CN114621934A - P450 reductase and application thereof - Google Patents

P450 reductase and application thereof Download PDF

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CN114621934A
CN114621934A CN202011455140.XA CN202011455140A CN114621934A CN 114621934 A CN114621934 A CN 114621934A CN 202011455140 A CN202011455140 A CN 202011455140A CN 114621934 A CN114621934 A CN 114621934A
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赵宗保
李青
王雪颖
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Abstract

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

Description

P450 reductase and application thereof
Technical Field
The application relates to a P450 reductase and application thereof, belonging to the technical field of biology.
Background
Nicotinamide cofactor (NAD (P)) and its reduced state NAD (P) H are important coenzymes in the life process, participate in the redox metabolism and other important biochemical processes in the life body, and any operation of changing the NAD concentration and its redox state can generate global influence on cells. And 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 have important significance on the research of biological catalysis and synthetic biology (Ji DB, et al. J Am Chem Soc,2011,133, 20857-. Several NAD analogues with good biocompatibility were reported by the present 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-. Meanwhile, some enzymes that recognize NAD analogs have been reported, such as NADH oxidase from Enterococcus faecalis (NOX, Genbank S45681), D-lactate dehydrogenase (DLDH, Gnebank CAA47255) V152R mutant, malic enzyme (ME, Genbank P26616) L310R/Q401C mutant, and malate dehydrogenase (MDH, Genbank CAA68326) L6R mutant. Regulation of intracellular metabolic reactions using Nicotinamide Cytosine Dinucleotide (NCD) has been achieved by using NTT4 derived from chlamydia or AtNDT2 protein derived from arabidopsis thaliana to transport NCD intracellularly, and specific biocatalytic regulation has been achieved by DLDH-V152R using NCD to reduce pyruvate to lactate (Wang L, et al. acs Catal,2017,7, 1977-.
The P450 enzyme is a mercaptide-heme enzyme protein with various functions, can selectively activate C-H bond, N-H bond, S-H bond and the like, thereby catalyzing more than 20 different types of reactions, realizing modification reactions such as selective hydroxylation, epoxidation, dealkylation and the like on a large number of substrates with different structures, and being known as a universal catalyst in the nature. The activity of P450 enzymes depends on the redox partners to transfer two electrons of NAD (P) H to the heme prosthetic group of the enzyme so as to complete the catalytic cycle of the enzyme and realize substrate conversion. P450 enzymes and redox chaperones can be classified into five categories according to 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 enzyme, three classes of Class I P450 proteins are soluble in bacteria, Class I P450 is only Fdx soluble in eukaryotic mitochondria, and FdR and P450 are anchored to the membrane. During the reaction, FdR transferred electrons from NAD (P) H to Fdx, Fdx in turn transferred electrons to the P450 enzyme. (2) Class II is a two-component system, present on the endoplasmic reticulum membrane of most eukaryotic microorganisms, and includes nad (P) HP450 reductase (nad (P) H cytochrome P450 reductase, CPR) and P450 enzymes, both anchored to the membrane, containing cofactors FAD and FMN. During the reaction, CPR transfers electrons from nad (P) H to the P450 enzyme. (3) The Class III is a monocomponent system, exists in bacteria, is a bifunctional peptide chain formed by fusing a P450 enzyme structural domain containing a heme prosthetic group and a P450 reductase structural domain containing cofactors FAD and FMN, does not need additional auxiliary electron transfer protein in the reaction process, and electrons are transferred in molecules, so that the Class III is an electron self-sufficient catalytic system. (4) Class IV is also a single component system with three components identical to Class I on a single fused polypeptide chain, with the N-terminal P450 domain linked to the FdR domain containing cofactor FMN, and further linked to the C-terminal Fdx domain containing the Fe-S cluster, and is also an electronic autarkic catalytic system. (5) Class V is an unusual nad (P) H independent P450 enzyme. (Bernhardt R.et al.J.Biotechnol,2006,124, 128-charge 145)
P450 BM3 is a fatty acid hydroxylase of Bacillus megaterium (Bacillus megaterium) belonging to Class III self-sufficient type P450, and comprises heme-containing P450 domain (BMP) and P450 reductase domain (BMR) utilizing NADPH and O2Catalyzing the hydroxylation of the inferior terminal methylene of the fatty acid to generate the hydroxy fatty acid. Is the P450 enzyme with the highest catalytic efficiency reported in the literature at present, and the catalytic efficiency is as high as 17000min-1Coupling efficiencies of up to 100% (Whitehouse, C.J.C.et al. chem Soc Rev,2012,41, 1218-. And the biotransformation systems of other free P450 enzymes have the problems of low overall efficiency, weak host substrate supply capacity, dependence on reduced coenzyme and the like, and seriously restrict the industrial application of the biotransformation systems. The construction of a self-sufficient bifunctional P450 enzyme by fusing the P450 BM3 reductase domain with different types of free P450 enzymes can improve the catalytic efficiency and coupling efficiency of the P450 enzyme by using a molecular engineering approach (Dodhia, V.R.et al.J Biol Inorg Chem 2006,11, 903-916).
The construction of the non-natural coenzyme-dependent P450 reductase is expected to realize the uncoupling of the non-natural coenzyme-mediated P450 reaction system and the supply of endogenous energy. The cofactor preference of the non-natural coenzyme-dependent P450 reductase constructed in the early stage is not strict, and the improvement of the cofactor preference of the P450 reductase to dihydronicotinamide cytosine dinucleotide (NCDH) is still a problem to be solved urgently.
Disclosure of Invention
According to one aspect of the application, a P450 reductase is provided, wherein the P450 reductase is a BMR subjected to protein directed evolution, takes a non-natural coenzyme NCDH as a cofactor and has reducing power, and transfers electrons to a P450 enzyme catalytic center to complete substrate catalytic conversion.
A P450 reductase having the 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 shown in 1: R967D, Q977E, Q1005E and W1047S.
The nucleotide sequence of SEQ ID NO: 1 is the amino acid sequence of BMR which is P450 reductase domain BMR containing flavin mononucleotide and flavin adenine dinucleotide of P450 BM3 derived from Bacillus megaterium, namely the amino acid sequence of P450 BM3 from 472 th to 1049 th.
The P450 reductase provided by the application is a multi-site mutant obtained by genetic engineering modification of BMR; the mutation sites of the multi-site mutant comprise at least one of R967D, Q977E, Q1005E and W1047S. The mutant recognizes NCDH and relies on NCDH for reduction.
Optionally, the mutations include R967D, Q977E, and W1047S; or
The mutations include R967D, Q977E, Q1005E, and W1047S.
Alternatively, the P450 reductase has the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
Optionally, the P450 reductase is a non-native coenzyme NCDH-dependent P450 reductase having a structure according to formula I:
Figure BDA0002827893740000021
the non-natural coenzyme NCDH is obtained by reducing non-natural coenzyme NCD by using a regeneration substrate through a non-natural coenzyme NCDH regeneration enzyme;
the non-natural coenzyme NCD has a structure represented by formula II:
Figure BDA0002827893740000022
optionally, the non-native coenzyme NCDH regeneration enzyme comprises at least one of malic 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 compounds, phosphorous acid compounds, D-lactic acid compounds, formic acid compounds 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 a D-lactate;
the phosphorous acid compound comprises phosphorous acid and/or a phosphite salt;
the formic acid compound includes formic acid and/or a formate salt.
According to another aspect of the present application, there is provided the P450 reductase as K3[Fe(CN)6]Reductase, cytochrome c reductase, and thiazole blue reductase.
Alternatively, P450 reductase as K3[Fe(CN)6]The reaction system of the application of the reductase is as follows: in pH 5-9 buffer system, non-natural coenzyme NCDH 0.01mM-10mM, K3[Fe(CN)6]0.2mM-3mM, non-native coenzyme NCDH-dependent P450 reductase 2 nM-100. mu.M.
Alternatively, the reaction system for the application of P450 reductase as cytochrome c reductase is: in a pH 5-9 buffer system, the non-native coenzyme NCDH is 0.01mM-10mM, the cytochrome c is 0.005mM-0.5mM, and the non-native coenzyme NCDH-dependent P450 reductase is 2 nM-100. mu.M.
Alternatively, the reaction system of the application of the P450 reductase as the thiazole blue reductase is as follows: in a pH 5-9 buffer system, 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 2 nM-100. mu.M.
According to another aspect of the application, there is provided a use of the P450 reductase enzyme in the catalysis of a substrate conversion by a P450 enzyme.
Optionally, in the application of the P450 reductase in the conversion of a P450 enzyme-catalyzed substrate, the reaction system is: in a pH 5-9 buffer system, non-native coenzyme NCDH 0.05mM-50mM, P450 enzyme 2 nM-100. mu.M, P450 enzyme substrate 0.05mM-50mM, non-native coenzyme NCDH-dependent P450 reductase 2 nM-100. mu.M.
According to another aspect of the present application, there is provided a fusion enzyme having an amino acid sequence comprising the amino acid sequence of the P450 reductase enzyme of any one of the above and the amino acid sequence of the P450 enzyme or P450 enzyme domain.
Optionally, 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 Class I Class P450 enzymes and Class II Class P450 enzymes;
the P450 enzyme domain is selected from any one of the Class III Class P450 enzyme domains.
Optionally, the Class I P450 enzyme is CYP101a1 or CYP152L 1.
Optionally, the Class II P450 enzyme is CYP53a 15.
Alternatively, the CYP53a15 does not include the N-terminal 35 amino acid transmembrane sequence.
Optionally, the Class III P450 enzyme domain is the P450 domain of CYP102a 1.
Optionally, the substrate of the P450 enzyme or P450 enzyme domain is selected from any one of D-camphor, C12-C18 saturated fatty acids, benzoic acid.
According to another aspect of the present application there is provided an enzymatic catalysis system comprising a P450 reductase, a P450 enzyme or a P450 enzyme domain of any of the above, a non-native coenzyme NCDH regenerating enzyme; or
Comprising the fusion enzyme, the non-natural coenzyme NCDH regeneration enzyme of any one of the above.
According to another aspect of the present application, there is provided a nucleic acid encoding any one of the P450 reductase of any one of the above, 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 according to any one of the above.
Optionally, the vector further comprises an expression cassette comprising a nucleic acid encoding a non-native coenzyme NCDH regenerating enzyme.
Optionally, the vector further comprises an expression cassette comprising a nucleic acid encoding a nucleotide transporter.
Optionally, 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 from chlamydia and/or an AtNDT2 nucleotide transporter from arabidopsis thaliana.
According to another aspect of the present application, there is provided a host cell comprising the vector of any one of the above.
Optionally, 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 present application there is provided a P450 reductase enzyme according to any of the above, a fusion enzyme according to any of the above, an enzymatic catalytic system according to any of the above, a nucleic acid according to any of the above, a vector according to any of the above, or a host cell according to any of the above, for use in a biocatalytic reaction mediated by a non-native coenzyme NCD.
The P450 reductase, the fusion enzyme formed by fusing the P450 reductase and the P450 enzyme, which is provided by the application, is further co-expressed with nucleotide transport protein, and has important significance for realizing energy consumption of the P450 enzyme and decoupling of endogenous NADPH supply.
The beneficial effects that this application can produce include:
(1) the P450 reductase provided by the application is a multi-site mutant obtained by genetic engineering transformation of BMR, and has higher K3[ Fe (CN)6], cytochrome c and thiazole blue reductase activities.
(2) The P450 reductase provided by the application takes the non-natural coenzyme NCDH as a cofactor and a reducing power, transfers electrons to a P450 enzyme catalytic center, and can improve the catalytic efficiency and the electron transfer efficiency of the P450 enzyme.
(3) The enzyme catalysis system provided by the application has the advantages of 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 bifunctional P450 enzyme, the reducing power of the chemical energy of the micromolecule is selectively transmitted to the substrate molecule through the mediation of the non-natural coenzyme NCD, the problem of the dependence on the endogenous energy is overcome without depending on the intracellular natural coenzyme NAD (P) H, the constructed bio-orthogonal metabolic pathway mediated by the non-natural coenzyme NCD realizes the decoupling of the energy consumption catalyzed by the P450 enzyme and the endogenous energy metabolism.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
The NCDH described in the specific embodiments of the application is a non-natural coenzyme dihydronicotinamide cytosine dinucleotide; the NCD is non-natural coenzyme nicotinamide cytosine dinucleotide. The NCD is 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 purpose of expression and purification of proteins, in constructing an expression vector, the initial amino acid methionine M and the amino acid sequence HHHHH (his tag) are added before arginine R at the N-terminus of the amino acid sequence thereof, that is, the amino acid sequence mhhhh is added.
SEQ ID NO: 2 represents the amino acid sequence of BMR-R967D/Q977E/W1047S; for the purpose of expression and purification of proteins, in constructing an expression vector, the initial amino acid methionine M and the amino acid sequence HHHHH (his tag) are added before arginine R at the N-terminus of the amino acid sequence thereof, that is, the amino acid sequence mhhhh is added.
SEQ ID NO: 3 represents the amino acid sequence of BMR-R967D/Q977E/Q1005E/W1047S, and when constructing an expression vector, the initial amino acid methionine M and the amino acid sequence HHHHHHHHHHH (his tag) are added before the arginine R at the N-terminal of the amino acid sequence, namely the amino acid sequence MHHHHHH, for expressing and purifying the protein.
SEQ ID NO: 4 represents the amino acid sequence of P450 BM3 (i.e., CYP102A1), and in order to express and purify the protein, when constructing an expression vector, the initial amino acid methionine M and the amino acid sequence HHHHHHH (his tag) are added before the initial amino acid methionine M at the N-terminal of the amino acid sequence, i.e., the amino acid sequence MHHHH is added.
SEQ ID NO: 5 represents the nucleic acid sequence of BMR (with amino acid sequence MHHHHHHH).
SEQ ID NO: 6 shows the nucleic acid sequence of BMR-R967D/Q977E/W1047S (with amino acid sequence MHHHHHHHHH).
SEQ ID NO: 7 denotes the nucleic acid sequence BMR-R967D/Q977E/Q1005E/W1047S (with the amino acid sequence MHHHHHHHHH).
SEQ ID NO: 8 denotes the nucleic acid sequence of P450 BM3, i.e.CYP 102A1 (with amino acid sequence MHHHHHHHH).
SEQ ID NO: 9 shows the nucleic acid sequence of CYP101A1 (his tag sequence HHHHH was added 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 HHHHHHH 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 HHHHHHH was 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 an amino acid sequence HHHHH consisting of 6 histidine residues.
As a specific embodiment, the application provides a non-natural coenzyme dihydronicotinamide-cytosine dinucleotide (NCDH) -dependent P450 reductase and application thereof, and particularly relates to a method for catalyzing and reducing P450 enzyme to catalyze and convert P450 enzyme to substrate by using NCDH as a cofactor and reducing power of BMR subjected to protein directed evolution. And providing a fusion enzyme, namely: disclosed is a method for constructing an NCDH-dependent self-sufficient bifunctional P450 enzyme by fusion expression of an NCDH-dependent P450 reductase and a P450 enzyme. The two-element system comprises NCDH-dependent P450 reductase, different P450 enzymes and NCDH regenerative enzyme, or comprises NCDH-dependent self-sufficient bifunctional P450 enzyme and NCDH regenerative enzyme, and can be co-expressed with nucleotide transport protein in microbial cells, used for construction of bioorthogonal metabolic pathways, and realizing decoupling of P450 enzyme-catalyzed energy consumption and endogenous energy metabolism. Therefore, the method can be applied to the fields of biological catalysis and biological conversion and has important value.
Specifically, an NCDH-dependent P450 reductase which is characterized in that:
1) the NCDH-dependent P450 reductase is a P450 reductase with NCDH as a cofactor, and K can be catalytically reduced by using NCDH3[Fe(CN)6]Cytochrome c and thiazole blue are K4[Fe(CN)6]Reduced cytochrome c and blue formazan; the NCDH is obtained by reducing NCD by using a regeneration substrate through NCDH regeneration enzyme;
2) the chemical structures of the NCDH and the NCD are respectively shown as a formula I and a formula II:
Figure BDA0002827893740000051
the NCDH-dependent P450 reductase, 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 shown in SEQ ID NO: 2, and the BMR-R967D/Q977E/Q1005E/W1047S has an amino acid sequence shown as SEQ ID NO: 3.
The NCDH-dependent P450 reductase can catalyze the reduction of K using NCDH3[Fe(CN)6]And cytochrome c thiazole blue is K4[Fe(CN)6]Reduced cytochrome c and blue formazan, and the reaction system is as follows: NCDH 0.01mM-10mM, K in pH 5-9 buffer system3[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 transmit two electrons of NCDH to different types of P450 enzyme catalytic centers to complete catalytic conversion of substrates, and the reaction system is as follows: NCDH 0.05mM-50mM, P450 enzyme 2 nM-100. mu.M, P450 enzyme substrate 0.05mM-50mM, NCDH-dependent P450 reductase 2 nM-100. mu.M in pH 5-9 buffer.
The NCDH is obtained by reducing NCD by using a regeneration substrate through NCDH regeneration enzyme; wherein NCDH regeneration enzyme includes but is not limited to malic 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 124, the corresponding regeneration substrate of NCDH regeneration enzyme includes but is not limited to malic compound, phosphorous acid compound, D-lactic acid compound, formic acid compound, methanol compound or two or more; wherein the malic acid compound is one or more of malic acid and malate; the D-lactic acid compound is one or more than two of D-lactic acid and D-lactate; the phosphorous acid 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 expressed with different types of P450 enzymes through fusion of connecting peptides to construct NCDH-dependent self-sufficient bifunctional P450 enzymes, wherein the connecting peptides include but are not limited to Linker 1: KKIPLGGIPSPSTEQSAKKV or Linker 2-5 (G)4S)nWherein n is an integer of 1-4 respectively, namely, the amino acid sequence of Linker 2 is as follows: GGGGS; the amino acid sequence of Linker3 is: GGGGSGGGGS; the amino acid sequence of Linker 4 is: GGGGSGGGGSGGS; the amino acid sequence of Linker5 is: GGGGSGGGGSGGGGSGGGGS. The self-sufficient dual-function means that electrons are transmitted in the molecule without adding auxiliary electron transfer protein in the reaction process.
The different types of P450 enzymes include, but are not limited to, Class I P450 enzymes, Class II P450 enzymes, and Class III P450 enzyme domains. Wherein the Class I P450 enzymes include but are not limited to CYP101A1 or CYP152L 1; class II P450 enzymes include, but are not limited to, CYP53A 15; class III Class P450 enzyme domains include, but are not limited to, the P450 domain of CYP102A 1; the P450 enzyme corresponding substrate includes but is not limited to D-camphor, C12-C18 saturated fatty acid and benzoic acid.
The NCDH dependent P450 reductase, different types of P450 enzymes, NCDH regenerative enzyme and nucleotide transport protein are co-expressed in microbial cells, and P450 substrates are catalyzed by the microbial cells to be converted into products; wherein NCDH is transported intracellularly via NTT4 derived from chlamydia or AtNDT2 nucleotide transporter derived from arabidopsis; microbial cells include, but are not limited to, prokaryotic E.coli and eukaryotic Saccharomyces cerevisiae.
The NCDH-dependent self-sufficient bifunctional P450 enzyme and NCDH regenerative enzyme can construct a double-enzyme coupled body catalytic system, and the reaction system is as follows: in a pH 5-9 buffer system, NCD 0.01mM-1mM, NCDH regeneration enzyme 2U/mL-100U/mL, regeneration substrate 0.1mM-100mM, NCDH dependent self-sufficient bifunctional P450 enzyme 1. mu.M-100. mu.M, P450 enzyme substrate 0.05mM-50 mM.
The NCDH dependent self-sufficient bifunctional P450 enzyme, the NCDH regeneration enzyme and the nucleotide transport protein are co-expressed in microbial cells, and the P450 substrate is catalyzed by the microbial cells to be converted into a product; wherein NCDH is transported into cells through NTT4 derived from chlamydia or 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 bifunctional P450 enzyme, which has mild catalysis conditions and high reaction efficiency; the product selectivity is high; the NCDH-dependent self-sufficient bifunctional P450 enzyme improves the catalytic efficiency and the electron transfer efficiency of the P450 enzyme; based on a whole-cell catalytic conversion system of NCDH-dependent P450 reductase and NCDH-dependent self-sufficient bifunctional P450 enzyme, the reducing power of the chemical energy of the small molecule is selectively transmitted to a substrate molecule through the mediation of non-natural coenzyme NCD, the problem of endogenous energy dependence is overcome without depending on intracellular natural coenzyme NAD (P) H, the constructed non-natural coenzyme NCD-mediated bioorthogonal metabolic pathway realizes the decoupling of the energy consumption catalyzed by the P450 enzyme and the endogenous energy metabolism.
The NCD reference method (Ji DB, et al. Sci China Chem,2013,56,296-300) in the specific examples of the present application was prepared and prepared as an aqueous solution with a concentration of 20mM for use.
The electrotransformation method for transformation of prokaryotic organisms such as Escherichia coli refers to molecular cloning guide, third edition, and the transformation method for eukaryotic organisms such as Saccharomyces cerevisiae refers to the lithium acetate transformation method in Gietz, R.D., et al, Nature Protocols 2007,2, 31.
The BMR described in the specific examples of the present application is the P450 reductase domain BMR containing flavin mononucleotide and flavin adenine dinucleotide derived from P450 BM3 of Bacillus megaterium, i.e., the amino acid sequence from position 472 to position 1049 of P450 BM 3.
In a specific example of the present application, NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S and BMR-R967D/Q977E/Q1005E/W1047S were mutants obtained by genetic engineering BMR, wherein BMR-R967D/Q977E/W1047S has the amino acid sequence as shown in SEQ ID NO: 2, BMR-R967D/Q977E/Q1005E/W1047S has the amino acid sequence shown in SEQ ID NO: 3.
In the present application, the mutation sites of the polypeptide mutant are represented by the following methods: amino acids before mutation, mutated amino acid numbers, and amino acids after mutation. For example, R967D indicates that the amino acid at position 967 in the polypeptide has been changed from the original arginine R to the subsequent aspartic acid D.
The polypeptide mutant is expressed by the following method: the original polypeptide-amino acid before mutation, mutated amino acid number, amino acid after mutation/… …, "/" is used to separate each mutated amino acid. For example, BMR-R967D/Q977E/W1047S represents a mutant in which BMR represents a pro-polypeptide by 3 amino acid mutations, and R967D represents a change from the original arginine R to the subsequent aspartic acid D at amino acid position 967; Q977E indicates that amino acid 977 has been changed from glutamine Q to glutamic acid E; W1047S, which shows the change of the original tryptophan W to serine S at amino acid position 1047. With respect to the numbering of the mutated amino acids, the numbering of the amino acids in BMR-R967D/Q977E/W1047S and BMR-R967D/Q977E/Q1005E/W1047S corresponds to that of P450 BM3, i.e. the numbering is carried out sequentially backwards with the initial amino acid methionine of P450 BM3 numbered 1. The remaining mutants were numbered the same as the polypeptide before mutation, i.e., the initial amino acid methionine of the polypeptide before mutation was numbered 1, and they were numbered sequentially in the order named after. In the artificial construction of expression vectors, neither the initial amino acid methionine M or histidine H in his tag, which is added for the purpose of protein expression or purification, is counted.
The NCDH-dependent self-sufficient bifunctional P450 enzyme used in the embodiment of the application is constructed by fusing C-terminal of amino acid sequence of P450 enzyme from different sources with N-terminal of amino acid sequence of NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S), constructing P450 gene with connecting peptide sequence between N-terminal his-tag of NCDH-dependent P450 reductase expression vector and NCDH-dependent P450 reductase gene sequence by RF cloning method (unlimited cloning method), and obtaining the NCDH-dependent self-sufficient bifunctional P450 enzyme expression vector. Wherein the N-terminal 35 amino acid transmembrane sequence of P450 was deleted upon fusion to Class II CYP53A 15.
The sequence of the linker peptide used in the specific examples of this application is:
Linker 1：KKIPLGGIPSPSTEQSAKKV；
Linker 3：GGGGSGGGGS；
Linker 5：GGGGSGGGGSGGGGSGGGGS
in the specific examples of the present application, CYP101A1 used is derived from Pseudomonas putida (UniProt code P00183), CYP152L1 used is derived from Jeotgaliccus sp.ATCC 8456(UniProt code E9NSU2), CYP53A15 used is derived from Curvularia lunata (UniProt code B8QM33), CYP102A1 (i.e., P450 BM3) used is derived from Bacillus megaterium (UniProt code P14779), and NCDH-dependent P450 BM3 enzyme (CYP102A 1-R967/Q977E/W7 1048 or CYP102A 1-R637D/Q977/Q1005 7378/Q1005E/W104S) is obtained by mutating CYP102A1(UniProt code P14779).
Malic Enzyme (ME) used in the present application is derived from Escherichia coli K12(Uniprot code P26616), D-lactate dehydrogenase (DLDH) from Lactobacillus helveticus (Uniprot code P30901), Phosphite Dehydrogenase (PDH) from Ralstonia sp strain 4506(Uniprot code G4XDR8), Formate Dehydrogenase (FDH) from Pseudomonas sp.101(Uniprot code P33160), and Methanol Dehydrogenase (MDH) from Bacillus stearothermophilus DSM2334(Uniprot code P42327).
The mutant dehydrogenases used in the examples of the present application are those utilizing
Figure BDA0002827893740000071
The single site mutation kit is obtained by introducing amino acid mutation into a mutant malic enzyme (ME-L310R/Q401C) obtained by mutating Malic Enzyme (ME) (UniProt code P26616), a mutant lactate dehydrogenase (DLDH-V152R/N213E or DLDH-V152R/I177K/N213I) obtained by mutating D-lactate dehydrogenase (DLDH) (UniProt code P30901), a mutant phosphite dehydrogenase (PDH-I151R/P176R/M207A or PDH-I151R/P176E/M207A) obtained by mutating Phosphite Dehydrogenase (PDH) (Uniproct code G4XDR8), a mutant formate dehydrogenase (FDH-V198I/C256I/P260S/E261P/S381N/S383/F) obtained by mutating Formate Dehydrogenase (FDH) (Uniproct code G4XDR8), a mutant Methanol Dehydrogenase (MDH) R/I240/MDH 468/MDH R) obtained by mutating methanol dehydrogenase (MDH 26/6852/26/L26/I26/P383) obtained by mutating Formate Dehydrogenase (FDH) (Uniproct dehydrogenase (Uniproct code P240/2) and the mutant malate dehydrogenase (MDH 26/L26) obtained by mutating P42327) was mutated.
Expression and purification of enzyme: the NCDH-dependent P450 reductase and the self-sufficient bifunctional P450 enzyme were purified for protein overexpression by Ni affinity chromatography according to literature methods (Wang JX, et al. Protein content determination: bovine serum albumin ABS was used as a standard protein and the measurement was performed by the 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 like and corresponding products in the reaction solution is analyzed and determined by utilizing an ICS-2500 ion chromatography system of the company Daian in the United states under an ED50 pulse electrochemical detection mode. IonPac AS11-HC anion exchange analytical column (200 mm. times.4 mm), IonPac AG11-HC anion exchange guard column (50 mm. times.4 mm) were used. Analysis conditions were as follows: mobile phase 1mM-15mM NaOH gradient, flow rate 1mL/min, column temperature: the sample size was 10. mu.L at 30 ℃. Qualitative by comparing with standard sample, and quantitative by standard curve method.
C12-C18 fatty acids and hydroxylated products were analyzed by GC-MS using N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) silane derivatized products, D-camphor and hydroxylated products with angiont 7890-5700D equipped with capillary flow distribution plates and FID on an HP-5MS capillary column (30 m.times.0.32 mm.times.0.4 μm); the temperature of a sample injector is 250 ℃, the initial temperature of a column incubator is 100 ℃, the temperature is 2min, the temperature is increased to 300 ℃ at a speed of 10 ℃/min, and the temperature is kept for 3 min; detector (FID) temperature 280 ℃; carrier gas He 40mL/min, H230mL/min and 300mL/min of air; the sample injection amount is 1 mu L, and the split ratio is 20: 1; the solvent delay of the MS detector is 4 min; MS is used for qualitative analysis, FID is used for quantitative analysis, and relative content is determined by adopting an external standard method.
Analyzing benzoic acid, p-hydroxybenzoic acid and 3, 4-dihydroxybenzoic acid by using a high performance liquid chromatography-diode array detector, wherein an analytical column is a C18 reverse silica gel column; using a solvent A: 5% acetonitrile, 0.1% H2SO 4; solvent B: acetonitrile 90% w/v, H2SO40.1% w/v gradient elution, 0-10min 100% A, 10-12min reduction to 60% A, 12-24min 100% A; the flow rate is 1 mL/min; the sample volume is 20 mu L; signals were detected at 215nm and 250 nm; the relative content is determined by an external standard method.
Example 1: NCDH-dependent P450 reductase BMR-R967D/Q977E/1047W1047S catalytic reduction of K Using NCDH3[Fe(CN)6]Is K4[Fe(CN)6]
100 μ L of 100mM Tris-HCl buffer pH 5: 0.2mM-3mM K3[Fe(CN)6]10mM NCDH, 1. mu.M NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S, reacted at 30 ℃ and the change in absorbance at 420nm was measured to determine K3[Fe(CN)6]Is reduced to K4[Fe(CN)6]，K3[Fe(CN)6]Molar absorptivity at 420nm of 1.020mM- 1cm-1. The results are shown in Table 1, where the enzyme activity is expressed in moles per minute per mole of enzyme-catalyzed product formation or substrate consumption in mol (mol enzyme)-1min-1。
TABLE 1 reduction of K by NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S using NCDH3[Fe(CN)6]Enzyme activity of (2)
Figure BDA0002827893740000081
Example 2: NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S uses NCDH to catalyze the reduction of cytochrome c to reduced cytochrome c
100 μ L of 300mM pH 7.5 potassium phosphate buffer: 5 μ M-500 μ M cytochrome C, 200 μ M NCDH, 2nM NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S, reaction at 30 ℃ and determination of absorbance at 550nM to determine the formation of reduced cytochrome C having a molar absorptivity of 21.1mM-1cm-1. The results are shown in Table 2, where the enzyme activity is expressed in moles per minute per mole of enzyme-catalyzed product formation or substrate consumption in mol (mol enzyme)-1min-1。
TABLE 2 enzymatic activities of NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S reduction of cytochrome c by NCDH
Figure BDA0002827893740000082
Example 3: NCDH dependent P450 reductase for catalytic reduction of thiazole blue to blue formazan using NCDH
100 μ L of 50mM Tris-HCl buffer pH 9: 0.01mM-10mM thiazole blue, 10mM NCDH, 100. mu.M NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q97 977E/Q1005E/W1047S), reacted at 30 ℃, and then its absorbance at 570nm was measured to determine the formation of reduced formazan having a molar absorption coefficient of 16.2mM formazan-1cm-1. The results are shown in Table 3, where the enzyme activity is expressed in moles per minute per mole of enzyme-catalyzed product formation or substrate consumption in mol (mol enzyme)-1min-1。
TABLE 3 enzymatic Activity of NCDH-dependent P450 reductase with NCDH to reduce Thiazolyl blue
Figure BDA0002827893740000083
Example 4: NCDH dependent P450 reductase and NCDH regenerative enzyme coupled catalytic reduction K3[Fe(CN)6]
100 μ L of 100mM Tris-HCl buffer pH 6.8: 2.5mM K3[Fe(CN)6]0.5mM NCD, 50U/mL ME-L310R/Q401C, 5mM L-malic acid, 5mM MgCl2100. mu.M NCDH-dependent P450 reductase (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S) was reacted at 30 ℃ to determine the decrease in absorbance at 420nm and to determine K3[Fe(CN)6]Reduction of (a) K3[Fe(CN)6]Molar absorptivity at 420nm of 1.020mM-1cm-1. The results are shown in Table 4, where the enzyme activity is expressed in moles per minute per mole of enzyme-catalyzed product formation or substrate consumption in mol (mol enzyme)-1min-1。
TABLE 4 reduction of K by NCDH-dependent P450 reductase coupled with NCDH-regenerating enzyme3[Fe(CN)6]Enzyme activity
Enzyme Enzyme activity (U)
BMR-R967D/Q977E/W1047S 4431±101
BMR-R967D/Q977E/Q1005E/W1047S 4609±72
Example 5: NCDH dependent P450 reductase and NCDH regenerative enzyme coupling catalytic reduction cytochrome c
100 μ L of 200mM pH 7.5 potassium phosphate buffer: 40 μ M cellsPigment c, 200. mu.M NCD, 100U/mL PDH-I151R/P176R/M207A, 20mM sodium phosphite, 2nM NCDH-dependent P450 reductase (BMR-R967D/Q97 977E/W1047S or BMR-R967D/Q977E/Q1005E/W104 1047S), reaction at 30 ℃ and determination of 550nM absorbance to determine the formation of reduced cytochrome c with a molar absorption coefficient of 21.1mM reduced cytochrome c-1cm-1. The results are shown in Table 5, where the enzyme activity is expressed in moles per minute per mole of enzyme-catalyzed product formation or substrate consumption in mol (mol enzyme)-1min-1。
TABLE 5 enzymatic Activity of NCDH-dependent P450 reductase coupled with NCDH-regenerating enzyme for reduction of cytochrome c
Enzyme Enzyme activity (U)
BMR-R967D/Q977E/W1047S 2891±38
BMR-R967D/Q977E/Q1005E/W1047S 3143±127
Example 6: NCDH dependent P450 reductase and NCDH regenerative enzyme coupled catalytic reduction thiazole blue
100 μ L of 100mM Tris-HCl buffer 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), reaction at 30 ℃ and determination of 570nM absorbance to determine the formation of reduced formazan having a molar absorption coefficient of 16.2mM formazan-1cm-1. The results are shown in Table 6, where the enzyme activity is expressed in moles of enzyme-catalyzed product formation or substrate consumption per minute per mole of enzymeIn mol (mol enzyme)-1min-1。
TABLE 6 enzymatic Activity of NCDH-dependent P450 reductase coupled with NCDH-regenerating enzyme for reduction of thiazole blue
Enzyme 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 μ L of 50mM Tris-HCl buffer pH 7.0: 0.1mM NCDH, 0.05mM D-camphor, 2nM CYP101A1, 2nM NCDH dependent P450 reductase BMR-R967D/Q977E/W1047S, reacting for 24h at 30 ℃, adding 5 uL 4M HCl to stop the reaction, extracting D-camphor and hydroxylation product with equal volume of ethyl acetate, centrifuging for 5min at 10000g, detecting the organic phase by GC-MS analysis, and the conversion rate of D-camphor is 20.3%.
Example 8: catalytic system of NCDH dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S and CYP152L1
100 μ L of 50mM Tris-HCl buffer 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, reacting for 4h at 30 ℃, adding 5 mu L4M HCl to terminate the reaction, extracting the stearic acid and the decarboxylation products by using chloroform with the same volume, centrifuging for 5min at 10000g, derivatizing the stearic acid and the decarboxylation products by BSTFA, and analyzing by GC-MS, wherein the stearic acid conversion rate is 18.8%.
Example 9: NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S and CYP53A15 catalytic system
100 μ L of 50mM Tris-HCl buffer pH 8.8: 50mM NCDH, 50mM benzoic acid, 100. mu.M CYP53A15 and 100. mu.M NCDH-dependent P450 reductase BMR-R967D/Q977E/W1047S, reacting for 4h at 30 ℃, adding 5. mu.L 4M HCl to stop the reaction, adding equal volume of acetonitrile to the sample, centrifuging at 10000g for 5min, and analyzing by HPLC, the benzoic acid conversion rate is 4.6%.
Example 10: catalytic system of NCDH dependent P450 reductase and different P450 enzymes
100 μ L of 50mM Tris-HCl buffer 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 CYP53A15) and NCDH-dependent reductase equimolar to free P450 (BMR-R967D/Q977E/W1047S or BMR-R967D/Q977E/Q1005E/W1047S), reaction at 30 ℃ for 4h, termination by addition of 5. mu.L 4M HCl, extraction of D-camphor and hydroxylated products with an equal volume of ethyl acetate (stearic acid and decarboxylated products are extracted with equal volume of chloroform; benzoic acid samples are added directly to acetonitrile), 10000g of ethyl acetate are centrifuged for 5min, and the organic phase is then assayed by GC-MS analysis (TFA and GC-MS after derivatization, products by GC-MS analysis, benzoic acid and HPLC analysis), the reaction results are shown in Table 7.
TABLE 7 substrate conversion rates of different P450 enzymes and NCDH-dependent P450 reductase catalytic systems
Figure BDA0002827893740000101
Example 11: catalytic system coupling NCDH dependent self-sufficient bifunctional P450 enzyme and NCDH regenerative enzyme
300 μ L of a 100mM MES buffer system pH 8.0: 0.1mM NCD, 0.4mg/mL FDH-V198/C256/P260/E261/S381/S383, 20mM sodium formate, 1.1mg/mL NCDH-dependent self-sufficient bifunctional P450 enzymes (CYP 101A-Linker-BMR 967/Q977/W1047 or CYP 101A-Linker-BMR 967/Q977/Q1005/W1047 or CYP 152L-Linker-BMR 967/Q977/Q1005/W1047 or CYP 53A-Linker-BMR 967/Q977/Q1005/W1047), 2mM D-1047 (or palmitic acid or benzoic acid), react at 30 ℃ for 4 h. 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 ratio of NCDH-dependent self-sufficient bifunctional P450 enzyme-coupled NCDH-regenerating enzyme system
Figure BDA0002827893740000102
Example 12: NCDH-dependent CYP102A1 enzyme and NCDH-regenerating enzyme coupled catalytic system
300 μ 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 (CYP102A1-R967D/Q977E/W1047S or CYP102A1-R967D/Q977E/Q1005E/W104 1047S), 2mM fatty acid (C102A 1-R967/Q977/Q1005E/W1043656)12:0Or C14:0Or C16:0Or C18:0) And reacting at 30 ℃ for 4 h. 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-regenerant enzyme System
Figure BDA0002827893740000103
Example 13: NCDH (N-methyl-D-hydroxy-dehydrogenase) -dependent P450 reductase-mediated prokaryotic cell catalytic transformation system
The NCDH dependent P450 reductase, the free P450 enzyme, the NCDH regenerative enzyme and the NCD transport protein are co-expressed in a host to construct an NCD mediated biological catalysis system. The biocatalytic system is initiated when the regenerative substrate and NCD in the culture medium enter the host cell. Thus, the use of NCDH-dependent P450 reductases will be independent of intracellular NADPH levels, and can be selectively delivered to P450 substrates using extracellular regenerative substrate reducing power, enabling decoupling of the P450 enzyme-catalyzed energy expenditure from the endogenous NADPH supply.
The construction of an engineered strain that catalyzes the decarboxylation of fatty acids using Escherichia coli BL21(DE3) as a host strain is described below as an example.
The NAD transporter AtNDT2(Accession NO. NC-003070) has a broader substrate spectrum (Palmieri F, et al. J Biol Chem,2009,284,31249-31259) and can transport NCD. The gene of AtNDT2 expressing the transporter was expressed under the control of gapAP1 promoter (Charpentier B, et al. J Bacteriol,1994,176,830-839), the gene encoding BMR-R967D/Q977E/W1047S, the gene encoding CYP152L1, the gene encoding malic enzyme ME-L310R/Q401C were controlled by lac, trc and T7 promoters, respectively, Isopropylthiogalactose (IPTG) was induced, and the above four expression cassettes were constructed by enzyme digestion and ligation into the same plasmid pUC18(bla:: kan) to obtain an engineered plasmid.
The above engineering plasmid was introduced into e.coli BL21(DE3) to obtain the engineering strain e.coli QL 001. The engineering strain E.coli QL001 is induced in LB culture medium to express the above four functional proteins, and 50. mu.g/mL kanamycin, 0.5mM IPTG, 1mM 5-aminolevulinic acid, 1mM vitamin B1, 0.04mM FeCl are added into the culture medium3Culturing in a shaker at 25 deg.C and 200rpm for 48 hr to OD600The cells were collected by centrifugation at 2000 Xg for 6min at 4.5.
The suspended cells were washed with MOPS medium at pH 7.5, and the cell density OD was determined600Adjusted to 9. 2mM lauric acid, 5% DMSO cosolvent, 10mM L-malic acid and 3mM NCD are added into the engineering bacteria suspension, the total volume is 1mL, the reaction is carried out in a shaker at 30 ℃ and 200rpm for 4 hours, 500 microliter of 4M HCl and 0.5mL chloroform are added, after vortex oscillation for 3min, centrifugation is carried out, the lower organic phase is taken out, 0.22 microliter of organic filter membrane is used for filtration, 150 microliter of silylation reagent N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) is added into the mixture, and after derivatization, GC-MS analysis is carried out, the conversion rate of lauric acid is 18.4%. In a control experiment without the addition of NCD, the conversion of lauric acid was 4.3%.
The experimental result shows that in the catalysis process of the whole cells of the escherichia coli, NCDH dependent P450 reductase BMR-R967D/Q977E/W1047S utilizes NCDH regenerated by malic enzyme ME-L310R/Q401C to transfer electrons to CYP152L1 enzyme to catalyze the decarboxylation reaction of lauric acid.
Example 14: a NCDH-dependent P450 reductase-mediated eukaryotic cell catalytic transformation system.
The NCDH dependent P450 reductase, the free P450 enzyme, the NCDH regenerative enzyme and the NCD transport protein are co-expressed in a host to construct an NCD mediated biological catalysis system. The biocatalytic system is initiated when the regenerative substrate and NCD in the culture medium enter the host cell. Thus, the use of NCDH-dependent P450 reductases will allow selective transfer to P450 substrates using extracellular regenerative substrate reducing power independent of intracellular NADPH levels, enabling decoupling of the P450 enzyme-catalyzed energy expenditure from the endogenous NADPH supply.
The construction of an engineered strain that catalyzes the hydroxylation of benzoic acid using Saccharomyces cerevisiae BY4741 as a host strain will be described below as an example.
The NAD transporter NTT4(Haferkamp I, et al. Nature,2004,432, 622-. The gene coding for CYP53A15 is controlled by TEF1 promoter and CYC1 terminator, the gene coding for BMR-R967D/Q977E/Q1005E/W1047S is controlled by TDH3 promoter and ADH1 terminator, the gene coding for FDH-V198I/C256I/P260S/E261P/S381N/S383F is controlled by PGK1 promoter and PHO5 terminator, the gene coding for NTT4 is controlled by URA3 promoter and TDH1 terminator, and the above four expression cassettes are integrated into P416 yeast free shuttle expression vector to obtain engineering plasmid.
And introducing the engineering plasmid into saccharomyces cerevisiae to obtain an engineering strain S. Inducing engineering bacteria S.cerevisiae QL002 to express the two functional proteins with YEPD culture medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone of pH 6.0, culturing in shaker at 25 deg.C and 200rpm for 48h to thallus density OD600Centrifugation at 2000 Xg for 6min to collect the cells at 4.5, washing the resuspended cells with Tris-Cl at 50mM, pH 7.5, and OD600Adjusted to 9. Preparing permeabilized cells: thawing 5mL of frozen cells in a water bath at room temperature, adding 5mM EDTA and 1% toluene by volume, performing warm bath at 30 ℃ in a shaker at 200rpm for 30min, and then standing at 4 ℃ for 1 h. Centrifuging at 2000g for 6min to remove supernatant containing EDTA and toluene, and concentratingThe cell was washed twice with 50mM Tris-Cl, pH 7.5, and then resuspended in 5mL Tris-Cl, 50mM Tris-Cl, pH 7.5, to obtain permeabilized cells.
5mM benzoic acid, 10mM sodium formate and 1mM NCD are added into the permeable engineering bacteria suspension which is resuspended by 50mM Tris-Cl and pH 7.5, the total volume is 1mL, the mixture is reacted for 4 hours in a shaker at 30 ℃ and 200rpm, 25 muL of 4M HCl and 0.5mL acetonitrile are added into 500 muL, vortex and shake for 1min, then the mixture is filtered by a 0.22 muM organic filter membrane, and the conversion rate of the benzoic acid is 41.3 percent by HPLC analysis. In a control experiment without the addition of NCD, the conversion of benzoic acid was 3.7%.
The experimental result shows that in the whole cell catalysis process of saccharomyces cerevisiae, NCDH-dependent P450 reductase BMR-R967D/Q977E/Q1005E/W1047S utilizes NCDH regenerated by formate dehydrogenase FDH-V198I/C256I/P260S/E261P/S381N/S383F to transfer electrons to CYP53A15 enzyme to catalyze the decarboxylation reaction of lauric acid.
Example 15: NCDH dependent self-sufficient bifunctional P450 enzyme mediated prokaryote cell catalytic conversion system
The NCDH dependent self-sufficient bifunctional P450 enzyme, NCDH regenerating enzyme and NCD transport protein are co-expressed in host cells to form a NCD dependent biocatalysis system. The biocatalytic system is initiated when the regenerative substrate and NCD in the culture medium enter the host cell. Thus, the use of NCDH-dependent self-sufficient bifunctional P450 enzymes will be independent of intracellular NADPH levels, and can be selectively transferred to P450 enzyme substrates using extracellular regenerative substrate reducing power, enabling decoupling of the energy consumption of the P450 enzyme from the endogenous NADPH supply.
The construction of an engineered strain that catalyzes the hydroxylation of D-camphor using Escherichia coli BW25113 as a host strain will be described below as an example.
The NAD transporter NTT4(Haferkamp I, et al. Nature,2004,432, 622-. The gene for NTT4 expressing the transporter was expressed from the gapAP1 promoter (Charpentier B, et al. J Bacteriol,1994,176, 830-839). The genes encoding CYP101A1-Linker1-BMR-R967D/Q977E/Q1005E/W1047S and the genes encoding phosphite dehydrogenase PDH-I151R/P176E/M207A are controlled by the lac and trc promoters induced by isopropyl thiogalactose (IPTG), and the three expression cassettes are cloned to the same plasmid by replacing the LacZ gene of pUC18(bla:: kan) to obtain an engineered plasmid.
The engineering plasmid is introduced into E.coli BW25113 to obtain an engineering strain E.coli QL 003. The engineering strain E.coli QL003 is induced in LB culture medium to express the above three functional proteins, and 50. mu.g/mL kanamycin, 0.5mM IPTG, 1mM 5-aminolevulinic acid, 1mM vitamin B1 and 0.04mM FeCl are added into the culture medium3Culturing in a shaker at 25 deg.C and 200rpm for 48 hr to OD600The cells were collected by centrifugation at 2000 Xg for 6min at 4.5.
Washing with MOPS medium of pH 7.5, resuspending the cells, and determining cell density OD600Adjusted to 9. 10mM D-camphor (5% v/v DMSO for assisting dissolution), 20mM sodium phosphite and 5mM NCD are added into the engineering bacteria suspension, the total volume is 1mL, the reaction is carried out in a shaker at 30 ℃ and 200rpm for 4 hours, 500 mu L of the engineering bacteria suspension is added with 25 mu L of 4M HCl and 0.5mL of ethyl acetate, after vortex oscillation is carried out for 3 minutes, the upper organic phase is centrifugally taken out, the organic phase is filtered by a 0.22 mu M organic filter membrane, 500 mu L of the organic phase is analyzed by GC-MS, and the conversion rate of the D-camphor is 30.3%. In the control experiment without addition of NCD, the conversion of D-camphor was 2.6%.
The experimental result shows that in the whole cell catalytic process of the Escherichia coli, the NCDH-dependent self-sufficient bifunctional P450 enzyme CYP101A1-Linker1-BMR-R967D/Q977E/Q1005E/W1047S utilizes NCDH regenerated by phosphorous acid dehydrogenase PDH-I151R to complete the phosphorous acid-driven NCD-camphor hydroxylation reaction.
Example 16: NCDH dependent self-sufficient bifunctional P450 enzyme mediated eukaryotic cell catalytic conversion system
The NCDH-dependent self-sufficient bifunctional P450 enzyme and the NCDH regenerating enzyme are co-expressed in an NCD-independent Escherichia coli host to form a NCD-dependent biocatalysis system. The biocatalytic system is activated when a regenerative substrate in the culture medium enters the host cell. Thus, the use of NCDH-dependent self-sufficient bifunctional P450 enzymes will be independent of intracellular NADPH levels, and can be selectively transferred to P450 enzyme substrates using extracellular regenerative substrate reducing power, enabling decoupling of the energy consumption of the P450 enzyme from the endogenous NADPH supply.
The construction of an engineered strain that catalyzes the hydroxylation of fatty acids using Saccharomyces cerevisiae BY4742 as a host strain will be described below as an example.
The gene coding CYP102A1-R967D/Q977E/Q1005E/W1047S is controlled by TEF1 promoter and CYC1 terminator, the gene coding DLDH-V152R/I177K/N213I is controlled by PGK1 promoter and PHO5 terminator, the gene coding NTT4 is controlled by URA3 promoter and TDH1 terminator, and the three expression cassettes are integrated into a p416 yeast free shuttle expression vector to obtain an engineering plasmid.
And introducing the engineering plasmid into saccharomyces cerevisiae to obtain an engineering strain S.cerevisiae QL 004. Inducing engineering bacteria S.cerevisiae QL004 to express the two functional proteins by using YEPD culture medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone and having pH of 6.0, and culturing in a shaker at 25 ℃ and 200rpm for 48h to obtain the strain density OD600The cells were centrifuged at 2000 Xg for 6min at 4.5 to collect the cells, 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. Preparing permeabilized cells: thawing 5mL of frozen cells in water bath at room temperature, adding 5mM EDTA and 1% toluene by volume, performing warm bath at 30 deg.C and 200rpm in a shaker for 30min, and standing at 4 deg.C for 1 h. The supernatant containing EDTA and toluene was removed by centrifugation at 2000g for 6min, washed twice with Tris-Cl at a concentration of 50mM and pH 7.5, and then resuspended in 5mL of Tris-Cl at a concentration of 50mM and pH 7.5 to obtain permeabilized cells.
1mM lauric acid (5% v/v DMSO solubilization), 10mM sodium D-lactate and 3mM NCD with a total volume of 1mL are added into the permeable engineering bacteria suspension which is resuspended by 50mM Tris-Cl and has a pH value of 7.5, the mixture is reacted for 4 hours in a shaking table at 30 ℃ and 200rpm, 25 mu L of 4M HCl and 0.5mL of chloroform are added into 500 mu L of the mixture, vortex and shake for 3min, the lower organic phase is centrifuged, the organic phase is filtered by a 0.22 mu M organic filter membrane, and the conversion rate of lauric acid hydroxylation reaction is 85.3% by GC-MS analysis of 500 mu L of the mixture. In the control experiment without adding NCD, the conversion rate of lauric acid hydroxylation reaction was 5.2%.
The experimental result shows that in the whole cell catalysis process of saccharomyces cerevisiae, the NCDH-dependent self-sufficient bifunctional P450 enzyme CYP102A1-R967D/Q977E/Q1005E/W1047S utilizes the NCDH regenerated by D-lactate dehydrogenase DLDH-V152R/I177K/N213I to complete the NCD-mediated fatty acid hydroxylation reaction.
The three letter abbreviations, one letter abbreviations correspondence for amino acids in this application are shown in table 10 below:
TABLE 10 one-letter abbreviations, three-letter abbreviations corresponding relation of amino acids in the present application
Chinese translation name One letter abbreviation Three letter abbreviations
Glycine G Gly
Alanine A Ala
Valine V Val
Leucine L Leu
Isoleucine I Ile
Phenylalanine F Phe
Tryptophan W Trp
Tyrosine Y Tyr
Aspartic acid D Asp
Histidine H His
Asparagine N Asn
Glutamic acid E Glu
Lysine K Lys
Glutamine amides Q Gln
Methionine M Met
Arginine R Arg
Serine S Ser
Threonine T Thr
Cysteine C Cys
Proline P Pro
Although the present invention has been described with reference to a few preferred embodiments, it should be understood that various changes and modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> institute of chemistry and physics, large connection of Chinese academy of sciences
<120> P450 reductase and application thereof
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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 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
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Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp
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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
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Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe
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Gly Cys Arg Ser Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu
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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
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Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile
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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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<212> DNA
<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 (13)

1. A P450 reductase, wherein the amino acid sequence of the P450 reductase is set forth in SEQ ID NO: 1, and at least one of the following mutations occurs on the basis of the amino acid sequence shown in the formula (1): R967D, Q977E, Q1005E and W1047S.
2. The P450 reductase of claim 1, wherein the mutations comprise R967D, Q977E, and W1047S; or
The mutations include R967D, Q977E, Q1005E, and W1047S;
preferably, the P450 reductase has the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3;
preferably, the P450 reductase is a non-native coenzyme NCDH-dependent P450 reductase having the structure of formula I:
Figure FDA0002827893730000011
preferably, the non-natural-coenzyme NCDH is obtained by reducing a non-natural-coenzyme NCD with a regeneration substrate by a non-natural-coenzyme NCDH regenerating enzyme;
the non-natural coenzyme NCD has a structure represented by formula II:
Figure FDA0002827893730000012
preferably, the non-natural coenzyme NCDH regeneration enzyme comprises at least one of malic 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 compounds, phosphorous acid compounds, D-lactic acid compounds, formic acid compounds and methanol;
preferably, the malic acid compound comprises malic acid and/or a malate salt;
the D-lactic acid compound comprises D-lactic acid and/or a D-lactate;
the phosphorous acid compound comprises phosphorous acid and/or a phosphite salt;
the formic acid compound includes formic acid and/or a formate salt.
3. A P450 reductase as claimed in any one of claims 1 to 2 as K3[Fe(CN)6]Reductase, cytochrome c reductase, and thiazole blue reductase.
4. Use of a P450 reductase enzyme according to any one of claims 1 to 2 in the catalytic conversion of a P450 enzyme substrate.
5. A fusion enzyme having an amino acid sequence comprising the amino acid sequence of the P450 reductase of any one of claims 1 to 2 and the amino acid sequence of the P450 enzyme or the P450 enzyme domain.
6. The fusion enzyme according to claim 5, wherein 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 enzyme by a linker peptide;
preferably, the linker peptide has the amino acid sequence as set forth in SEQ ID NO: 12 to 16;
preferably, the P450 enzyme is selected from any one of P450 enzymes of Class I and Class II;
the P450 enzyme domain is selected from any one of Class III Class P450 enzyme domains;
preferably, the Class I P450 enzyme is CYP101A1 or CYP152L 1;
preferably, the Class II P450 enzyme is CYP53a 15;
preferably, the Class III Class P450 enzyme domain is the P450 domain of CYP102a 1;
preferably, the substrate of the P450 enzyme or P450 enzyme domain is selected from any one of D-camphor, C12-C18 saturated fatty acids, benzoic acid.
7. An enzyme catalysis system, which is characterized in that the enzyme catalysis system comprises the P450 reductase, the P450 enzyme or the P450 enzyme structure domain of any one of 1 to 2 and an unnatural coenzyme NCDH regenerative enzyme; or
Comprising the fusion enzyme of any one of claims 5 to 6, a non-native coenzyme NCDH-regenerating enzyme.
8. A nucleic acid encoding the P450 reductase of any one of claims 1 to 2 or the fusion enzyme of any one of claims 5 to 6.
9. A vector comprising an expression cassette comprising the nucleic acid of claim 8.
10. The vector of claim 9, wherein the vector further comprises an expression cassette comprising a nucleic acid encoding a non-native coenzyme NCDH regenerating enzyme;
preferably, the vector further comprises an expression cassette comprising a nucleic acid encoding a nucleotide transporter;
preferably, the nucleotide transporter in the expression cassette comprising a nucleic acid encoding a nucleotide transporter comprises an NTT4 nucleotide transporter from chlamydia and/or an AtNDT2 nucleotide transporter from arabidopsis thaliana.
11. A host cell comprising the vector of any one of claims 9 to 10.
12. The host cell according to claim 11, wherein the host cell is selected from the group consisting of prokaryotes and/or eukaryotes;
preferably, the prokaryote is escherichia coli; the eukaryote is saccharomyces cerevisiae.
13. A P450 reductase as claimed in any one of claims 1 to 2, a fusion enzyme as claimed in any one of claims 5 to 6, an enzymatic catalytic system as claimed in claim 7, a nucleic acid as claimed in claim 8, a vector as claimed in any one of claims 9 to 10, use of a host cell as claimed in any one of claims 11 to 12 in a biocatalytic reaction mediated by non-native coenzyme NCD.
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