WO2017132924A1 - 一种用于生物合成异戊二烯和异戊烯的酶及其突变体 - Google Patents

一种用于生物合成异戊二烯和异戊烯的酶及其突变体 Download PDF

Info

Publication number
WO2017132924A1
WO2017132924A1 PCT/CN2016/073413 CN2016073413W WO2017132924A1 WO 2017132924 A1 WO2017132924 A1 WO 2017132924A1 CN 2016073413 W CN2016073413 W CN 2016073413W WO 2017132924 A1 WO2017132924 A1 WO 2017132924A1
Authority
WO
WIPO (PCT)
Prior art keywords
ferredoxin
polypeptide
cell
reductase
methyl
Prior art date
Application number
PCT/CN2016/073413
Other languages
English (en)
French (fr)
Inventor
马延和
葛德永
薛燕芬
Original Assignee
中国科学院微生物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院微生物研究所 filed Critical 中国科学院微生物研究所
Priority to PCT/CN2016/073413 priority Critical patent/WO2017132924A1/zh
Priority to US15/578,767 priority patent/US10287569B2/en
Priority to CN201680012121.5A priority patent/CN107531763B/zh
Publication of WO2017132924A1 publication Critical patent/WO2017132924A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03027—Isoprene synthase (4.2.3.27)

Definitions

  • the invention relates to the field of molecular biology technology, in particular to the field of biosynthesis of isoprene.
  • the present invention provides a polypeptide capable of producing isoprene using 4-hydroxy-3-methyl-2-butenyl-pyrophosphate (HMBPP) as a substrate, and a nucleic acid encoding the polypeptide, A vector and a cell containing the nucleic acid.
  • HMBPP 4-hydroxy-3-methyl-2-butenyl-pyrophosphate
  • the invention also provides methods of using the polypeptides to produce isoprene, and methods of making the polypeptides.
  • Isoprene is an important chemical raw material, mainly used in the production of rubber, and can also be used in the synthesis of fine chemicals such as isopentenyl chloride, linalool, myrcene and vitamins A, E, K, etc. .
  • fine chemicals such as isopentenyl chloride, linalool, myrcene and vitamins A, E, K, etc.
  • isoprene and isoamylene used in industrial applications are derived from petroleum derived raw materials.
  • isoprene and isoamylene used in industrial applications are derived from petroleum derived raw materials.
  • the biosynthesis of isoprene has attracted widespread attention and research due to its non-polluting and reproducible advantages, and preliminary progress has been made.
  • DMAPP dimethylallyl pyrophosphate
  • IPP isopentenyl pyrophosphate
  • the DXP pathway involves first condensing pyruvate and glyceraldehyde-3-phosphate to 1-deoxy-D-xylulose 5-phosphate (DXP); then converting DXP to 4-hydroxy-3-yl by five enzymatic reactions Benz-2-butenyl-pyrophosphate (HMBPP); HMBPP is then converted to DMAPP and IPP by the action of HMBPP reductase (IspH).
  • HMBPP Benz-2-butenyl-pyrophosphate
  • IspH HMBPP reductase
  • the MVA approach is Acetyl-CoA is a single material and DMAPP is produced by seven consecutive enzyme reactions.
  • IspS isoprene synthase
  • the present inventors have discovered a novel enzyme in Bacillus sp. N16-5 (Bacillus sp. N16-5) which has various activities including: (a) generating DMAPP and IPP using HMBPP as a substrate; b) isoprene is produced using HMBPP as a substrate; and, (c) isoprene (2-methyl-2-butene and 3-methyl-1-butene) is formed as a DMAPP substrate. Further, the inventors have modified the enzyme to obtain two mutants (H131N and E133Q).
  • the present invention provides a polypeptide having activity of producing isoprene using 4-hydroxy-3-methyl-2-butenyl-pyrophosphate (HMBPP) as a substrate, and Having an amino acid sequence selected from the group consisting of:
  • the amino acid sequence of the polypeptide of the invention has at least 90% identity to SEQ ID NO: 2, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% Identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.
  • the amino acid sequence of the polypeptide of the invention differs from SEQ ID NO: 2 by one or more (eg, 1, 2, 3, 4, 5, 6, 7) Replacement, deletion or addition of one, eight or nine) amino acid residues.
  • amino acid sequence of the polypeptide of the invention differs from SEQ ID NO: 2 by one or more (eg, 1, 2, 3, 4, 5, 6, 7) Conservative substitution of one, eight or nine) amino acid residues.
  • the amino acid sequence of a polypeptide of the invention is SEQ ID NO: 2 or is different from SEQ ID NO: 2 by substitution of one or several (eg, 1) amino acid residues.
  • the amino acid sequence of a polypeptide of the invention differs from SEQ ID NO: 2 by an amino acid substitution at position 131 or 133 of SEQ ID NO: 2.
  • the amino acid sequence of the polypeptide of the invention differs from SEQ ID NO: 2 in that the histidine at position 131 of SEQ ID NO: 2 is mutated to asparagine; The glutamic acid at position 133 of ID NO: 2 was mutated to glutamine.
  • polypeptides of the invention have an amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 3, and 4.
  • the polypeptide of the invention further has 2-methyl-2-butene and 3-methyl-1-butene produced using dimethylallyl pyrophosphate (DMAPP) as a substrate. active.
  • DMAPP dimethylallyl pyrophosphate
  • the polypeptide of the invention does not have the production of 2-methyl-2-butene and 3-methyl-1-butene using dimethylallyl pyrophosphate (DMAPP) as a substrate. active.
  • DMAPP dimethylallyl pyrophosphate
  • the invention provides an isolated nucleic acid encoding as described above Peptide.
  • the invention provides a vector comprising the isolated nucleic acid.
  • the isolated nucleic acids of the invention encode polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 4.
  • Vectors useful for insertion of a polynucleotide of interest are well known in the art and include, but are not limited to, cloning vectors and expression vectors.
  • the vector is, for example, a plasmid, a cosmid, a phage, and the like.
  • the invention also relates to a host cell comprising the isolated nucleic acid or vector described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells and Bacillus cells (eg, Bacillus alkalophilus, Bacillus subtilis), and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells.
  • the isolated nucleic acid is heterologous to the cell. In certain preferred embodiments, the isolated nucleic acid is foreign to the cell.
  • the cell further comprises a nucleic acid or expression encoding an electron transporter (eg, ferredoxin) and/or an enzyme required for electron transport (eg, ferredoxin reductase). Electron transfersomes (eg, ferredoxin) and/or electron transferers are required to deliver electrons (eg, ferredoxin reductase).
  • the cell may further comprise a nucleic acid encoding a ferredoxin and a ferredoxin reductase, or a ferredoxin and a ferredoxin reductase.
  • the electron transporter and/or electron transporter is required to deliver electrons with an enzyme that is endogenous to the cell.
  • the electron transporter and/or electron transporter is required to deliver electrons to the exogenous enzyme relative to the cell.
  • the ferredoxin and/or ferredoxin reductase is endogenous or exogenous to the cell.
  • the cell further comprises an exogenously introduced nucleic acid encoding a ferredoxin, and/or an exogenously introduced nucleic acid encoding a ferredoxin reductase.
  • the ferredoxin reductase is ferredoxin-NADP + reductase (EC 1.18.1.2).
  • the cell further expresses a polypeptide of the DXP pathway.
  • the polypeptide of the DXP pathway is selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reduction Isomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphatidyltransferase (MCT; EC 2.7.7.60), 4-(cytidine 5'-di Phospho)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS; EC 4.6.1.12), 4-Hydroxy-3-methyl-2-(E)-butenyl diphosphate synthase (DXS; EC
  • the cell also expresses isoprene synthase (IspS; EC 4.2.3.27). In certain preferred embodiments, the cell does not express isoprene synthase (IspS; EC 4.2.3.27).
  • the cell is a prokaryotic cell, such as E. coli or Bacillus or cyanobacteria.
  • the present invention is also a composition
  • a composition comprising a polypeptide of the present invention, HMBPP, NADPH or NADH, an electron transporter (eg, ferredoxin), and an enzyme required for electron transporter to deliver electrons (eg, Iron redox protein reductase).
  • the composition comprises the polypeptide, HMBPP, NADPH, ferredoxin, and ferredoxin reductase (eg, ferredoxin-NADP + reductase).
  • ferredoxin reductase eg, ferredoxin-NADP + reductase
  • the present invention is also a composition
  • a composition comprising a polypeptide having the amino acid sequence of SEQ ID NO: 2, DMAPP, NADPH or NADH, an electron transporter (eg, ferredoxin), and electron transport An enzyme required for the delivery of electrons (eg, ferredoxin reductase).
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, DMAPP, NADPH, ferredoxin, and ferredoxin reductase (eg, ferredoxin- NADP + reductase).
  • Such compositions of the invention can be used to synthesize isopentene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene) in vitro.
  • the invention provides a method of producing isoprene comprising converting HMBPP to isoprene using a polypeptide of the invention.
  • the method comprises (a) an enzyme required to deliver the polypeptide, HMBPP, NADPH or NADH, an electron transporter (eg, a ferredoxin), and an electron transporter (eg, For example, ferredoxin reductase) is mixed and incubated (for example, incubation at 20-40 ° C, such as incubation at room temperature or 37 ° C); and, (b) collection of isoprene produced in step (a) .
  • an enzyme required to deliver the polypeptide, HMBPP, NADPH or NADH an electron transporter (eg, a ferredoxin), and an electron transporter (eg, For example, ferredoxin reductase) is mixed and incubated (for example, incubation at 20-40 ° C, such as incubation at room temperature or 37 ° C); and, (b) collection of isoprene produced in step (a) .
  • an enzyme required to deliver the polypeptide, HMBPP, NADPH or NADH
  • the polypeptide, HMBPP, NADPH, ferredoxin, and ferredoxin reductase are mixed and Incubation, for example, incubation at 20-40 ° C (eg, incubation at room temperature or 37 ° C) to produce isoprene.
  • the polypeptide has the amino acid sequence set forth in SEQ ID NO: 3 or 4.
  • the method does not involve the use of isoprene synthase (IspS; EC 4.2.3.27). In certain preferred embodiments, the method is used to produce isoprene in vitro.
  • IspS isoprene synthase
  • the invention provides a method of producing isoprene, comprising: (a) cultivating cells expressing an exogenously introduced polypeptide of the invention; and, (b) collecting step (a) ) isoprene produced.
  • the cells are cultured under conditions suitable for the production of isoprene.
  • the cells may be provided with any one or more of the following: (1) a medium that maintains or promotes cell growth; (2) a substrate for the polypeptide of the present invention, HMBPP; 3) an electron transporter (e.g., ferredoxin); (4) an enzyme required for electron transport to deliver electrons (e.g., ferredoxin reductase); and (5) NADPH or NADH.
  • the cell also expresses an electron transporter (e.g., ferredoxin) and/or an enzyme required for electron transport (e.g., iron redox protein reductase).
  • an electron transporter e.g., ferredoxin
  • an enzyme required for electron transport e.g., iron redox protein reductase
  • the cells also express ferredoxin and ferredoxin reductase.
  • the electron transporter and/or electron transporter is required to deliver electrons with an enzyme that is endogenous to the cell.
  • the electron transporter and/or electron transporter is required to deliver electrons to the exogenous enzyme relative to the cell.
  • the ferredoxin and/or ferredoxin reductase may be endogenous or exogenous relative to the cell.
  • the ferredoxin reductase is ferredoxin-NADP + reductase (EC 1.18.1.2).
  • the cells naturally express ferredoxin and ferredoxin reductase.
  • a nucleic acid encoding a ferredoxin and a ferredoxin reductase may be introduced exogenously.
  • a nucleic acid encoding a ferredoxin and a ferredoxin reductase is further introduced to increase expression of ferredoxin and ferredoxin reductase, thereby further enhancing the present invention.
  • the activity of the polypeptide In some cases, the cells do not express ferredoxin and ferredoxin reductase.
  • HMBPP can be added to the cell culture medium to provide the desired substrate for the polypeptides of the invention.
  • the cells are caused to synthesize HMBPP by establishing a DXP pathway in the cell, and thereby provide the substrate required for the polypeptide of the invention.
  • cells can be made to synthesize HMBPP by expressing one or more polypeptides of the DXP pathway in a cell, thereby providing the substrate required for the polypeptide of the invention.
  • the cell also expresses a polypeptide of the DXP pathway.
  • the polypeptide of the DXP pathway is selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylone Sugar-5-phosphate reductoisomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphatidyltransferase (MCT; EC 2.7.7.60), 4-( Cytidine 5'-diphosphate)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate Synthase (MCS; EC 4.6.1.12), 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate synthase (I
  • the polypeptide of the DXP pathway is endogenous to the cell. In certain preferred embodiments, the polypeptide of the DXP pathway is foreign to the cell.
  • the cell also expresses isoprene synthase (IspS; EC 4.2.3.27).
  • IspS isoprene synthase
  • the cells can biosynthesize isoprene by a variety of routes.
  • the cell does not express isoprene synthase (IspS; EC 4.2.3.27).
  • the cell is selected from the group consisting of prokaryotic cells such as E. coli cells and Bacillus cells (eg, Bacillus alkalophilus, Bacillus subtilis), and eukaryotic cells such as yeast cells, insect cells, plants Cell and animal cells.
  • prokaryotic cells such as E. coli cells and Bacillus cells (eg, Bacillus alkalophilus, Bacillus subtilis)
  • eukaryotic cells such as yeast cells, insect cells, plants Cell and animal cells.
  • the cells are prokaryotic cells, such as E. coli or Bacillus or cyanobacteria.
  • the method is for the biosynthesis of isoprene.
  • the invention provides a method of producing isoamylene (eg, 3-methyl-1-butene and/or 2-methyl-2-butene) comprising: using SEQ ID NO A polypeptide of the amino acid sequence shown in 2, which converts DMAPP to isoamylene.
  • isoamylene eg, 3-methyl-1-butene and/or 2-methyl-2-butene
  • the method comprises (a) an enzyme required to deliver electrons to the polypeptide, DMAPP, NADPH or NADH, electron transporters (eg, ferredoxin), and electron transporters (eg, Ferricoxin reductase) is mixed and incubated; and, (b) isolating the isopentene produced in step (a).
  • an enzyme required to deliver electrons to the polypeptide, DMAPP, NADPH or NADH, electron transporters (eg, ferredoxin), and electron transporters (eg, Ferricoxin reductase) is mixed and incubated; and, (b) isolating the isopentene produced in step (a).
  • step (a) the polypeptide, DMAPP, NADPH, ferredoxin, and ferredoxin reductase (eg, ferredoxin-NADP + reductase) are mixed and Incubation, for example, incubation at 20-40 ° C (eg, incubation at room temperature or 37 ° C) to produce isoamylene.
  • the method is used to produce isopentene in vitro.
  • the invention provides a method of producing isopentenyl (eg, 3-methyl-1-butene and/or 2-methyl-2-butene), comprising: (a) culturing cells Cell table An exogenously introduced polypeptide having the amino acid sequence of SEQ ID NO: 2; and, (b) collecting isopentene produced in step (a).
  • isopentenyl eg, 3-methyl-1-butene and/or 2-methyl-2-butene
  • the cultivation is carried out under conditions suitable for the production of isopentene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene).
  • cell for example, to promote the production of isopentenes by the cells, the cells may be provided with any one or more of the following: (1) a medium that maintains or promotes cell growth; (2) a substrate for the polypeptide of the present invention, DMAPP; An electron transporter (eg, ferredoxin); (4) an enzyme required for electron transport to deliver electrons (eg, ferredoxin reductase); and (5) NADPH or NADH.
  • the cell also expresses an electron transporter (eg, ferredoxin) and/or an enzyme required for electron transport (eg, ferredoxin reductase).
  • an electron transporter eg, ferredoxin
  • an enzyme required for electron transport eg, ferredoxin reductase
  • the cells also express ferredoxin and ferredoxin reductase.
  • the electron transporter and/or electron transporter is required to deliver electrons with an enzyme that is endogenous to the cell.
  • the electron transporter and/or electron transporter is required to deliver electrons to the exogenous enzyme relative to the cell.
  • the ferredoxin and/or ferredoxin reductase may be endogenous or exogenous relative to the cell.
  • the ferredoxin reductase is ferredoxin-NADP + reductase (EC 1.18.1.2).
  • the cells naturally express ferredoxin and ferredoxin reductase.
  • a nucleic acid encoding a ferredoxin and a ferredoxin reductase may be introduced exogenously.
  • a nucleic acid encoding a ferredoxin and a ferredoxin reductase is further introduced to increase expression of ferredoxin and ferredoxin reductase, thereby further enhancing the present invention.
  • the activity of the polypeptide In some cases, the cells do not express ferredoxin and ferredoxin reductase.
  • DMAPP can be added to the cell culture medium to provide the desired substrate for the polypeptides of the invention.
  • the cells are caused to synthesize DMAPP by establishing a DXP pathway in the cell, and thereby provide the substrate required for the polypeptide of the invention.
  • cells can be stimulated to synthesize DMAPP by expressing one or more polypeptides of the DXP pathway in a cell, thereby providing the substrate required for the polypeptide of the invention.
  • the cell also expresses a polypeptide of the DXP pathway.
  • the polypeptide of the DXP pathway is selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylone Sugar-5-phosphate reductoisomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphatidyltransferase (MCT; EC 2.7.7.60), 4-( Cytidine 5'-diphosphate)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate Synthase (MCS; EC 4.6.1.12), 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate synthase (I
  • the polypeptide of the DXP pathway is endogenous to the cell. In certain preferred embodiments, the polypeptide of the DXP pathway is foreign to the cell.
  • the cell further expresses isopentenyl diphosphate isomerase (IDI; EC 5.3.3.2).
  • IDI isopentenyl diphosphate isomerase
  • the isopentenyl diphosphate isomerase is endogenous to the cell.
  • the isopentenyl diphosphate isomerase is foreign to the cell.
  • the cell is selected from the group consisting of prokaryotic cells such as E. coli cells and Bacillus cells (eg, Bacillus alkalophilus, Bacillus subtilis), and eukaryotic cells such as yeast cells, insect cells, plants Cell and animal cells.
  • prokaryotic cells such as E. coli cells and Bacillus cells (eg, Bacillus alkalophilus, Bacillus subtilis)
  • eukaryotic cells such as yeast cells, insect cells, plants Cell and animal cells.
  • the cells are prokaryotic cells, such as E. coli or Bacillus or cyanobacteria.
  • the method is used to biosynthesize isoamylene (eg, 3-methyl-1-butene and/or 2-methyl-2-butene).
  • the present invention provides a method of preparing a polypeptide of the present invention, which comprises And (a) cultivating a host cell comprising and expressing a nucleic acid encoding the polypeptide; and, (b) collecting the polypeptide expressed by the cell.
  • host cells for protein expression include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammals). Cells, such as mouse cells, human cells, etc.). Particularly preferably, the host cell is Escherichia coli.
  • HMBPP 4-hydroxy-3-methyl-2-butenyl-pyrophosphate having the structural formula shown below for Formula I:
  • DMAPP dimethylallyl pyrophosphate having the structural formula shown below in Formula II:
  • the term "2M2B” refers to 2-methyl-2-butene, the structural formula of which is As used herein, the term “3M1B” refers to 3-methyl-1-butene, the structural formula of which is As used herein, the term “isoprene” refers to 2-methyl-1,3-butadiene, the structural formula of which is
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which is conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds Substitution of residues by force, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • conservative substitutions generally refer to the replacement of the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al, Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12 (10): 879-884 (1999). And Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which is incorporated herein by reference.
  • the IspH protein as shown in SEQ ID NO: 2 is capable of converting HMBPP to isoprene.
  • methods for mutating known polypeptides to obtain variants thereof have been described in detail in the prior art and are exemplarily described in the present application.
  • an embodiment of the present specification provides a method of preparing a mutant of the IspH protein, and two mutants (H131N and E133Q) are obtained.
  • methods for determining whether a polypeptide has the activity of converting HMBPP to isoprene are known to those skilled in the art and are also described in detail in the present application.
  • the examples of the present specification provide a method for testing whether H131N and E133Q are capable of producing isoprene with HMBPP as a substrate, and it has been confirmed that both H131N and E133Q have an activity of converting HMBPP to isoprene.
  • H131N and E133Q have an activity of converting HMBPP to isoprene.
  • polypeptide of the present invention is not limited to the IspH protein as shown in SEQ ID NO: 2 and its mutants H131N and E133Q, and is intended to encompass all other mutants of the IspH protein, as long as such mutants still retain the conversion of HMBPP to different The activity of pentadiene.
  • nucleic acid/polypeptide is heterologous to a cell
  • nucleic acid/polypeptide is endogenous to a cell
  • nucleic acid/polypeptide is foreign to a cell
  • nucleic acid/polypeptide is introduced into the cell exogenously by artificial means. It will be understood that such nucleic acids/polypeptides may not be naturally present in the cell (ie, heterologous to the cell) for introducing a heterologous nucleic acid/polypeptide into the cell; or, The endogenous nucleic acid/polypeptide naturally present in the cell is the same, which is used to increase the copy number or expression of the endogenous nucleic acid/polypeptide in the cell.
  • electron transfersome refers to a protein that participates in electron transport in an electron transport chain.
  • an electron mediator can accept electrons as an electron acceptor and electrons as an electron donor, thereby enabling electron transfer.
  • electron transporters are well known to those skilled in the art and include, but are not limited to, ferredoxin and flavin redox protein.
  • the participation of oxidoreductases, which are referred to herein as enzymes required for electron transporters to deliver electrons is generally required.
  • ferredoxin reductase typically requires the participation of ferredoxin reductase
  • flavin redox protein typically requires the participation of flavin redox reductase
  • Enzymes required for electron transport to transport electrons are also well known to those skilled in the art, such as, but not limited to, ferredoxin reductase and flavin redox reductase.
  • ferredoxin reductase refers to an enzyme capable of catalyzing the redox reaction of iron redox proteins, which includes two types:
  • the ferredoxin reductase is preferably ferredoxin-NADP + reductase (EC 1.18.1.2).
  • polypeptide of the DXP pathway refers to a polypeptide that is involved in the DXP pathway.
  • the DXP pathway refers to a pathway for synthesizing DMAPP from pyruvic acid and glyceraldehyde-3-phosphate as a raw material in an organism, which comprises the following steps: (1) condensation of pyruvic acid and glyceraldehyde-3-phosphate to 1-deoxy- D-xylulose 5-phosphate (DXP); (2) conversion of DXP to 2-C-methyl-D-erythritol 4-phosphate (MEP); (3) conversion of MEP to 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME); (4) conversion of CDP-ME to 2-phospho-4-(cytidine 5'-diphosphate)- 2-C-methyl-D-erythritol (CDP-ME2P); (5) conversion of CDP-ME
  • DXP pathway A detailed description of the DXP pathway can be found, for example, in Tomohisa Kuzuyama, Biosci. Biotechnol. Biochem., 66(8), 1619-1627, 2002; Thomas D. Sharkey et al., Plant Physiology, February 2005, Vol. 137, pp. .700-712; and US8507235B2.
  • Polypeptides involved in the DXP pathway include, but are not limited to, 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductive isomerization Enzyme (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphatidyltransferase (MCT; EC 2.7.7.60), 4-(cytidine 5'-diphosphate) -2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS; EC 4.6.
  • DXS 1-deoxy-D-xylulose-5-phosphate synthase
  • DXR 1-deoxy-D-xylulose-5-phosphate reductive iso
  • IspS isoprene synthase
  • IPI isopentenyl diphosphate isomerase
  • the polypeptide (enzyme) of the present invention is capable of directly producing isoprene using HMBPP as a substrate. Compared with the method and enzyme for producing isoprene based on the MEP pathway disclosed in the prior art, the technical scheme of the present invention has the following beneficial effects:
  • the method for producing isoprene of the present invention can avoid the accumulation of DMAPP and IPP which are toxic to the growth of host cells in a host cell without the participation of DMAPP;
  • the method for producing isoprene of the present invention reduces the one-step enzymatic reaction without the participation of isoprene synthase (IspS), simplifies the process, and improves the efficiency.
  • IspS isoprene synthase
  • Figure 1 shows the results of SDS-PAGE detection of the expressed and purified protein in Example 2, wherein lane M: molecular weight marker; lane 1: ferredoxin-NADP + reductase; lane 2: IspH protein; lane 3: Iron redox protein.
  • the results show that the IspH protein of the present invention is highly expressed in E. coli, has a molecular weight of about 35 kD, and has high purity after purification, as shown by a single electrophoresis band in lane 2 of Figure 1.
  • Figure 2 shows the results of a full-wavelength broad-spectrum scan of purified IspH protein, where the abscissa represents the wavelength and the ordinate represents the absorbance.
  • the results show that the IspH protein of the present invention has a maximum absorption peak at 410 nm and a shoulder peak at 320 nm. This indicates that the protein contains There is an iron and sulfur center.
  • Figure 3 shows the results of SDS-PAGE electrophoresis of the mutant proteins H131N and E133Q expressed and purified in Example 3, wherein lane M: molecular weight marker; lane 1: mutant protein H131N; lane 2: mutant protein E133Q.
  • the results showed that the mutant proteins H131N and E133Q of the present invention can be highly expressed in E. coli, have a molecular weight of about 35 kD, and have high purity after purification.
  • Figure 4 shows the Michaelis-Menten curve of the enzymatic reaction of IspH protein to convert DMAPP to isoamylene, wherein the abscissa indicates the concentration of DMAPP (mM) and the ordinate indicates the reaction rate (nmol min -1 mg -1 ).
  • the panel in Figure 4 shows the Lineweaver-Burk curve for the enzymatic reaction, wherein the abscissa represents the reciprocal of the concentration of DMAPP and the ordinate represents the reciprocal of the reaction rate.
  • the results showed that the wild-type IspH protein with DMAPP as a substrate to form isopentenes had a maximum reactivity of about 6.2 nmol min -1 mg -1 and a Km of about 275 ⁇ M.
  • Figure 5 is a schematic diagram showing three activities of the IspH protein of the present invention, wherein Glucose: glucose; MVA pathway: MVA pathway; DXP pathway: DXP pathway; HMBPP: 4-hydroxy-3-methyl-2-butenyl- Pyrophosphoric acid; DMAPP: dimethylallyl pyrophosphate; IPP: isopentenyl pyrophosphate; IDI: isopentenyl diphosphate isomerase; IspS: isoprene synthase; 2M2B: 2-methyl- 2-butene; 3M1B: 3-methyl-1-butene.
  • Glucose glucose
  • MVA pathway MVA pathway
  • DXP pathway DXP pathway
  • HMBPP 4-hydroxy-3-methyl-2-butenyl- Pyrophosphoric acid
  • DMAPP dimethylallyl pyrophosphate
  • IPP isopentenyl pyrophosphate
  • IDI isopentenyl diphosphate isomerase
  • Figure 6 shows the GC profile of the supernatant air of cultured wild type B. aeruginosa N16-5 (WT) and recombinant cell WNH (pMH), wherein 3-methyl-1-butene (3M1B), The residence times of 2-methyl-2-butene (2M2B) and isoprene were 4.5 min, 5.5 min, and 11.3 min, respectively. The results showed that wild type B.
  • WT aeruginosa N16-5
  • pMH recombinant cell WNH
  • alkalophilus N16-5 was able to produce isoprene, as well as trace amounts of 2-methyl-2-butene and 3-methyl-1-butene; and, overexpression of IspH protein overexpression
  • the yield of 2-methyl-2-butene and 3-methyl-1-butene of the cell WNH was significantly increased, but the production of isoprene was only slightly increased.
  • Figure 7 shows a comparison of isoprene production of wild type B. alkalophilus N16-5 (WT), recombinant cells WNH, H131N and E133Q.
  • WT alkalophilus N16-5
  • the results showed that the isoprene production of recombinant cell WNH was slightly increased compared with wild-type B. alkalophilus N16-5, but the isoprene production of recombinant cells H131N and E133Q was significantly increased, respectively, about wild type. 3 times the strain and 4 times.
  • Figure 8a shows the growth of wild-type strains and mutant strains ⁇ fer and ⁇ fld of B. alkalophilus N16-5. The results showed that all three strains were able to grow normally in Horikoshi-I medium, and there was no significant difference in growth.
  • Figure 8b shows a comparison of the isoprene production of the wild type strain of B. alkalophilus N16-5 and the mutant strains ⁇ fer and ⁇ fld. The results showed that the isoprene production of the mutant strain ⁇ fer was significantly lower than that of the wild type strain (only about 50% of the wild type strain); while the isoprene production of the mutant strain ⁇ fld was slightly lower than that of the wild type strain.
  • Figure 9 shows the GC profile of the supernatant air of cultured recombinant E. coli cells STV165HF (Fig. 9A), and the standard (3-methyl-1-butene, 2-methyl-2-butene, 2 GC profile of -methyl-1-butene and isoprene (Fig. 9B), wherein the GC detection conditions used were: inlet temperature 180 ° C, column temperature 100 ° C, detector temperature 200 ° C And, peak a is a characteristic peak of 3-methyl-1-butene; peak b is a characteristic peak of 2-methyl-2-butene; and peak c is a characteristic peak of 2-methyl-1-butene The peak d is a characteristic peak of isoprene.
  • Figure 10 shows the peak 1 component of Figure 9A and the standard 3-methyl-1-butene (Figure 10A), the peak 2 component and the standard 2-methyl-2-butene (Figure 10B), peak Comparison of the mass spectrometry results of the 3 component with the standard isoprene (Fig. 10C); wherein, in Figs. 10A-10C, the graph above the abscissa axis shows the components to be tested (peak 1, peak 2, peak) 3) Mass spectrometry results, and the graph below the abscissa axis shows the mass spectrometry results of the standards (3-methyl-1-butene, 2-methyl-2-butene, isoprene).
  • Figure 11 shows the GC profile of the headspace of cultured recombinant E. coli cells STV165HF-H131N (Figure 11A) and STV165HF-E133Q ( Figure 11B);
  • the GC detection conditions were: inlet temperature 180 ° C, column temperature 130 ° C, detector temperature 200 ° C; and, by mass spectrometry confirmed, the headspace gas measured in Figures 11A-11B contained isoprene, but did not contain Isopentenol.
  • Sequence 7 (SEQ ID NO: 7):
  • Sequence 8 (SEQ ID NO: 8):
  • Sequence 9 (SEQ ID NO: 9):
  • the molecular biology experimental methods used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al.
  • the method of editing the Guide to Molecular Biology, 3rd edition, described in John Wiley & Sons, Inc., 1995; the detection methods used are all conventional detection methods in the art, according to the steps described in the relevant literature or according to Follow the steps recommended by the manufacturer of the instrument used.
  • the invention is described by way of example, and is not intended to limit the scope of the invention.
  • Bacillus subtilis N16-5 Bacillus subtilis N16-5 (Bacillus sp. N16-5) (obtained from China General Microorganisms Collection and Management Center, CGMCC) was cultured in Horikoshi-I medium (pH 10.0) containing 2% NaCl (see, Horikoshi) K., Microbiol Mol Biol Rev. 1999; 63:735-50). Using a DNA extraction kit (EZNA TM Bacterial DNA Isolation Kit, Omega, USA), extracted alkalophilic Bacillus genomic DNA N16-5 of.
  • EZNA TM Bacterial DNA Isolation Kit EZNA TM Bacterial DNA Isolation Kit, Omega, USA
  • upstream primer 275F 5'-CATG CCATGG AGGTTTTGAAAATTTCC-3' (SEQ ID NO: 5), wherein the underlined portion indicates the NcoI cleavage site
  • Downstream primer 275R 5'-CCG CTCGAG TGGCCCGCTCCGCTTTTTTCTTTC-3' (SEQ ID NO: 6), wherein the underlined portion indicates the XhoI cleavage site.
  • the PCR reaction system was 50 ⁇ L, and the reaction conditions were: pre-denaturation at 94 ° C for 3 min; 30 cycles (denaturation at 94 ° C for 1 min, annealing at 58 ° C for 30 s, extension at 72 ° C for 1.5 min); extension at 72 ° C for 5 min.
  • PCR products were kit (EZNA TM Bacterial DNA Isolation Kit, Omega, USA) was purified and digested with NcoI and XhoI, was then ligated into NcoI and XhoI digested vector pET28a (TaKaRa Co.), and ultimately transformed into E. coli DH5 ⁇ .
  • the transformed Escherichia coli DH5 ⁇ was cultured in LB medium, a plasmid was extracted therefrom, and the plasmid was subjected to sequencing verification.
  • the constructed plasmid was named pET-H.
  • the protein encoded by the isc (iron-Sulfur cluster) operon of E. coli contributes to the correct construction of the [4Fe-4S] center of the IspH protein of the present invention in the protein. Therefore, in order to make the expressed IspH protein highly active, the isc operon was first highly expressed in E. coli. Briefly, the isc operon was ligated into the pSTV28 vector and transformed into E. coli BL21 (DE) to increase the copy number of the operon in the host cell and increase the expression of the protein encoded by the operon (Grawert, T. et al. IspH protein of Escherichia coli: studies on iron-Sulfur cluster implementation and catalysis. Journal of the American Chemical Society 126, 12847-12855 (2004)). Subsequently, the pET-H plasmid constructed as above was transferred into Escherichia coli which highly expressed the isc operon.
  • the transformed Escherichia coli was cultured in LB medium containing 25 mg/L of chloramphenicol and 50 mg/L of kanamycin, and cultured at 37 ° C until the OD value was about 0.6. Subsequently, added to the culture cysteine (1mM), FeCl 3 (0.1mM ) and 0.1mM IPTG, and culturing the host cell to continue for 16h at 18 °C, low speed oscillation conditions. After the completion of the culture, the cells were collected by centrifugation, washed twice with distilled water, and stored at -80 ° C until use.
  • the protein expressed in the cells is purified under anaerobic conditions. Briefly, the collected cells were lysed with a BugBuster Protein Extraction Reagent (Merck, Germany). Subsequently, the bacterial lysate was centrifuged at 16000 g for 20 min, and the supernatant was collected, and the precipitate was discarded.
  • the expressed IspH protein contains six histidine tags at the amino terminus (the histidine tag is encoded by the sequence carried by the pET28a vector itself and is located at the N-terminus of the protein expressed by the recombinant vector), therefore, according to the manufacturer
  • the IspH protein in the supernatant was purified using a Ni-NTA column (Merck, Germany) and a desalting column (Sangon Biotech, China). The purified IspH protein was shown to be brown and stored at -80 °C until use.
  • ferredoxin ferredoxin
  • ferredoxin-NADP + reductase ferredoxin-NADP + -reductase, FNR
  • DNA fragments encoding ferredoxin and ferredoxin-NADP + reductase were separately cloned into vector pET28a and transformed into E. coli for expression.
  • E. coli was lysed, and the protein expressed by E. coli was purified using a Ni-NTA column (Merck, Germany) and a desalting column (Sangon Biotech, China).
  • the purified ferredoxin and ferredoxin-NADP + reductase were separately stored at -80 ° C and used.
  • the purified protein was detected using SDS-PAGE.
  • the results are shown in Figure 1.
  • the results show that the IspH protein of the present invention is highly expressed in E. coli, has a molecular weight of about 35 kD, and has high purity after the above purification step, as shown by a single electrophoresis band in lane 2 of Figure 1.
  • the results of Figure 1 also show that high purity ferredoxin (having a molecular weight of about 11 kD, as shown by the single electrophoresis band in lane 3 of Figure 1) and ferredoxin-NADP + were obtained by the above method .
  • Reductase mo weight about 36 kD, as shown by the single electrophoresis band in lane 1 of Figure 1).
  • H131N and E133Q Two mutants of IspH protein, H131N and E133Q, were prepared by site-directed mutagenesis, wherein the mutant H131N (the sequence of which is shown in SEQ ID NO: 3) differs from the IspH protein in that it is located in SEQ ID NO: 2
  • the histidine at position 131 was mutated to asparagine; the mutant E133Q (the sequence of which is shown in SEQ ID NO: 4) and the region of the IspH protein
  • the glutamic acid at position 133 of SEQ ID NO: 2 was mutated to glutamine.
  • H131N-F CTACATTGGTAAGAAAGGAaACCCCGAGCCAGAAGGCG (SEQ I D NO: 7),
  • H131N-R CGCCTTCTGGCTCGGGGTtTCCTTTCTTACCAATGTAG (SEQ ID NO: 8);
  • E133Q-F GTAAGAAAGGACACCCCcAGCCAGAAGGCGCTATC (SEQ ID NO: 9),
  • E133Q-R GATAGCGCCTTCTGGCTgGGGGTGTCCTTTCTTAC (SEQ ID NO: 10).
  • the amplified product was treated with DpnI enzyme at 37 ° C for 2 h and then transformed into E. coli DMT strain (Beijing Quanjin Biotechnology Co., Ltd.).
  • E. coli DMT strain was cultured in LB medium, and a plasmid was extracted therefrom, and the plasmid was subjected to sequencing verification. Subsequently, the plasmid with the correct sequence was transformed into E. coli which highly expressed the isc operon to express the mutant protein.
  • the method of expression and purification of the mutant protein is as described in Example 2.
  • mutant proteins H131N and E133Q were detected using SDS-PAGE. The results are shown in Figure 3. The results showed that the mutant proteins H131N and E133Q of the present invention can be highly expressed in E. coli, have a molecular weight of about 35 kD, and have high purity after the above purification step.
  • the activity of IspH protein and its mutant was detected under anaerobic conditions in a sealed 5 ml chromatography tube.
  • the reaction system contained: 150 mM NaCl, 20 mM Tris-HCl (pH 7.8), 2 mM DTT, 1 mM NADPH, 1 mM HMBPP, 20 ⁇ M ferredoxin, 5 ⁇ M ferredoxin-NADP + reductase, and 0.5 ⁇ M of IspH protein obtained above or mutants thereof (H131N and E133Q); total reaction volume is 500 ⁇ l; reaction conditions are at 37 ° C Incubate for 1 h.
  • GC-MS gas chromatography-mass spectrometry
  • the reaction solution in the chromatographic tube was also analyzed after the reaction to determine whether IPP and DMAPP were produced. Since DMAPP can spontaneously form isoprene under acidic conditions, the amount of DMAPP in the reaction solution can be determined by measuring the amount of isoprene produced in the reaction solution under acidic conditions. Briefly, under anaerobic conditions, take 50 ⁇ L of the reaction solution in another 5 ml chromatography tube, dilute to 500 ⁇ L with cold water, and add an equal volume of 8 M H 2 SO 4 ; then, immediately seal the chromatographic tube and place it in Incubate at 37 ° C for 30 min. After the incubation, the amount of isoprene in the headspace gas was determined by the GC-MS method as described above to determine whether DMAPP was present in the reaction solution.
  • the experimental results are shown in Table 2.
  • the results showed that wild-type IspH protein and its mutants (H131N and E133Q) were able to catalyze the conversion of HMBPP to isoprene, and the catalytic performance of the three was not significantly different.
  • the results also showed that in the reaction system containing the wild-type IspH protein, isopentenyl (3-methyl-1-butene and 2-methyl-2-butene) was formed; and the mutant protein was contained. In the reaction system of (H131N and E133Q), the formation of isopentene was not detected.
  • the results also showed that the reaction solution containing the wild-type IspH protein produced isoprene under acidic conditions (this indicates the presence of DMAPP in the reaction solution); conversely, the reaction solution containing the mutant proteins (H131N and E133Q) was There is substantially no isoprene formation under acidic conditions (this indicates that DMAPP is substantially absent in the reaction solution).
  • Table 2 Quantitative analysis of the headspace of the reaction system and the headspace of the reaction solution after acid treatment
  • mutant proteins H131N and E133Q are only capable of converting HMBPP to isoprene, and basic Isopentene, IPP and DMAPP could not be produced.
  • the activity of IspH protein and its mutant was detected under anaerobic conditions in a sealed 5 ml chromatography tube.
  • the reaction system contained: 150 mM NaCl, 20 mM Tris-HCl (pH 7.8), 2 mM DTT, 1 mM NADPH, 1 mM DMAPP, 20 ⁇ M ferredoxin, 5 ⁇ M ferredoxin-NADP + reductase, and 0.5 ⁇ M of IspH protein obtained above or mutants thereof (H131N and E133Q); total reaction volume is 500 ⁇ l; reaction conditions are at 37 ° C Incubate for 1 h.
  • the wild-type IspH protein of the present invention has three catalytic activities (Fig. 5): (a) DMAPP and IPP are produced by using HMBPP as a substrate; (b) isoprene is produced by using HMBPP as a substrate. ; and (c) to form isoamylene (2-methyl-2-butene and 3-methyl-1-butene) as a DMAPP substrate.
  • the mutant proteins H131N and E133Q retained only the ability to produce isoprene with HMBPP as a substrate, while essentially losing other activities (ie, substantially unable to convert HMBPP to IPP and DMAPP, nor DMAPP was converted to 2-methyl-2-butene and 3-methyl-1-butene).
  • the B. alkalophilus N16-5 strain from which the IspH protein of the present invention is derived is selected as a host cell in which IspH protein and its mutants (H131N and E133Q) are overexpressed, and the gas generated by the host cell is detected, The intracellular activity of the IspH protein and its mutants was determined.
  • a DNA fragment encoding an IspH protein or a mutant thereof was fused to pLDH (the promoter of lactate dehydrogenase) and cloned into the pMK4 vector to construct an expression vector; subsequently, through the protoplast Transformation method
  • the constructed expression vector was transferred into B. alkalophilus N16-5 to construct a recombinant host cell.
  • Three recombinant cells were constructed by the above method: recombinant cell WNH, which is Bacillus alkalophilus N16-5 overexpressing wild-type IspH protein; recombinant cell H131N, which is an A. albicans N16- overexpressing mutant H131N 5; and recombinant cell E133Q, which is a Bacillus alkalophilus N16-5 overexpressing the expression mutant E133Q.
  • the constructed recombinant cells were inoculated into a sealed culture flask and incubated at 37 ° C for 12 h. Subsequently, 1 mL of the headspace gas in the flask was taken for GC-MS analysis. The detailed method of GC-MS analysis is as described above. In addition, in this experiment, wild type B. alkalophilus N16-5 (WT) was used as a control.
  • WT wild type B. alkalophilus N16-5
  • Figure 6 shows the cultured wild type alkalophilic GC profile of the headspace of Bacillus sp. N16-5 (WT) and recombinant cell WNH (pMH), wherein 3-methyl-1-butene (3M1B), 2-methyl-2-butene (2M2B)
  • WT Bacillus sp. N16-5
  • pMH recombinant cell WNH
  • 3M1B 3-methyl-1-butene
  • 2M2B 2-methyl-2-butene
  • aeruginosa N16-5 was able to produce isoprene, as well as trace amounts of 2-methyl-2-butene and 3-methyl-1-butene; and overexpressing wild-type IspH protein
  • the yield of 2-methyl-2-butene and 3-methyl-1-butene of the recombinant cell WNH was significantly increased, but the production of isoprene was only slightly increased.
  • the results also showed that the production of isopentenes in the recombinant cells H131N and E133Q did not change significantly compared to the wild type B. alkalophilus N16-5 (Table 3).
  • Figure 7 shows a comparison of isoprene production of wild type B. alkalophilus N16-5 (WT), recombinant cells WNH, H131N and E133Q.
  • WT alkalophilus N16-5
  • the results showed that the isoprene production of recombinant cell WNH was slightly increased compared with wild-type B. alkalophilus N16-5, but the isoprene production of recombinant cells H131N and E133Q was significantly increased, respectively, about wild type. 3 and 4 times the strain.
  • Table 3 shows a comparison of isopenten production and isoprene production of wild type B. alkalophilus N16-5 (WT), recombinant cells WNH, H131N and E133Q.
  • WT alkalophilus N16-5
  • the results showed that compared with wild-type B. alkalophilus N16-5, the production of isopentenes in recombinant cells was significantly increased, while the production of isoprene was slightly increased; the production of isoprene in recombinant cells H131N and E133Q was significantly improved. (3 and 4 times, respectively, of the wild-type strain), but the production of isoamene did not change significantly.
  • B. alkalophilus N16-5 is capable of expressing electron mediators, ferredoxin and flavin redox protein.
  • ⁇ fer It lacks the fer gene encoding ferredoxin; and, ⁇ fld, which lacks the fld gene encoding flavin redox protein.
  • ⁇ fld which lacks the fld gene encoding flavin redox protein.
  • Figure 8a shows the growth of wild-type strains and mutant strains ⁇ fer and ⁇ fld of B. alkalophilus N16-5. The results showed that all three strains were able to grow normally in Horikoshi-I medium, and there was no significant difference in growth.
  • Figure 8b shows a comparison of the isoprene production of the wild type strain of B. alkalophilus N16-5 and the mutant strains ⁇ fer and ⁇ fld. The results showed that the isoprene production of the mutant strain ⁇ fer was significantly lower than that of the wild type strain (only about 50% of the wild type strain); while the isoprene production of the mutant strain ⁇ fld was slightly lower than that of the wild type strain.
  • E. coli was also used to detect the intracellular activity of IspH proteins and their mutants (H131N and E133Q).
  • a DNA fragment encoding IspH protein and ferredoxin was cloned into the pSTV28 vector to construct the expression vector pSTV165HF.
  • the constructed expression vector was transformed into Escherichia coli Trans109 strain (TrahsGen Biotech) to construct recombinant cell STV165HF, which is an Escherichia coli overexpressing IspH protein and ferredoxin.
  • a recombinant cell STV165HF-H131N which is an Escherichia coli overexpressing the mutant protein H131N and ferredoxin, was constructed using a similar method; and recombinant cell STV165HF-E133Q, which is an overexpressed mutant protein E133Q and iron oxide Reduced protein of E. coli.
  • the constructed recombinant cells were inoculated into a sealed culture flask and incubated at 37 ° C for 10 h. Subsequently, 1 mL of the headspace gas in the flask was taken for GC-MS analysis. The detailed method of GC-MS analysis is as described above.
  • Figure 9 shows the GC profile of the supernatant air of cultured recombinant E. coli cells STV165HF (Fig. 9A), and the standard (3-methyl-1-butene, 2-methyl-2-butene, 2 Gc characteristic spectrum of -methyl-1-butene and isoprene (Fig. 9A).
  • peak a is a characteristic peak of 3-methyl-1-butene
  • peak b is a characteristic peak of 2-methyl-2-butene
  • peak c is a characteristic peak of 2-methyl-1-butene
  • peak d is a characteristic peak of isoprene.
  • Figure 10 shows the peak 1 component of Figure 9A and the standard 3-methyl-1-butene (Figure 10A), the peak 2 component and the standard 2-methyl-2-butene (Figure 10B), peak Comparison of the mass spectrometry results of the 3 component with the standard isoprene (Fig. 10C), wherein, in Figs. 10A-10C, the graph above the abscissa axis shows the components to be tested (peak 1, peak 2, peak) 3) Mass spectrometry results, and the graph below the abscissa axis shows the mass spectrometry results of the standards (3-methyl-1-butene, 2-methyl-2-butene, isoprene).
  • Figure 11 shows the GC profile of the headspace of cultured recombinant E. coli cells STV165HF-H131N (Figure 11A) and STV165HF-E133Q (Figure 11B), wherein the GC detection conditions used were: inlet temperature 180 °C, column temperature 130 ° C, detector temperature 200 ° C. Further, the components in the respective peaks of Figures 11A-11B were identified by mass spectrometry. The results show that the headspace air measured in Figures 11A-11B contains isoprene but does not contain isopentene. Therefore, these experimental results indicate that recombinant E. coli cells overexpressing the mutant protein H131N or E133Q are capable of producing a significant amount of isoprene, and neither produces isopentene.
  • the IspH protein of the present invention and mutants thereof can be used to construct recombinant engineered bacteria capable of biosynthesizing isoprene. Furthermore, the IspH protein of the present invention can also be used to construct recombinant engineered bacteria capable of biosynthesizing isoamylene (2-methyl-2-butene and 3-methyl-1-butene).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供一种能够以4－羟基－3－甲基－2－丁烯基－焦磷酸（HMBPP）为底物产生异戊二烯的多肽、编码该多肽的核酸、含有该核酸的载体和细胞。还提供使用该多肽来生产异戊二烯的方法，以及制备该多肽的方法。

Description

一种用于生物合成异戊二烯和异戊烯的酶及其突变体 技术领域
本发明涉及分子生物学技术领域，特别是异戊二烯的生物合成领域。具体而言，本发明提供了一种能够以4-羟基-3-甲基-2-丁烯基-焦磷酸(HMBPP)为底物产生异戊二烯的多肽，编码所述多肽的核酸，含有所述核酸的载体和细胞。此外，本发明还提供了使用所述多肽来生产异戊二烯的方法，以及制备所述多肽的方法。
背景技术
异戊二烯是一种重要的化工原料，其主要用于生产橡胶，并且还可用于精细化学品(例如异戊烯氯、芳樟醇、月桂烯以及维生素A、E、K等)的合成。目前，工业应用的异戊二烯及异戊烯几乎全部来源于石油衍生原料。然而，随着工业上对异戊二烯需求的日益增加以及世界石油资源的逐渐枯竭，迫切需要一种可用于生产异戊二烯的替代来源。在此背景下，异戊二烯的生物合成法由于其无污染、可再生等优点引起人们的广泛关注及研究，并且也取得了初步的进展。在2010年，美国杰能科公司及固特异公司合作开发出了一种生物合成异戊二烯的方法，其核心就是，构建工程菌，并利用该工程菌把可再生的糖类转化为异戊二烯；并且，该方法的异戊二烯产量达到了60g/L。
目前的研究表明，生物主要是通过下述途径来合成异戊二烯：先通过代谢途径合成异戊二烯的前体二甲烯丙基焦磷酸(DMAPP)，然后在异戊二烯合酶(IspS；EC 4.2.3.27)的催化下，将DMAPP转化为异戊二烯。DMAPP及其同分异构体异戊烯基焦磷酸(IPP)是目前发现的所有异戊二烯类化合物的前体，并且它们在生物体内可通过两种不同的途径来合成：DXP途径和MVA途径。DXP途径包括，首先把丙酮酸和甘油醛-3-磷酸缩合为1-脱氧-D-木酮糖5-磷酸(DXP)；随后通过5个酶反应将DXP转化为4-羟基-3-甲基-2-丁烯基-焦磷酸(HMBPP)；然后在HMBPP还原酶(IspH)的作用下，将HMBPP转化为DMAPP和IPP。MVA途径则以 乙酰辅酶A为单一原料，并且通过连续的7个酶反应来生成DMAPP。有关异戊二烯的生物合成(特别是DXP途径和MVA途径)的详细介绍还参见例如Tomohisa Kuzuyama，Biosci.Biotechnol.Biochem.，66(8)，1619-1627，2002；Thomas D.Sharkey et al.，Plant Physiology，February 2005，Vol.137，pp.700-712；和US8507235B2。
通常认为，异戊二烯合酶(IspS；EC4.2.3.27)是生物合成异戊二烯最后一步所需要的酶。目前，已分离并鉴定了一种植物来源的IspS。该酶以DMAPP为底物，通过催化消除底物的焦磷酸基团来生成异戊二烯。尽管很多植物中都存在IspS，但它们的同源性都很高，且Km值都比较大，达到mM级。此外，研究还发现，IspS的底物(DMAPP及IPP)在细胞中的高浓度积累对工程菌的生长具有毒性作用。因此，使用IspS来生物合成异戊二烯的方法仍存在着不足。
因此，本领域需要开发新的生物合成异戊二烯的方法。
发明内容
本发明人在嗜碱芽孢杆菌N16-5(Bacillus sp.N16-5)中发现了一种新的酶，其具有多种活性，包括：(a)以HMBPP为底物生成DMAPP和IPP；(b)以HMBPP为底物生成异戊二烯；和，(c)以DMAPP底物生成异戊烯(2-甲基-2-丁烯和3-甲基-1-丁烯)。进一步，本发明人对该酶进行了改造，获得了两种突变体(H131N和E133Q)。这两种突变体丧失了以HMBPP为底物生成DMAPP和IPP的活性以及生成异戊烯的活性，但均保留了以HMBPP为底物生成异戊二烯的活性，并且其在细胞内生产异戊二烯的能力强于野生型酶(异戊二烯的产量提高了3-4倍)。基于此，本发明人开发了新的合成异戊二烯和异戊烯的方法。
因此，在一个方面，本发明提供了一种多肽，其具有以4-羟基-3-甲基-2-丁烯基-焦磷酸(HMBPP)为底物产生异戊二烯的活性，并且其具有选自下列的氨基酸序列：
(1)如SEQ ID NO：2所示的氨基酸序列；
(2)与SEQ ID NO：2具有至少90％同一性的氨基酸序列；和
(3)与SEQ ID NO：2相异在于一个或几个氨基酸残基的置换、缺失或添加的氨基酸序列。
在某些优选的实施方案中，本发明多肽的氨基酸序列与SEQ ID NO：2具有至少90％同一性，优选至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在某些优选的实施方案中，本发明多肽的氨基酸序列与SEQ ID NO：2相异在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的置换、缺失或添加。
在某些优选的实施方案中，本发明多肽的氨基酸序列与SEQ ID NO：2相异在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的保守置换。
在某些优选的实施方案中，本发明多肽的氨基酸序列为SEQ ID NO：2或者与SEQ ID NO：2相异在于一个或几个(例如1个)氨基酸残基的置换。例如，在某些优选的实施方案中，本发明多肽的氨基酸序列与SEQ ID NO：2相异在于，位于SEQ ID NO：2的第131位或第133位的氨基酸置换。在某些优选的实施方案中，本发明多肽的氨基酸序列与SEQ ID NO：2相异在于：位于SEQ ID NO：2的第131位的组氨酸被突变为天冬酰胺；或，位于SEQ ID NO：2的第133位的谷氨酸被突变为谷氨酰胺。
在某些优选的实施方案中，本发明多肽具有选自SEQ ID NO：2、3和4的氨基酸序列。
在某些优选的实施方案中，本发明的多肽还具有以二甲烯丙基焦磷酸(DMAPP)为底物产生2-甲基-2-丁烯和3-甲基-1-丁烯的活性。
在某些优选的实施方案中，本发明的多肽不具有以二甲烯丙基焦磷酸(DMAPP)为底物产生2-甲基-2-丁烯和3-甲基-1-丁烯的活性。
在另一个方面，本发明提供了一种分离的核酸，其编码如上所述 的多肽。在另一个方面，本发明提供了一种载体，其包含所述分离的核酸。在某些优选的实施方案中，本发明的分离的核酸编码具有选自SEQ ID NO：2、3和4的氨基酸序列的多肽。可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个实施方案中，载体是例如质粒，粘粒，噬菌体等等。
在另一个方面，本发明还涉及包含上述分离的核酸或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞和芽孢杆菌细胞(例如嗜碱性芽孢杆菌，枯草芽孢杆菌)，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞。
在某些优选的实施方案中，所述分离的核酸相对于所述细胞而言是异源的。在某些优选的实施方案中，所述分离的核酸相对于所述细胞而言是外源的。
在某些优选的实施方案中，所述细胞还包含编码电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)的核酸或者表达电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)。例如，所述细胞可以还包含编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸，或者表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在某些优选的实施方案中，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是内源的。在某些优选的实施方案中，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是外源的。例如，所述铁氧化还原蛋白和/或铁氧化还原蛋白还原酶相对于所述细胞而言是内源的或外源的。在某些优选的实施方案中，所述细胞还包含外源导入的编码铁氧化还原蛋白的核酸，和/或，外源导入的编码铁氧化还原蛋白还原酶的核酸。在某些优选的实施方案中，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶(EC 1.18.1.2)。
在某些优选的实施方案中，所述细胞还表达DXP途径的多肽。优 选地，所述DXP途径的多肽选自，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG；EC 1.17.7.1)，以及其任何组合。在某些优选的实施方案中，所述DXP途径的多肽相对于所述细胞而言是内源的。在某些优选的实施方案中，所述DXP途径的多肽相对于所述细胞而言是外源的。
在某些优选的实施方案中，所述细胞还表达异戊二烯合酶(IspS；EC 4.2.3.27)。在某些优选的实施方案中，所述细胞不表达异戊二烯合酶(IspS；EC 4.2.3.27)。
在某些优选的实施方案中，所述细胞为原核细胞，例如大肠杆菌或芽孢杆菌或蓝藻。
在另一个方面，本发明还涉及一种组合物，其包含本发明的多肽、HMBPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)，以及电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)。在某些优选的实施方案中，所述组合物包含所述多肽、HMBPP、NADPH、铁氧化还原蛋白和铁氧化还原蛋白还原酶(例如铁氧化还原蛋白-NADP+还原酶)。本发明的此类组合物可用于在体外合成异戊二烯。
在另一个方面，本发明还涉及一种组合物，其包含具有SEQ ID NO：2所示的氨基酸序列的多肽、DMAPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)，以及电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)。在某些优选的实施方案中，所述组合物包含具有SEQ ID NO：2所示的氨基酸序列的多肽、DMAPP、NADPH、铁氧化还原蛋白和铁氧化还原蛋白还原酶(例如铁氧化还原蛋白-NADP+还原酶)。本发明的此类组合物可用于在体外合成异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)。
在另一个方面，本发明提供了一种生产异戊二烯的方法，其包括，使用本发明的多肽，将HMBPP转化为异戊二烯。
在某些优选的实施方案中，所述方法包括，(a)将所述多肽、HMBPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)，以及电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)混合并温育(例如在20-40℃进行温育，例如在室温或37℃进行温育)；和，(b)收集步骤(a)产生的异戊二烯。在某些优选的实施方案中，在步骤(a)中，将所述多肽、HMBPP、NADPH、铁氧化还原蛋白和铁氧化还原蛋白还原酶(例如铁氧化还原蛋白-NADP+还原酶)混合并温育，例如在20-40℃进行温育(例如在室温或37℃进行温育)，以产生异戊二烯。在某些优选的实施方案中，所述多肽具有SEQ ID NO：3或4所示的氨基酸序列。
在某些优选的实施方案中，所述方法不涉及异戊二烯合酶(IspS；EC 4.2.3.27)的使用。在某些优选的实施方案中，所述方法用于体外生产异戊二烯。
在另一个方面，本发明提供了一种生产异戊二烯的方法，其包括，(a)培养细胞，所述细胞表达外源导入的本发明的多肽；和，(b)收集步骤(a)产生的异戊二烯。
在某些优选的实施方案中，在步骤(a)中，在适合产生异戊二烯的条件下培养细胞。例如，为了促进细胞产生异戊二烯，可以给细胞提供下列中的任一项或多项：(1)维持或促进细胞生长的培养基；(2)本发明多肽的底物，HMBPP；(3)电子传递体(例如铁氧化还原蛋白)；(4)电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)；和(5)NADPH或NADH。
用于培养细胞的各种合适的培养基是本领域技术人员公知的，并且是市售可得的。
在某些优选的实施方案中，所述细胞还表达电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋 白还原酶)。例如，所述细胞还表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在某些优选的实施方案中，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是内源的。在某些优选的实施方案中，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是外源的。例如，所述铁氧化还原蛋白和/或铁氧化还原蛋白还原酶相对于所述细胞而言可以是内源的或外源的。在某些优选的实施方案中，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶(EC 1.18.1.2)。
在某些情况下，所述细胞天然表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在此类细胞中，可以无需外源导入编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸。然而，特别优选地，在此类细胞中，进一步导入编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸，以提高铁氧化还原蛋白和铁氧化还原蛋白还原酶的表达，从而进一步提高本发明多肽的活性。在某些情况下，所述细胞不表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在此类细胞中，特别优选地，导入编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸，以为本发明多肽提供高效的电子供体。
在某些优选的实施方案中，可以在细胞培养基中添加HMBPP，以提供本发明多肽所需的底物。在某些优选的实施方案中，通过在细胞中建立DXP途径来促使细胞合成HMBPP，并由此提供本发明多肽所需的底物。例如，可通过在细胞中表达DXP途径的一种或多种多肽来促使细胞合成HMBPP，从而提供本发明多肽所需的底物。
因此，在某些优选的实施方案中，所述细胞还表达DXP途径的多肽。在某些优选的实施方案中，所述DXP途径的多肽选自，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG； EC 1.17.7.1)，以及其任何组合。在某些优选的实施方案中，所述细胞表达上述DXP途径多肽中的1种，2种，3种，4种，5种或6种。
在某些优选的实施方案中，所述DXP途径的多肽相对于所述细胞而言是内源的。在某些优选的实施方案中，所述DXP途径的多肽相对于所述细胞而言是外源的。
在某些优选的实施方案中，所述细胞还表达异戊二烯合酶(IspS；EC 4.2.3.27)。在此类细胞中，所述细胞可以通过多种途径来生物合成异戊二烯。在某些优选的实施方案中，所述细胞不表达异戊二烯合酶(IspS；EC 4.2.3.27)。
在某些优选的实施方案中，所述细胞选自，原核细胞例如大肠杆菌细胞和芽孢杆菌细胞(例如嗜碱性芽孢杆菌，枯草芽孢杆菌)，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞。然而，特别优选地，所述细胞为原核细胞，例如大肠杆菌或芽孢杆菌或蓝藻。
在某些优选的实施方案中，所述方法用于生物合成异戊二烯。
在另一个方面，本发明提供了一种生产异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)的方法，其包括，使用具有SEQ ID NO：2所示的氨基酸序列的多肽，将DMAPP转化为异戊烯。
在某些优选的实施方案中，所述方法包括，(a)将所述多肽、DMAPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)和电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)混合并温育；和，(b)收集步骤(a)产生的异戊烯。在某些优选的实施方案中，在步骤(a)中，将所述多肽、DMAPP、NADPH、铁氧化还原蛋白和铁氧化还原蛋白还原酶(例如铁氧化还原蛋白-NADP+还原酶)混合并温育，例如在20-40℃进行温育(例如在室温或37℃进行温育)，以产生异戊烯。在某些优选的实施方案中，所述方法用于体外生产异戊烯。
在另一个方面，本发明提供了一种生产异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)的方法，其包括，(a)培养细胞，所述细胞表 达外源导入的、具有SEQ ID NO：2所示的氨基酸序列的多肽；和，(b)收集步骤(a)产生的异戊烯。
在某些优选的实施方案中，在步骤(a)中，在适合产生异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)的条件下培养细胞。例如，为了促进细胞产生异戊烯，可以给细胞提供下列中的任一项或多项：(1)维持或促进细胞生长的培养基；(2)本发明多肽的底物，DMAPP；(3)电子传递体(例如铁氧化还原蛋白)；(4)电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)；和(5)NADPH或NADH。
用于培养细胞的各种合适的培养基是本领域技术人员公知的，并且是市售可得的。
在某些优选的实施方案中，所述细胞还表达电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)。例如，所述细胞还表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在某些优选的实施方案中，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是内源的。在某些优选的实施方案中，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是外源的。例如，所述铁氧化还原蛋白和/或铁氧化还原蛋白还原酶相对于所述细胞而言可以是内源的或外源的。在某些优选的实施方案中，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶(EC 1.18.1.2)。
在某些情况下，所述细胞天然表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在此类细胞中，可以无需外源导入编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸。然而，特别优选地，在此类细胞中，进一步导入编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸，以提高铁氧化还原蛋白和铁氧化还原蛋白还原酶的表达，从而进一步提高本发明多肽的活性。在某些情况下，所述细胞不表达铁氧化还原蛋白和铁氧化还原蛋白还原酶。在此类细胞中，特别优选地，导入编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸，以为本发明多肽提供高效的电子供体。
在某些优选的实施方案中，可以在细胞培养基中添加DMAPP，以提供本发明多肽所需的底物。在某些优选的实施方案中，通过在细胞中建立DXP途径来促使细胞合成DMAPP，并由此提供本发明多肽所需的底物。例如，可通过在细胞中表达DXP途径的一种或多种多肽来促使细胞合成DMAPP，从而提供本发明多肽所需的底物。
因此，在某些优选的实施方案中，所述细胞还表达DXP途径的多肽。在某些优选的实施方案中，所述DXP途径的多肽选自，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG；EC 1.17.7.1)，以及其任何组合。在某些优选的实施方案中，所述细胞表达上述DXP途径多肽中的1种，2种，3种，4种，5种或6种。
在某些优选的实施方案中，所述DXP途径的多肽相对于所述细胞而言是内源的。在某些优选的实施方案中，所述DXP途径的多肽相对于所述细胞而言是外源的。
在某些优选的实施方案中，所述细胞还表达异戊烯基二磷酸异构酶(IDI；EC 5.3.3.2)。在某些优选的实施方案中，所述异戊烯基二磷酸异构酶相对于所述细胞而言是内源的。在某些优选的实施方案中，所述异戊烯基二磷酸异构酶相对于所述细胞而言是外源的。
在某些优选的实施方案中，所述细胞选自，原核细胞例如大肠杆菌细胞和芽孢杆菌细胞(例如嗜碱性芽孢杆菌，枯草芽孢杆菌)，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞。然而，特别优选地，所述细胞为原核细胞，例如大肠杆菌或芽孢杆菌或蓝藻。
在某些优选的实施方案中，所述方法用于生物合成异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)。
在另一个方面，本发明提供了一种制备本发明多肽的方法，其包 括，(a)培养宿主细胞，所述宿主细胞包含并表达编码所述多肽的核酸；和，(b)收集所述细胞表达的所述多肽。
用于蛋白表达的各种宿主细胞是本领域技术人员公知的，其包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。特别优选地，所述宿主细胞为大肠杆菌。
相关术语的定义和解释
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的相关实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“HMBPP”是指4-羟基-3-甲基-2-丁烯基-焦磷酸，其结构式如下面的式I所示：
Figure PCTCN2016073413-appb-000001
如本文中所使用的，术语“DMAPP”是指二甲烯丙基焦磷酸，其结构式如下面的式II所示：
Figure PCTCN2016073413-appb-000002
如本文中所使用的，术语“2M2B”是指2-甲基-2-丁烯，其结构式为
Figure PCTCN2016073413-appb-000003
如本文中所使用的，术语“3M1B”是指3-甲基-1-丁烯，其结构式为
Figure PCTCN2016073413-appb-000004
如本文中所使用的，术语“异戊二烯” 是指2-甲基-1，3-丁二烯，其结构式为
Figure PCTCN2016073413-appb-000005
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar，Inc)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4：11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在WWW.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能 力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，保守置换通常是指，用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32：1180-1187(1993)；Kobayashi等人Protein Eng.12(10)：879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94：412-417(1997)，其通过引用并入本文)。
在本申请中，已证实如SEQ ID NO：2所示的IspH蛋白能够将HMBPP转化为异戊二烯。与此同时，对已知多肽进行突变以获得其变体的方法已在现有技术中进行了详细描述，并且示例性地描述于本申请中。例如，本说明书的实施例提供了制备所述IspH蛋白的突变体的方法，并制备获得了2种突变体(H131N和E133Q)。另外，测定某一多肽是否具有将HMBPP转化为异戊二烯的活性的方法是本领域技术人员已知的，并且也详细描述于本申请中。例如，本说明书的实施例提供了测试H131N和E133Q是否能够以HMBPP为底物生成异戊二烯的方法，并且已证实，H131N和E133Q均具有将HMBPP转化为异戊二烯的活性。因此，通过重复本发明的这些方法，可以制备所述IspH蛋白的具有将HMBPP转化为异戊二烯的活性的其他突变体。因此，本发明的多肽不限于如SEQ ID NO：2所示的IspH蛋白以及其突变体H131N和E133Q，并且意欲涵盖IspH蛋白的所有其他突变体，只要此类突变体仍然保留将HMBPP转化为异戊二烯的活性。
如本文中使用的，表述“核酸/多肽相对于细胞是异源的”意指，所述核酸/多肽不天然存在于所述细胞中。也即，所述细胞在天然状态 下不包含或表达所述核酸/多肽。
如本文中使用的，表述“核酸/多肽相对于细胞是内源的”意指，所述核酸/多肽天然存在于所述细胞中。也即，所述细胞在天然状态下包含或表达所述核酸/多肽。
如本文中使用的，表述“核酸/多肽相对于细胞是外源的”意指，所述核酸/多肽是通过人工方式外源导入所述细胞中。应当理解，此类核酸/多肽可以不天然存在于所述细胞中(即，相对于所述细胞是异源的)，其用于在细胞中引入异源的核酸/多肽；或者，也可以与天然存在于所述细胞中的内源核酸/多肽相同，其用于增加所述细胞中的内源核酸/多肽的拷贝数或表达。
如本文中使用的，术语“电子传递体”是指，在电子传递链中参与电子传递的蛋白。通常，电子传递体既能够作为电子受体接受电子，又能够作为电子供体提供电子，从而能够实现电子的传递。此类电子传递体是本领域技术人员公知的，并且包括但不限于，铁氧化还原蛋白和黄素氧化还原蛋白。此外，在电子传递体传递电子的过程中，通常需要氧化还原酶(其在本文中被称为，电子传递体传递电子所需的酶)的参与。例如，铁氧化还原蛋白的电子传递通常需要铁氧化还原蛋白还原酶的参与；黄素氧化还原蛋白的电子传递通常需要黄素氧化还原蛋白还原酶的参与。电子传递体传递电子所需的酶也是本领域技术人员公知的，例如但不限于铁氧化还原蛋白还原酶和黄素氧化还原蛋白还原酶。
如本文中使用的，术语“铁氧化还原蛋白还原酶”是指能够催化铁氧化还原蛋白的氧化还原反应的酶，其包括两种类型：
(1)铁氧化还原蛋白-NADP+还原酶，其IntEnz登录号为EC 1.18.1.2，并且能够催化下述反应：
2还原型铁氧化还原蛋白+NADP++H+
Figure PCTCN2016073413-appb-000006
2氧化型铁氧化还原蛋白+NADPH；和
(2)铁氧化还原蛋白-NAD+还原酶，其IntEnz登录号为EC 1.18.1.3，并且能够催化下述反应：
2还原型铁氧化还原蛋白+NAD++H+
Figure PCTCN2016073413-appb-000007
2氧化型铁氧化还原蛋白+NADH。
在本发明中，铁氧化还原蛋白还原酶优选为铁氧化还原蛋白-NADP+还原酶(EC 1.18.1.2)。
如本文中使用的，术语“DXP途径的多肽”是指，参与DXP途径的多肽。DXP途径是指，生物体中以丙酮酸和甘油醛-3-磷酸为原料合成DMAPP的途径，其包括下述步骤：(1)将丙酮酸和甘油醛-3-磷酸缩合为1-脱氧-D-木酮糖5-磷酸(DXP)；(2)将DXP转化为2-C-甲基-D-赤藓醇4-磷酸(MEP)；(3)将MEP转化为4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇(CDP-ME)；(4)将CDP-ME转化为2-磷酸-4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇(CDP-ME2P)；(5)将CDP-ME2P转化为2-C-甲基-D-赤藓醇2，4-环二磷酸(MECDP)；(6)将MECDP转化为HMBPP；和(7)将HMBPP转化为DMAPP和IPP。关于DXP途径的详细描述可参见例如，Tomohisa Kuzuyama，Biosci.Biotechnol.Biochem.，66(8)，1619-1627，2002；Thomas D.Sharkey et al.，Plant Physiology，February 2005，Vol.137，pp.700-712；和US8507235B2。
参与DXP途径的多肽包括但不限于，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG；EC 1.17.7.1)。关于这些多肽(酶)的详细描述可参见公共数据库IntEnz(http：//www.ebi.ac.uk/intenz/)。
如本文中使用的，术语“异戊二烯合酶(IspS)”是指，能够以DMAPP为底物产生异戊二烯的酶，其IntEnz登录号为EC 4.2.3.27。
如本文中使用的，术语“异戊烯基二磷酸异构酶(IDI)”是指，能够催化DMAPP和IPP之间的异构反应的酶，其也称为IPP异构酶，IntEnz登录号为EC 5.3.3.2。
发明的有益效果
本发明的多肽(酶)能够以HMBPP为底物直接生成异戊二烯。与现有技术中公开的基于MEP途径生产异戊二烯的方法和酶相比，本发明的技术方案具有以下有益效果：
(1)本发明的生产异戊二烯的方法无需DMAPP的参与，能够避免对宿主细胞的生长具有毒性的DMAPP和IPP在宿主细胞内的积累；
(2)本发明的生产异戊二烯的方法减少了一步酶促反应，无需异戊二烯合酶(IspS)的参与，简化了流程，提高了效率。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例2中表达和纯化的蛋白的SDS-PAGE检测结果，其中，泳道M：分子量标记；泳道1：铁氧化还原蛋白-NADP+还原酶；泳道2：IspH蛋白；泳道3：铁氧化还原蛋白。结果显示，本发明的IspH蛋白能够在大肠杆菌中高表达，其分子量为约35kD，并且在纯化后具有高纯度，如图1泳道2中的单一电泳条带所示。此外，图1的结果还显示，通过实施例2的方法获得了高纯度的铁氧化还原蛋白(分子量为约11kD，如图1泳道3中的单一电泳条带所示)和铁氧化还原蛋白-NADp+还原酶(分子量为约36kD，如图1泳道1中的单一电泳条带所示)。
图2显示了经纯化的IspH蛋白的全波长广谱扫描结果，其中，横坐标表示波长，纵坐标表示吸光度。结果显示，本发明的IspH蛋白在410nm处有一最大吸收峰，在320nm处有一肩峰。这表明，该蛋白含 有铁硫中心。
图3显示了实施例3中表达和纯化的突变体蛋白H131N和E133Q的SDS-PAGE电泳检测结果，其中泳道M：分子量标记；泳道1：突变体蛋白H131N；泳道2：突变体蛋白E133Q。结果显示，本发明的突变体蛋白H131N和E133Q能够在大肠杆菌中高表达，其分子量均为约35kD，并且在纯化后具有高纯度。
图4显示了IspH蛋白将DMAPP转化为异戊烯的酶促反应的Michaelis-Menten曲线，其中，横坐标表示DMAPP的浓度(mM)，纵坐标表示反应速率(nmol min-1mg-1)。图4中的小图显示了所述酶促反应的Lineweaver-Burk曲线，其中，横坐标表示DMAPP的浓度的倒数，纵坐标表示反应速率的倒数。结果显示，野生型IspH蛋白以DMAPP为底物生成异戊烯的最大反应活性为约6.2nmol min-1mg-1，Km为约275μM。
图5为显示本发明IspH蛋白的三种活性的示意图，其中，Glucose：葡萄糖；MVA pathway：MVA途径；DXP pathway：DXP途径；HMBPP：4-羟基-3-甲基-2-丁烯基-焦磷酸；DMAPP：二甲烯丙基焦磷酸；IPP：异戊烯基焦磷酸；IDI：异戊烯基二磷酸异构酶；IspS：异戊二烯合酶；2M2B：2-甲基-2-丁烯；3M1B：3-甲基-1-丁烯。
图6显示了经培养的野生型嗜碱芽孢杆菌N16-5(WT)和重组细胞WNH(pMH)的顶空气体的GC特征谱，其中，3-甲基-1-丁烯(3M1B)，2-甲基-2-丁烯(2M2B)和异戊二烯的滞留时间分别为4.5min，5.5min，和11.3min。结果显示，野生型嗜碱芽孢杆菌N16-5能够产生异戊二烯，以及微量的2-甲基-2-丁烯和3-甲基-1-丁烯；并且，过表达IspH蛋白的重组细胞WNH的2-甲基-2-丁烯和3-甲基-1-丁烯的产量显著提高，但异戊二烯的产量仅略有提升。
图7显示了野生型嗜碱芽孢杆菌N16-5(WT)、重组细胞WNH、H131N和E133Q的异戊二烯产量的比较。结果显示，与野生型嗜碱芽孢杆菌N16-5相比，重组细胞WNH的异戊二烯产量略有提升，但重组细胞H131N和E133Q的异戊二烯产量均显著提高，分别约为野生型菌株的3倍和 4倍。
图8a显示了嗜碱芽孢杆菌N16-5的野生型菌株和突变体菌株Δfer和Δfld的生长情况。结果显示，这3种菌株均能够在Horikoshi-I培养基中正常生长，其生长情况无显著差异。图8b显示了嗜碱芽孢杆菌N16-5的野生型菌株和突变体菌株Δfer和Δfld的异戊二烯产量的比较。结果显示，突变体菌株Δfer的异戊二烯产量显著低于野生型菌株(仅为野生型菌株的约50％)；而突变体菌株Δfld的异戊二烯产量则略微低于野生型菌株。这些实验结果表明，铁氧化还原蛋白和黄素氧化还原蛋白均可用作IspH蛋白生产异戊二烯所需的电子供体(即，当缺失铁氧化还原蛋白时，黄素氧化还原蛋白可用作电子供体；反之亦然)；但是，铁氧化还原蛋白对于IspH蛋白的该催化活性而言是更优选、更高效的。
图9显示了经培养的重组大肠杆菌细胞STV165HF的顶空气体的GC特征谱(图9A)，以及标准品(3-甲基-1-丁烯，2-甲基-2-丁烯，2-甲基-1-丁烯和异戊二烯)的GC特征谱(图9B)，其中，所使用的GC检测条件为：进样口温度180℃，柱温100℃，检测器温度200℃；并且，峰a为3-甲基-1-丁烯的特征峰；峰b为2-甲基-2-丁烯的特征峰；峰c为2-甲基-1-丁烯的特征峰；峰d为异戊二烯的特征峰。
图10显示了图9A中峰1组分与标准品3-甲基-1-丁烯(图10A)、峰2组分与标准品2-甲基-2-丁烯(图10B)、峰3组分与标准品异戊二烯(图10C)的质谱分析结果的比较；其中，在图10A-10C中，横坐标轴上方的图显示了待测组分(峰1、峰2、峰3)的质谱分析结果，而横坐标轴下方的图显示了标准品(3-甲基-1-丁烯、2-甲基-2-丁烯、异戊二烯)的质谱分析结果。
图9-10的实验结果显示，过表达IspH蛋白的重组大肠杆菌细胞STV165HF能够产生显著量的异戊二烯(峰3)，2-甲基-2-丁烯(峰2)和3-甲基-1-丁烯(峰1)。
图11显示了经培养的重组大肠杆菌细胞STV165HF-H131N(图11A)和STV165HF-E133Q(图11B)的顶空气体的GC特征谱；其中，所使用 的GC检测条件为：进样口温度180℃，柱温130℃，检测器温度200℃；并且，经质谱分析确认，图11A-11B所测定的顶空气体包含异戊二烯，但不包含异戊烯。这些实验结果表明，过表达突变体蛋白H131N或E133Q的重组大肠杆菌细胞均能够产生显著量的异戊二烯，且均不产生异戊烯。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2016073413-appb-000008
序列1(SEQ ID NO：1)：
Figure PCTCN2016073413-appb-000009
Figure PCTCN2016073413-appb-000010
序列2(SEQ ID NO：2)：
Figure PCTCN2016073413-appb-000011
序列3(SEQ ID NO：3)：
Figure PCTCN2016073413-appb-000012
序列4(SEQ ID NO：4)：
Figure PCTCN2016073413-appb-000013
序列5(SEQ ID NO：5)：
Figure PCTCN2016073413-appb-000014
序列6(SEQ ID NO：6)：
Figure PCTCN2016073413-appb-000015
序列7(SEQ ID NO：7)：
Figure PCTCN2016073413-appb-000016
序列8(SEQ ID NO：8)：
Figure PCTCN2016073413-appb-000017
序列9(SEQ ID NO：9)：
Figure PCTCN2016073413-appb-000018
序列10(SEQ ID NO：10)：
Figure PCTCN2016073413-appb-000019
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；所使用的检测方法均是本领域的常规检测方法，其按照相关文献中记载的步骤或者按照所使用仪器的制造商推荐的步骤来进行。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.基因的克隆
将嗜碱芽孢杆菌N16-5(Bacillus sp.N16-5)(获自中国普通微生物菌种保藏管理中心，CGMCC)培养于含有2％NaCl的Horikoshi-I培养基(pH 10.0)(参见，Horikoshi K.，Microbiol Mol Biol Rev.1999；63：735-50)中。使用DNA提取试剂盒(E.Z.N.A.TM Bacterial DNA Isolation Kit，Omega，USA)，提取嗜碱芽孢杆菌N16-5的基因组DNA。以提取的基因组DNA为模板，使用下列引物进行PCR扩增：上游引物275F：5’-CATGCCATGGAGGTTTTGAAAATTTCC-3’(SEQ ID NO：5)，其中划线部分表示NcoI酶切位点；以及，下游引物275R：5’-CCGCTCGAGTGGCCCGCTCCGCTTTTTTCTTTC-3’(SEQ ID NO：6)，其中划线部分表示XhoI酶切位点。PCR反应体系为50μL，反应条件为：94℃预变性3min；30个循环的(94℃变性1min，58℃退火30s，72℃延伸 1.5min)；72℃延伸5min。
PCR产物用试剂盒(E.Z.N.A.TMBacterial DNA Isolation Kit，Omega，USA)进行纯化，并用NcoI和XhoI进行酶切，随后连接入经NcoI和XhoI酶切的载体pET28a(TaKaRa公司)中，并最终转化到大肠杆菌DH5α中。在LB培养基中培养经转化的大肠杆菌DH5α，从中提取质粒，并对质粒进行测序验证。测序结果显示，插入载体pET28a中的基因(IspH基因)具有如SEQ ID NO：1所示的序列，其编码的蛋白(IspH蛋白)具有如SEQ ID NO：2所示的氨基酸序列。将所构建的质粒命名为pET-H。
实施例2.多肽的表达与纯化
大肠杆菌的isc(iron-Sulfur cluster)操纵子所编码的蛋白有助于本发明IspH蛋白的[4Fe-4S]中心在蛋白质内的正确构建。因此，为了使表达的IspH蛋白具有高活性，首先在大肠杆菌中高表达isc操纵子。简言之，将isc操纵子连接到pSTV28载体中，并转化入大肠杆菌BL21(DE)，以增加该操纵子在宿主细胞中的拷贝数，提高该操纵子所编码的蛋白的表达(Grawert，T.et al.IspH protein of Escherichia coli：studies on iron-Sulfur cluster implementation and catalysis.Journal of the American Chemical Society126，12847-12855(2004))。随后，将如上构建的pET-H质粒转入到高表达isc操纵子的大肠杆菌中。
经转化的大肠杆菌培养于含有25mg/L的氯霉素和50mg/L的卡那霉素的LB培养基中，并在37℃培养至OD值为0.6左右。随后，向培养物中加入半胱氨酸(1mM)，FeCl3(0.1mM)和0.1mM IPTG，并在18℃、低速振荡的条件下继续培养宿主细胞16h。培养结束后，离心收集菌体，用蒸馏水洗涤2次，并于-80℃保存备用。
在厌氧条件下纯化菌体中表达的蛋白。简言之，用BugBuster Protein Extraction Reagent(Merck，Germany)裂解收集的菌体。随后，将菌体裂解液以16000g离心20min，并收集上清，弃去沉淀。 由于所表达的IspH蛋白在氨基端含有6个组氨酸标签(该组氨酸标签由pET28a载体自身携带的序列编码，且位于重组载体所表达的蛋白的N端)，因此，按照制造商的说明书，使用Ni-NTA柱(Merck，Germany)和脱盐柱(Sangon Biotech，China)来对上清中的IspH蛋白进行纯化。经纯化的IspH蛋白显示为褐色，保存于-80℃，备用。
另外，还使用与上述类似的方法表达和纯化了铁氧化还原蛋白(ferredoxin)和铁氧化还原蛋白-NADP+还原酶(ferredoxin-NADP+-reductase，FNR)。简言之，将编码铁氧化还原蛋白和铁氧化还原蛋白-NADP+还原酶的DNA片段分别克隆入载体pET28a中，然后转化入大肠杆菌进行表达。随后，将大肠杆菌裂解，并使用Ni-NTA柱(Merck，Germany)和脱盐柱(Sangon Biotech，China)对大肠杆菌所表达的蛋白进行纯化。经纯化的铁氧化还原蛋白和铁氧化还原蛋白-NADP+还原酶分别保存于-80℃，备用。
使用SDS-PAGE来对经纯化的蛋白进行检测。结果示于图1中。结果显示，本发明的IspH蛋白能够在大肠杆菌中高表达，其分子量为约35kD，并且经上述纯化步骤后具有高纯度，如图1泳道2中的单一电泳条带所示。类似地，图1的结果还显示，通过上述方法获得了高纯度的铁氧化还原蛋白(分子量为约11kD，如图1泳道3中的单一电泳条带所示)和铁氧化还原蛋白-NADP+还原酶(分子量为约36kD，如图1泳道1中的单一电泳条带所示)。
另外，还对经纯化的IspH蛋白进行全波长广谱扫描。结果示于图2中。图2的结果显示，本发明的IspH蛋白在410nm处有一最大吸收峰，在320nm处有一肩峰。这表明，该蛋白含有铁硫中心。
实施例3.IspH突变体的制备
采用定点突变的方法来制备IspH蛋白的两个突变体，H131N和E133Q，其中，突变体H131N(其序列如SEQ ID NO：3所示)与IspH蛋白的区别在于，位于SEQ ID NO：2第131位的组氨酸被突变为天冬酰胺；突变体E133Q(其序列如SEQ ID NO：4所示)与IspH蛋白的区 别在于，位于SEQ ID NO：2的第133位的谷氨酸被突变为谷氨酰胺。
简言之，以质粒pET-H为模板，使用下述引物进行全质粒PCR：
用于构建突变体H131N的引物对：
H131N-F：CTACATTGGTAAGAAAGGAaACCCCGAGCCAGAAGGCG(SEQ I D NO：7)，
H131N-R：CGCCTTCTGGCTCGGGGTtTCCTTTCTTACCAATGTAG(SEQ ID NO：8)；
用于构建突变体E133Q的引物对：
E133Q-F：GTAAGAAAGGACACCCCcAGCCAGAAGGCGCTATC(SEQ ID NO：9)，
E133Q-R：GATAGCGCCTTCTGGCTgGGGGTGTCCTTTCTTAC(SEQ ID NO：10)。
扩增产物用DpnI酶在37℃下处理2h，然后转化入大肠杆菌DMT菌株(北京全式金生物技术有限公司)中。在LB培养基中培养经转化的大肠杆菌DMT菌株，从中提取质粒，并对质粒进行测序验证。随后，将具有正确序列的质粒转化入高表达isc操纵子的大肠杆菌中，以表达突变体蛋白。突变体蛋白的表达及纯化方法如实施例2所述。
使用SDS-PAGE来对经纯化的突变体蛋白H131N和E133Q进行检测。结果示于图3中。结果显示，本发明的突变体蛋白H131N和E133Q能够在大肠杆菌中高表达，其分子量均为约35kD，并且经上述纯化步骤后具有高纯度。
实施例4.体外活性的检测
(1)以HMBPP为底物的活性检测
在厌氧条件下，在密封的5ml色谱管中，进行IspH蛋白及其突变体的活性检测，其中，反应体系包含：150mM NaCl，20mM Tris-HCl(pH7.8)，2mM DTT，1mM NADPH，1mM HMBPP，20μM铁氧化还原蛋白，5μM铁氧化还原蛋白-NADP+还原酶，和0.5μM如上获得的IspH蛋白或其突变体(H131N和E133Q)；总反应体积为500μl；反应条件为在37℃温育1h。
反应结束后，收集色谱管顶空的气体，并进行气相色谱-质谱分析(GC-MS)。简言之，使用配备有Agilent HP-PLOT A1203/S GC柱(25m，0.32mm，8.00μm，7inch cage)的Agilent Technologies 7890B  GC/5977A MSD来对气体进行分析，其中，使用氦气作为载体气体，其流速为2ml/min。随后，将获自测试样品的实验数据(峰滞留时间、峰面积和质谱图等)与标准品(异戊二烯、3-甲基-1-丁烯和2-甲基-2-丁烯)的实验数据进行比较，以鉴定气体成分并进行定量分析。
另外，还在反应后对色谱管中的反应液进行分析，以确定是否产生了IPP和DMAPP。由于DMAPP在酸性条件下可以自发生成异戊二烯，因此，可通过测定反应液在酸性条件下产生的异戊二烯的量来确定反应液中DMAPP的量。简言之，在厌氧条件下，取50μL反应液于另一5ml色谱管中，用冷水稀释至500μL，并加入等体积的8M H2SO4；随后，立即密封色谱管，并将其在37℃温育30min。温育后，通过如上所述的GC-MS法来测定顶空气体中异戊二烯的量，以确定反应液中是否存在DMAPP。
实验结果示于表2中。结果显示，野生型IspH蛋白及其突变体(H131N和E133Q)均能够催化HMBPP转化为异戊二烯，并且三者的催化能力无显著差异。此外，结果还显示，在包含野生型IspH蛋白的反应体系中，生成了异戊烯(3-甲基-1-丁烯和2-甲基-2-丁烯)；而在包含突变体蛋白(H131N和E133Q)的反应体系中，则检测不到异戊烯的生成。此外，结果还显示，包含野生型IspH蛋白的反应液在酸性条件下生成了异戊二烯(这表明反应液中存在DMAPP)；相反地，包含突变体蛋白(H131N和E133Q)的反应液在酸性条件下基本上无异戊二烯生成(这表明反应液中基本上不存在DMAPP)。
表2：反应体系的顶空气体以及反应液经酸处理后的顶空气体的定量分析
  野生型IspH IspH-H131N IspH-E133Q
反应体系产生的异戊二烯(μgL-1OD-1) 104.4 100.2 106.4
有无3-甲基-1-丁烯的生成 有 无 无
有无2-甲基-2-丁烯的生成 有 无 无
酸处理反应液而产生的异戊二烯(μg L-1OD-1) 696.3 10.1 6.1
这些结果表明，野生型IspH能够以HMBPP底物生成异戊二烯、异戊烯、IPP和DMAPP；相比之下，突变体蛋白H131N和E133Q只能够将HMBPP转化为异戊二烯，而基本上无法生成异戊烯、IPP和DMAPP。
(2)以DMAPP/IPP为底物的活性检测
在厌氧条件下，在密封的5ml色谱管中，进行IspH蛋白及其突变体的活性检测，其中，反应体系包含：150mM NaCl，20mM Tris-HCl(pH7.8)，2mM DTT，1mM NADPH，1mM DMAPP，20μM铁氧化还原蛋白，5μM铁氧化还原蛋白-NADP+还原酶，以及0.5μM如上获得的IspH蛋白或其突变体(H131N和E133Q)；总反应体积为500μl；反应条件为在37℃温育1h。
反应结束后，收集色谱管顶空的气体，并如上所述进行气相色谱-质谱分析(GC-MS)。实验结果显示，野生型IspH蛋白以DMAPP底物生成异戊烯，即，2-甲基-2-丁烯和3-甲基-1-丁烯(二者比例为约7∶1-8∶1)。相比之下，突变体蛋白H131N和E133Q已丧失了该活性，无法将DMAPP转化为异戊烯。
另外，还以IPP为底物重复了上述实验。实验结果显示，野生型IspH蛋白、突变体蛋白(H131N和E133Q)对于IPP均不具有催化活性。
为了测定野生型IspH蛋白的动力学参数，还使用不同浓度(0，0.1，0.2，0.3，0.4，0.5，1.0mM)的DMAPP来重复上述实验。实验结果示于图4中。实验结果显示，野生型IspH蛋白以DMAPP为底物生成异戊烯的最大反应活性为约6.2nmol min-1mg-1，Km为约275μM。
为了进一步确定实验(1)中的野生型IspH蛋白用于生成异戊烯的底物是HMBPP还是DMAPP，我们还测定了实验(1)中不同阶段，含有野生型IspH蛋白的反应体系的顶空气体的成分。结果显示，在反应早期，该反应体系的顶空气体中仅包含异戊二烯，而不包含异戊烯(2-甲基-2-丁烯和3-甲基-1-丁烯)。随着反应的进行，反应体系的顶空气体中才开始包含异戊烯。这一实验结果表明，(i)野生型IspH蛋白能够 将HMBPP转化为异戊二烯；和，(ii)野生型IspH蛋白并不具有将HMBPP直接转化为异戊烯的能力；但是，它能够将HMBPP转化为DMAPP，并进一步将DMAPP转化为异戊烯。
从上述实验结果来看，本发明的野生型IspH蛋白具有三种催化活性(图5)：(a)以HMBPP为底物生成DMAPP和IPP；(b)以HMBPP为底物生成异戊二烯；和，(c)以DMAPP底物生成异戊烯(2-甲基-2-丁烯和3-甲基-1-丁烯)。相比之下，突变体蛋白H131N和E133Q仅保留了以HMBPP为底物产生异戊二烯的能力，而基本上丧失了其他活性(即，基本上不能将HMBPP转化成IPP和DMAPP，也不能将DMAPP转化成2-甲基-2-丁烯和3-甲基-1-丁烯)。
实施例5.胞内活性的检测
将本发明IspH蛋白所源自的嗜碱芽孢杆菌N16-5菌株选择作为宿主细胞，在其中过表达IspH蛋白及其突变体(H131N和E133Q)，并检测由所述宿主细胞产生的气体，以确定IspH蛋白及其突变体的胞内活性。
简言之，将编码IspH蛋白或其突变体(H131N或E133Q)的DNA片段与pLDH(乳酸脱氢酶的启动子)融合，并克隆入pMK4载体中，以构建表达载体；随后，通过原生质体转化法将构建的表达载体转入到嗜碱芽孢杆菌N16-5中，以构建重组宿主细胞。通过上述方法，构建了3种重组细胞：重组细胞WNH，其为过表达野生型IspH蛋白的嗜碱芽孢杆菌N16-5；重组细胞H131N，其为过表达突变体H131N的嗜碱芽孢杆菌N16-5；及重组细胞E133Q，其为过表达表达突变体E133Q的嗜碱芽孢杆菌N16-5。
将所构建的重组细胞接种于密封的培养瓶中，并在37℃培养12h。随后，取培养瓶中的1mL顶空气体，进行GC-MS分析。GC-MS分析的详细方法如上文所述。另外，在该实验中，将野生型嗜碱芽孢杆菌N16-5(WT)用作对照。
实验结果示于图6-7和表3中。图6显示了经培养的野生型嗜碱 芽孢杆菌N16-5(WT)和重组细胞WNH(pMH)的顶空气体的GC特征谱，其中，3-甲基-1-丁烯(3M1B)，2-甲基-2-丁烯(2M2B)和异戊二烯的滞留时间分别为4.5min，5.5min，和11.3min。结果显示，野生型嗜碱芽孢杆菌N16-5能够产生异戊二烯，以及微量的2-甲基-2-丁烯和3-甲基-1-丁烯；并且，过表达野生型IspH蛋白的重组细胞WNH的2-甲基-2-丁烯和3-甲基-1-丁烯的产量显著提高，但异戊二烯的产量仅略有提升。另外，结果还显示，重组细胞H131N和E133Q的异戊烯产量与野生型嗜碱芽孢杆菌N16-5相比未发生显著变化(表3)。
图7显示了野生型嗜碱芽孢杆菌N16-5(WT)、重组细胞WNH、H131N和E133Q的异戊二烯产量的比较。结果显示，与野生型嗜碱芽孢杆菌N16-5相比，重组细胞WNH的异戊二烯产量略有提升，但重组细胞H131N和E133Q的异戊二烯产量均显著提高，分别约为野生型菌株的3倍和4倍。
表3显示了野生型嗜碱芽孢杆菌N16-5(WT)、重组细胞WNH、H131N和E133Q的异戊烯产量和异戊二烯产量的比较。结果显示，与野生型嗜碱芽孢杆菌N16-5相比，重组细胞WNH的异戊烯产量显著提高，而异戊二烯产量略有提升；重组细胞H131N和E133Q的异戊二烯产量显著提高(分别约为野生型菌株的3倍和4倍)，但异戊烯产量无显著变化。
表3：各种菌株的异戊烯产量和异戊二烯产量的比较
Figure PCTCN2016073413-appb-000020
另外，嗜碱芽孢杆菌N16-5能够表达电子传递体，铁氧化还原蛋白和黄素氧化还原蛋白。为了观察IspH蛋白的活性对这两种电子传递体的依赖性，我们制备了嗜碱芽孢杆菌N16-5的两个突变株：Δfer， 其缺失了编码铁氧化还原蛋白的fer基因；和，Δfld，其缺失了编码黄素氧化还原蛋白的fld基因。培养这两个突变株，并将其生长情况和异戊二烯的产量与野生型嗜碱芽孢杆菌N16-5进行比较。实验结果示于图8中。
图8a显示了嗜碱芽孢杆菌N16-5的野生型菌株和突变体菌株Δfer和Δfld的生长情况。结果显示，这3种菌株均能够在Horikoshi-I培养基中正常生长，其生长情况无显著差异。图8b显示了嗜碱芽孢杆菌N16-5的野生型菌株和突变体菌株Δfer和Δfld的异戊二烯产量的比较。结果显示，突变体菌株Δfer的异戊二烯产量显著低于野生型菌株(仅为野生型菌株的约50％)；而突变体菌株Δfld的异戊二烯产量则略微低于野生型菌株。这些实验结果表明，铁氧化还原蛋白和黄素氧化还原蛋白均可用作IspH蛋白生产异戊二烯所需的电子供体(即，当缺失铁氧化还原蛋白时，黄素氧化还原蛋白可用作电子供体；反之亦然)；但是，铁氧化还原蛋白对于IspH蛋白的该催化活性而言是更优选、更高效的。
另外，还使用大肠杆菌来检测IspH蛋白及其突变体(H131N和E133Q)的胞内活性。
简言之，将编码IspH蛋白和铁氧化还原蛋白的DNA片段克隆入pSTV28载体中，以构建表达载体pSTV165HF。随后，将所构建的表达载体转化入大肠杆菌Trans109菌株(TrahsGen Biotech)，以构建重组细胞STV165HF，其为过表达IspH蛋白和铁氧化还原蛋白的大肠杆菌。
另外，还使用类似方法构建了重组细胞STV165HF-H131N，其为过表达突变体蛋白H131N和铁氧化还原蛋白的大肠杆菌；以及，重组细胞STV165HF-E133Q，其为过表达突变体蛋白E133Q和铁氧化还原蛋白的大肠杆菌。
将所构建的重组细胞接种于密封的培养瓶中，并在37℃培养10h。随后，取培养瓶中的1mL顶空气体，进行GC-MS分析。GC-MS分析的详细方法如上文所述。
实验结果示于图9-11中。图9显示了经培养的重组大肠杆菌细胞STV165HF的顶空气体的GC特征谱(图9A)，以及标准品(3-甲基-1-丁烯，2-甲基-2-丁烯，2-甲基-1-丁烯和异戊二烯)的Gc特征谱(图9B)，其中，所使用的GC检测条件为：进样口温度180℃，柱温100℃，检测器温度200℃；并且，峰a为3-甲基-1-丁烯的特征峰；峰b为2-甲基-2-丁烯的特征峰；峰c为2-甲基-1-丁烯的特征峰；峰d为异戊二烯的特征峰。
图10显示了图9A中峰1组分与标准品3-甲基-1-丁烯(图10A)、峰2组分与标准品2-甲基-2-丁烯(图10B)、峰3组分与标准品异戊二烯(图10C)的质谱分析结果的比较，其中，在图10A-10C中，横坐标轴上方的图显示了待测组分(峰1、峰2、峰3)的质谱分析结果，而横坐标轴下方的图显示了标准品(3-甲基-1-丁烯、2-甲基-2-丁烯、异戊二烯)的质谱分析结果。
图9-10的实验结果显示，过表达IspH蛋白的重组大肠杆菌细胞STV165HF能够产生显著量的异戊二烯(峰3)，2-甲基-2-丁烯(峰2)和3-甲基-1-丁烯(峰1)。
图11显示了经培养的重组大肠杆菌细胞STV165HF-H131N(图11A)和STV165HF-E133Q(图11B)的顶空气体的GC特征谱，其中，所使用的GC检测条件为：进样口温度180℃，柱温130℃，检测器温度200℃。进一步，通过质谱分析来鉴定图11A-11B的各个峰中的组分。结果显示，图11A-11B所测定的顶空气体中包含异戊二烯，但不包含异戊烯。因此，这些实验结果表明，过表达突变体蛋白H131N或E133Q的重组大肠杆菌细胞均能够产生显著量的异戊二烯，且均不产生异戊烯。
上述实验结果表明，本发明的IspH蛋白及其突变体可用于构建能够生物合成异戊二烯的重组工程细菌。此外，本发明的IspH蛋白还可用于构建能够生物合成异戊烯(2-甲基-2-丁烯和3-甲基-1-丁烯)的重组工程细菌。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术 人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (11)

  1. 一种多肽，其具有以4-羟基-3-甲基-2-丁烯基-焦磷酸(HMBPP)为底物产生异戊二烯的活性，并且其具有选自下列的氨基酸序列：
    (1)如SEQ ID NO：2所示的氨基酸序列；
    (2)与SEQ ID NO：2具有至少90％同一性，优选至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性的氨基酸序列；和
    (3)与SEQ ID NO：2相异在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的置换、缺失或添加的氨基酸序列；
    优选地，所述多肽的氨基酸序列为SEQ ID NO：2或者与SEQ ID NO：2相异在于一个或几个(例如1个)氨基酸残基的置换；例如，所述多肽的氨基酸序列与SEQ ID NO：2相异在于，位于SEQ ID NO：2的第131位或第133位的氨基酸置换；例如，所述多肽的氨基酸序列与SEQ ID NO：2相异在于：位于SEQ ID NO：2的第131位的组氨酸被突变为天冬酰胺；或，位于SEQ ID NO：2的第133位的谷氨酸被突变为谷氨酰胺；
    优选地，所述多肽具有选自SBQ ID NO：2、3和4的氨基酸序列；
    任选地，所述多肽还具有以二甲烯丙基焦磷酸(DMAPP)为底物产生2-甲基-2-丁烯和3-甲基-1-丁烯的活性。
  2. 一种分离的核酸，其编码权利要求1的多肽。
  3. 包含权利要求2的分离的核酸的载体。
  4. 包含权利要求2的分离的核酸和/或权利要求3的载体的细胞，其中，所述分离的核酸相对于所述细胞而言是异源的或外源的；
    优选地，所述细胞还包含编码电子传递体(例如铁氧化还原蛋白)和 /或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)的核酸或者表达电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)；例如，所述细胞还包含编码铁氧化还原蛋白和铁氧化还原蛋白还原酶的核酸，或者表达铁氧化还原蛋白和铁氧化还原蛋白还原酶；
    优选地，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是内源的或外源的；例如，所述铁氧化还原蛋白和/或铁氧化还原蛋白还原酶相对于所述细胞而言是内源的或外源的；优选地，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶(EC1.18.1.2)；
    优选地，所述细胞还表达DXP途径的多肽；优选地，所述DXP途径的多肽选自，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG；EC 1.17.7.1)，以及其任何组合；
    优选地，所述DXP途径的多肽相对于所述细胞而言是内源的或外源的；
    任选地，所述细胞表达或者不表达异戊二烯合酶(IspS；EC4.2.3.27)；
    优选地，所述细胞为原核细胞，例如大肠杆菌或芽孢杆菌或蓝藻。
  5. 一种组合物，其包含权利要求1的多肽、HMBPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)，以及电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)；优选地，所述组合物包含所述多肽、HMBPP、NADPH、铁氧化还原蛋白和铁氧化还原蛋白还原酶(例如铁氧化还原蛋白-NADP+还原酶)。
  6. 一种组合物，其包含权利要求1的多肽、DMAPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)，以及电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)，其中，所述多肽具有SEQ ID NO：2所示的氨基酸序列；优选地，所述组合物包含所述多肽、DMAPP、NADPH、铁氧化还原蛋白和铁氧化还原蛋白还原酶(例如铁氧化还原蛋白-NADP+还原酶)。
  7. 一种生产异戊二烯的方法，其包括，使用权利要求1的多肽，将HMBPP转化为异戊二烯；
    优选地，所述方法包括，(a)将所述多肽、HMBPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)和电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)混合并温育(优选在20-40℃进行温育，例如在室温或37℃进行温育)；和，(b)收集步骤(a)产生的异戊二烯；
    优选地，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶。
  8. 一种生产异戊二烯的方法，其包括，(a)培养细胞，所述细胞表达外源导入的权利要求1的多肽；和，(b)收集步骤(a)产生的异戊二烯；
    优选地，所述细胞还表达电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)；例如，所述细胞还表达铁氧化还原蛋白和铁氧化还原蛋白还原酶；
    优选地，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是内源的或外源的；例如，所述铁氧化还原蛋白和/或铁氧化还原蛋白还原酶相对于所述细胞而言是内源的或外源的；优选地，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶(EC1.18.1.2)；
    优选地，所述细胞还表达DXP途径的多肽；优选地，所述DXP途径的多肽选自，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D- 赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG；EC 1.17.7.1)，以及其任何组合；
    优选地，所述DXP途径的多肽相对于所述细胞而言是内源的或外源的；
    任选地，所述细胞表达或者不表达异戊二烯合酶(IspS；EC4.2.3.27)；
    优选地，所述细胞为原核细胞，例如大肠杆菌或芽孢杆菌或蓝藻。
  9. 一种生产异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)的方法，其包括，使用权利要求1的多肽，将DMAPP转化为异戊烯；其中，所述多肽具有SEQ ID NO：2所示的氨基酸序列；
    优选地，所述方法包括，(a)将所述多肽、DMAPP、NADPH或NADH、电子传递体(例如铁氧化还原蛋白)和电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)混合并温育(优选在20-40℃进行温育，例如在室温或37℃进行温育)；和，(b)收集步骤(a)产生的异戊烯；
    优选地，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶。
  10. 一种生产异戊烯(例如3-甲基-1-丁烯和/或2-甲基-2-丁烯)的方法，其包括，(a)培养细胞，所述细胞表达外源导入的权利要求1的多肽，其中，所述多肽具有SEQ ID NO：2所示的氨基酸序列；和，(b)收集步骤(a)产生的异戊烯；
    优选地，所述细胞还表达电子传递体(例如铁氧化还原蛋白)和/或电子传递体传递电子所需的酶(例如铁氧化还原蛋白还原酶)；例如，所述细胞还表达铁氧化还原蛋白和铁氧化还原蛋白还原酶；
    优选地，所述电子传递体和/或电子传递体传递电子所需的酶相对于所述细胞而言是内源的或外源的；例如，所述铁氧化还原蛋白和/或 铁氧化还原蛋白还原酶相对于所述细胞而言是内源的或外源的；优选地，所述铁氧化还原蛋白还原酶为铁氧化还原蛋白-NADP+还原酶(EC1.18.1.2)；
    优选地，所述细胞还表达DXP途径的多肽；优选地，所述DXP途径的多肽选自，1-脱氧-D-木酮糖-5-磷酸合酶(DXS；EC 2.2.1.7)，1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR；EC 1.1.1.267)，2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶(MCT；EC 2.7.7.60)，4-(胞苷5′-二磷酸)-2-C-甲基-D-赤藓醇激酶(CMK；EC 2.7.1.148)，2-C-甲基-D-赤藓醇2，4-环二磷酸合酶(MCS；EC 4.6.1.12)，4-羟基-3-甲基-2-(E)-丁烯基二磷酸合酶(IspG；EC 1.17.7.1)，以及其任何组合；
    优选地，所述DXP途径的多肽相对于所述细胞而言是内源的或外源的；
    优选地，所述细胞还表达异戊烯基二磷酸异构酶(IDI；EC 5.3.3.2)；优选地，所述异戊烯基二磷酸异构酶相对于所述细胞而言是内源的或外源的；
    优选地，所述细胞为原核细胞，例如大肠杆菌或芽孢杆菌或蓝藻。
  11. 一种制备权利要求1的多肽的方法，其包括，(a)培养宿主细胞，所述宿主细胞包含并表达编码所述多肽的核酸；和，(b)收集所述细胞表达的所述多肽。
PCT/CN2016/073413 2016-02-04 2016-02-04 一种用于生物合成异戊二烯和异戊烯的酶及其突变体 WO2017132924A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/CN2016/073413 WO2017132924A1 (zh) 2016-02-04 2016-02-04 一种用于生物合成异戊二烯和异戊烯的酶及其突变体
US15/578,767 US10287569B2 (en) 2016-02-04 2016-02-04 Enzyme for biosynthesis of isoprene and isopentenyl, and mutant thereof
CN201680012121.5A CN107531763B (zh) 2016-02-04 2016-02-04 一种用于生物合成异戊二烯和异戊烯的酶及其突变体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/073413 WO2017132924A1 (zh) 2016-02-04 2016-02-04 一种用于生物合成异戊二烯和异戊烯的酶及其突变体

Publications (1)

Publication Number Publication Date
WO2017132924A1 true WO2017132924A1 (zh) 2017-08-10

Family

ID=59500504

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/073413 WO2017132924A1 (zh) 2016-02-04 2016-02-04 一种用于生物合成异戊二烯和异戊烯的酶及其突变体

Country Status (3)

Country Link
US (1) US10287569B2 (zh)
CN (1) CN107531763B (zh)
WO (1) WO2017132924A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009076676A2 (en) * 2007-12-13 2009-06-18 Danisco Us Inc. Compositions and methods for producing isoprene
CN103476926A (zh) * 2010-12-22 2013-12-25 丹尼斯科美国公司 使用两种类型的ispg酶改善异戊二烯产量的组合物和方法
CN104053765A (zh) * 2011-10-07 2014-09-17 丹尼斯科美国公司 利用磷酸酮醇酶产生甲羟戊酸、类异戊二烯前体和异戊二烯

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8686702B2 (en) 2012-02-15 2014-04-01 Linear Technology Corporation Negative slope compensation for current mode switching power supply

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009076676A2 (en) * 2007-12-13 2009-06-18 Danisco Us Inc. Compositions and methods for producing isoprene
CN103476926A (zh) * 2010-12-22 2013-12-25 丹尼斯科美国公司 使用两种类型的ispg酶改善异戊二烯产量的组合物和方法
CN104053765A (zh) * 2011-10-07 2014-09-17 丹尼斯科美国公司 利用磷酸酮醇酶产生甲羟戊酸、类异戊二烯前体和异戊二烯

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein [O] 26 May 2013 (2013-05-26), "4-hydroxy-3-methylbut-2-enyl diphosphate reductase [Bacillus cellulosilyticus", XP055405078, Database accession no. WP_013488222.1 *

Also Published As

Publication number Publication date
CN107531763B (zh) 2020-11-20
US20180087040A1 (en) 2018-03-29
CN107531763A (zh) 2018-01-02
US10287569B2 (en) 2019-05-14

Similar Documents

Publication Publication Date Title
JP6254728B2 (ja) イソプレンシンターゼおよびそれをコードするポリヌクレオチド、ならびにイソプレンモノマーの製造方法
JP2015516173A (ja) イソプレン生産用微生物およびプロセス
CN108251396B (zh) 5-氨基乙酰丙酸合成酶突变体及其宿主细胞和应用
US20220348974A1 (en) Biotin synthases for efficient production of biotin
Kaewbai-ngam et al. Production of glycogen, PHB, biohydrogen, NAD (P) H, and proteins in Synechocystis sp. PCC 6803 disrupted in metabolically linked biosynthetic pathway (s)
WO2021054441A1 (ja) サッカロミケス由来フェルラ酸デカルボキシラーゼ変異体、及びそれを用いた不飽和炭化水素化合物の製造方法
JP2017512460A (ja) イソプレンモノマーの製造方法
CN110607335B (zh) 一种烟酰胺腺嘌呤二核苷酸类化合物生物合成方法
JP5305440B2 (ja) 変異体ＳｅｐＲＳ及びこれを用いるタンパク質への部位特異的ホスホセリン導入法
Ogura et al. Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme
CN114686547B (zh) 一种以双醋瑞因为供体的酶促合成乙酰辅酶a的方法
WO2017132924A1 (zh) 一种用于生物合成异戊二烯和异戊烯的酶及其突变体
EP4116424A1 (en) Modified transglutaminase
RU2557299C1 (ru) Мутантная растительная формиатдегидрогеназа (варианты)
JP2009089649A (ja) クロストリジウム・クルベリのジアホラーゼ遺伝子およびその利用
Yeo et al. Production, purification, and characterization of soluble NADH-flavin oxidoreductase (StyB) from Pseudomonas putida SN1
JPWO2015080273A1 (ja) イソプレンモノマーの製造方法
CN112481231B (zh) 一种兼具酰基转移酶和谷丙转氨酶活性的双功能酶
JP4513967B2 (ja) Ｄ−アミノアシラーゼの活性向上方法
JP2020195375A (ja) ５−アミノレブリン酸シンテターゼ変異体およびその宿主細胞と応用
CN117025557A (zh) 热稳定性更高酶活更强的环氧化酶Lsd18变体及其应用
RU2522819C2 (ru) Мутантная формиатдегидрогеназа (варианты)
RU2545966C1 (ru) Мутантная формиатдегидрогеназа (варианты)
Brytan et al. Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27
CN114901814A (zh) 具有氢化酶活性的单体多肽，特别是具有氢化酶活性的重组单体多肽

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16888749

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15578767

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16888749

Country of ref document: EP

Kind code of ref document: A1