtattcc ttcacctagc actgaacagt ctgctaaaaa agtacgcaaa 1440
aaggcagaaa acgctcataa tacgccgctg cttgtgctat acggttcaaa tatgggaaca 1500
gctgaaggaa cggcgcgtga tttagcagat attgcaatga gcaaaggatt tgcaccgcag 1560
gtcgcaacgc ttgattcaca cgccggaaat cttccgcgcg aaggagctgt attaattgta 1620
acggcgtctt ataacggtca tccgcctgat aacgcaaagc aatttgtcga ctggttagac 1680
caagcgtctg ctgatgaagt aaaaggcgtt cgctactccg tatttggatg cggcgataaa 1740
aactgggcta ctacgtatca aaaagtgcct gcttttatcg atgaaacgct tgccgctaaa 1800
ggggcagaaa acatcgctga ccgcggtgaa gcagatgcaa gcgacgactt tgaaggcaca 1860
tatgaagaat ggcgtgaaca tatgtggagt gacgtagcag cctactttaa cctcgacatt 1920
gaaaacagtg aagataataa atctactctt tcacttcaat ttgtcgacag cgccgcggat 1980
atgccgcttg cgaaaatgca cggtgcgttt tcaacgaacg tcgtagcaag caaagaactt 2040
caacagccag gcagtgcacg aagcacgcga catcttgaaa ttgaacttcc aaaagaagct 2100
tcttatcaag aaggagatca tttaggtgtt attcctcgca actatgaagg aatagtaaac 2160
cgtgtaacag caaggttcgg cctagatgca tcacagcaaa tccgtctgga agcagaagaa 2220
gaaaaattag ctcatttgcc actcgctaaa acagtatccg tagaagagct tctgcaatac 2280
gtggagcttc aagatcctgt tacgcgcacg cagcttcgcg caatggctgc taaaacggtc 2340
tgcccgccgc ataaagtaga gcttgaagcc ttgcttgaaa agcaagccta caaagaacaa 2400
gtgctggcaa aacgtttaac aatgcttgaa ctgcttgaaa aatacccggc gtgtgaaatg 2460
aaattcagcg aatttatcgc ccttctgcca agcatacgcc cgcgctatta ctcgatttct 2520
tcatcacctc gtgtcgatga aaaacaagca agcatcacgg tcagcgttgt ctcaggagaa 2580
gcgtggagcg gatatggaga atataaagga attgcgtcga actatcttgc cgagctgcaa 2640
gaaggagata cgattacgtg ctttatttcc acaccgcagt cagaatttac gctgccaaaa 2700
gaccctgaaa cgccgcttat catggtcgga ccgggaacag gcgtcgcgcc gtttagaggc 2760
tttgtgcagg cgcgcaaaca gctaaaagaa caaggacagt cacttggaga agcacattta 2820
tacttcggct gccgttcacc tcatgaagac tatctgtatc aagaagagct tgaaaacgcc 2880
caaagcgaag gcatcattac gcttcatacc gctttttctc gcatgccaaa tcagccgaaa 2940
acatacgttc agcacgtaat ggaacaagac ggcaagaaat tgattgaact tcttgatcaa 3000
ggagcgcact tctatatttg cggagacgga agccaaatgg cacctgccgt tgaagcaacg 3060
cttatgaaaa gctatgctga cgttcaccaa gtgagtgaag cagacgctcg cttatggctg 3120
cagcagctag aagaaaaagg ccgatacgca aaagacgtgt gggctgggta a 3171
<210> 9
<211> 1266
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atgcaccacc accaccacca caccaccgag accatccaga gcaacgcgaa cctggcgccg 60
ttaccgccgc acgtcccgga acacctggtc ttcgacttcg acatgtacaa cccgagcaac 120
ctgagcgcgg gcgttcaaga agcctgggca gtcctgcagg agagcaacgt cccggacctg 180
gtttggaccc gctgcaacgg cggtcactgg attgcgaccc gcggtcagct gatccgcgag 240
gcgtacgagg actaccgcca cttctccagc gagtgcccgt tcattccgcg cgaagcaggc 300
gaggcgtacg acttcatccc gaccagcatg gacccgccgg aacagcgtca gtttcgcgca 360
ctggccaacc aagtcgtcgg catgccggtc gtcgacaaac tggagaaccg catccaggag 420
ctggcctgca gcctgatcga gagcctgcgt ccgcagggcc agtgcaactt caccgaggac 480
tacgcggagc cgttcccgat ccgcatcttc atgctgctgg cgggtctgcc ggaagaggac 540
atcccgcacc tgaaatacct gaccgaccag atgacccgcc cggacggcag catgaccttc 600
gcggaggcga aagaggcgct gtacgactac ctgatcccga tcatcgagca gcgccgccag 660
aaaccgggca ccgacgcgat cagcatcgtc gcgaacggcc aggtcaacgg tcgtccgatc 720
accagcgacg aggccaaacg catgtgcggc ctgctgctgg taggcggcct ggataccgtc 780
gtcaacttcc tgtccttcag catggagttc ctggcgaaaa gcccggagca ccgccaggaa 840
ctgatcgaac gcccggaacg catcccggca gcctgcgaag aactgctgcg ccgcttcagc 900
ctggttgcgg acggtcgcat cctgaccagc gactacgagt tccacggcgt ccagctgaaa 960
aaaggcgacc agatcctgct gccgcagatg ctgagcggtc tggacgaacg cgagaacgcc 1020
tgcccgatgc acgtcgactt cagccgccag aaagtcagcc acaccacctt cggccacggc 1080
tcccatctgt gcctgggcca gcatctggcg cgccgcgaga tcatcgtcac cctgaaagag 1140
tggctgaccc gcatcccgga cttcagcatc gcaccgggcg cgcagatcca gcacaaaagc 1200
ggcatcgtta gcggcgttca ggcactgccg ctggtctggg atccggcgac caccaaagcc 1260
gtctaa 1266
<210> 10
<211> 1278
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atgcaccatc atcatcatca tatggcaaca cttaagaggg ataagggctt agataatact 60
ttgaaagtat taaagcaagg ttatctttac acaacaaatc agagaaatcg tctaaacaca 120
tcagttttcc aaactaaagc actcggtggt aaaccattcg tagttgtgac tggtaaggaa 180
ggcgctgaaa tgttctacaa caatgatgtt gttcaacgtg aaggcatgtt accaaaacgt 240
atcgttaata cgctttttgg taaaggtgca atccatacgg tagatggtaa aaaacacgta 300
gacagaaaag cattgttcat gagcttgatg actgaaggta acttgaatta tgtacgagaa 360
ttaacgcgta cattatggca tgcgaacaca caacgtatgg aaagtatgga tgaggtaaat 420
atttaccgtg aatctatcgt actacttaca aaagtaggaa cacgttgggc aggcgttcaa 480
gcaccacctg aagatatcga aagaatcgca acagacatgg acatcatgat cgattcattt 540
agagcacttg gtggtgcctt taaaggttac aaggcatcaa aagaagcacg tcgtcgtgtt 600
gaagattggt tagaagaaca aattattgag actcgtaaag ggaatattca tccaccagaa 660
ggtacagcac tttacgaatt tgcacattgg gaagactact taggtaaccc aatggactca 720
agaacttgtg cgattgactt aatgaacaca ttccgcccat taatcgcaat caacagattc 780
gtttcattcg gtttacacgc gatgaacgaa aacccaatca cacgtgaaaa aattaaatca 840
gaacctgact atgcatataa attcgctcaa gaagttcgtc gttactatcc attcgttcca 900
ttccttccag gtaaagcgaa agtagacatc gacttccaag gcgttacaat tcctgcaggt 960
gtaggtcttg cattagatgt ttatggtaca acgcatgatg aatcactttg ggacgatcca 1020
aatgaattcc gcccagaaag attcgaaact tgggacggat caccatttga ccttattcca 1080
caaggtggtg gagattactg gacaaatcac cgttgtgcag gtgaatggat cacagtaatc 1140
atcatggaag aaacaatgaa atactttgca gaaaaaataa cttatgatgt tccagaacaa 1200
gatttagaag tggacttaaa cagtatccca ggatacgtta agagtggctt tgtaatcaaa 1260
aatgttcgcg aagtttaa 1278
<210> 11
<211> 1422
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atgcaccacc accaccacca ctctccgttc ccggcggctt ggaccaacct gtggctgctg 60
taccagtgcc gtcgcggtcg tcgtttcctg gcagtccacg aggcgcacca gaaactgggc 120
aaactggtcc gcatccagcc gaaccacgtg agcatcgcgg acgcggacgc gattacccag 180
gtctacggcc acggcaacgg cttcctgaaa agcgagtact acgacgcgtt cgtcagcatc 240
cgccgcggtc tgttcaacac ccgcgatcgc gcggagcaca cccgcaaacg caaaaccgtc 300
gcgcacacct tcagcgcgaa aagcgtcctg cagttcgagc agtacatcca ccacaacctg 360
caggagctgc agaaccagtg ggaccgtcgc gcagaaagcg tcaaaggcgg ctggtacgag 420
atggacgcgc tgaactggtt caactacctg gcgttcgacg tcatcggcga cctggcattc 480
ggcgagccgt tcggcatgct gaaaaaaggc cgcgacgaag cggaagtcgc acgcggcggc 540
aaaatcacct acgcgccggc gatcgaggtc ctgaaccgcc gcggcgaagt tagcggcacc 600
gtcggcatct tcccggcgat caaaccgtac gcgaaatact tcccggaccc gttcttctcc 660
cagggcatga aagcggtcga gaacctggcg ggcatcgcga ttgcgcgcgt taacgcccgc 720
ctggagaaac cgagcgatcg cgttgacctg ctggcccgtc tgatggaagg ccgcgacgag 780
aacggcaaca aactgggccg cgaagaactg accgcggaag cactgaccca gctgatcgcg 840
ggcagcgaca ccaccagcaa caccagctgc gcgctgctgt accactgcct gcagcacccg 900
gaggtcgtcc agaaactgca gaacgaactg gacgcggcac tgccgaatcc ggacgcggtc 960
ccgagctacg cgcaggtcaa agacctgccg tacgtcgacg cggtcatcaa agagaccatg 1020
cgcatccaca gcaccagcag cctgggtctg ccgcgcgtta ttccgccggg tccgggcgtt 1080
accattctgg gccgccactt cccgcagggt accgttctga gcgtcccggc gtacaccatc 1140
caccacagca ccgagatctg gggcccggac gcagatacct ttcgcccgga acgctgggag 1200
aaagtcaccg agcagcagaa agcggcgttc atcccgttca gctacggtcc gcgcgcctgc 1260
gttggccgca acgtcgcgga aatggagctg gcgctgatcg tcgcgaccgt cttccgccgc 1320
tacgagttcg aactgcgtca gggcgagatg gagacccgcg aaggcttcct gcgcaaaccg 1380
ctggcgctgc aggtcggtat gcgcaaacgc agcttcgcgt ga 1422
<210> 12
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu Gln Ser
1 5 10 15
Ala Lys Lys Val
20
<210> 13
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Gly Gly Gly Gly Ser
1 5
<210> 14
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 15
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 16
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20

Claims (14)

1. A P450 reductase, wherein the amino acid sequence of the P450 reductase is as set forth in SEQ ID NO:2 or SEQ ID NO: 3.
2. A P450 reductase as claimed in claim 1 as K 3 [Fe(CN) 6 ]The application of any one of reductase, cytochrome c reductase and thiazole blue reductase.
3. Use of the P450 reductase of claim 1 for the catalytic conversion of a substrate by a P450 enzyme.
4. A fusion enzyme, wherein the amino acid sequence of the fusion enzyme comprises the amino acid sequence of the P450 reductase of claim 1 and the amino acid sequence of the P450 enzyme or P450 enzyme domain;
the C-terminal of the amino acid sequence of the P450 enzyme or the P450 enzyme domain is connected with the N-terminal of the amino acid sequence of the P450 reductase through a connecting peptide;
the P450 enzyme is CYP101A1, CYP152L1 or CYP53A15;
the P450 enzyme domain is the P450 domain of CYP102 A1;
the connecting peptide is Linker1, linker3 or Linker5;
the fusion enzyme is selected from any one of the following:
CYP101A1 Linker3 BMR R967D/Q977E/W1047S；
CYP101A1 Linker1 BMR R967D/Q977E/Q1005E/W1047S；
CYP152L1 Linker1 BMR R967D/Q977E/W1047S；
CYP152L1 Linker1 BMR R967D/Q977E/Q1005E/W1047S；
CYP53A15 Linker5 BMR R967D/Q977E/W1047S；
CYP53A15 Linker1 BMR R967D/Q977E/Q1005E/W1047S；
CYP102A1 R967D/Q977E/W1047S；
CYP102A1 R967D/Q977E/Q1005E/W1047S；
the nucleic acid sequence of CYP101A1 is shown in SEQ ID NO: shown as 9;
the nucleic acid sequence of CYP152L1 is shown in SEQ ID NO:10 is shown in the figure;
the nucleic acid sequence of CYP53A15 is shown in SEQ ID NO: 11;
the amino acid sequence of CYP102A1 is shown in SEQ ID NO:4 is shown in the figure;
the amino acid sequence of the Linker1 is shown as SEQ ID NO: shown at 12;
the amino acid sequence of Linker3 is shown as SEQ ID NO: 14;
the amino acid sequence of Linker5 is shown as SEQ ID NO: shown at 16;
the amino acid sequence of BMR 967D/Q977E/W1047S is shown in SEQ ID NO:2 is shown in the figure;
the amino acid sequence of BMR 967D/Q977E/Q1005E/W1047S is shown in SEQ ID NO: 3.
5. An enzyme catalysis system comprising the P450 reductase, P450 enzyme or P450 enzyme domain, non-native coenzyme NCDH enzyme of claim 1; or (b)
Comprising the fusion enzyme according to claim 4, and an unnatural coenzyme NCDH regenerating enzyme.
6. A nucleic acid encoding any one of the P450 reductase of claim 1 and the fusion enzyme of claim 4.
7. A vector comprising an expression cassette comprising the nucleic acid of claim 6.
8. The vector of claim 7, further comprising an expression cassette comprising a nucleic acid encoding a non-native coenzyme NCDH regenerant enzyme.
9. The vector of claim 7, further comprising an expression cassette comprising a nucleic acid encoding a nucleotide transporter.
10. The vector of claim 9, wherein the nucleotide transporter in the expression cassette comprising a nucleic acid encoding a nucleotide transporter comprises an NTT4 nucleotide transporter derived from chlamydia and/or an AtNDT2 nucleotide transporter derived from arabidopsis thaliana.
11. A host cell comprising the vector of any one of claims 7 to 10.
12. The host cell of claim 11, wherein the host cell is selected from the group consisting of prokaryotes and eukaryotes.
13. The host cell of claim 12, wherein the prokaryote is escherichia coli; the eukaryote is Saccharomyces cerevisiae.
14. Use of a P450 reductase according to claim 1, a fusion enzyme according to claim 4, an enzyme catalytic system according to claim 5, a nucleic acid according to claim 6, a vector according to any one of claims 7 to 10, a host cell according to any one of claims 11 to 13 in a biocatalytic reaction mediated by non-native coenzyme NCD.
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