WO2020062977A1 - Gène codant une protéine de photosynthèse artificielle et utilisation associée - Google Patents

Gène codant une protéine de photosynthèse artificielle et utilisation associée Download PDF

Info

Publication number
WO2020062977A1
WO2020062977A1 PCT/CN2019/093277 CN2019093277W WO2020062977A1 WO 2020062977 A1 WO2020062977 A1 WO 2020062977A1 CN 2019093277 W CN2019093277 W CN 2019093277W WO 2020062977 A1 WO2020062977 A1 WO 2020062977A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
amino acid
carbon dioxide
psp2
artificial
Prior art date
Application number
PCT/CN2019/093277
Other languages
English (en)
Chinese (zh)
Inventor
王江云
刘晓红
康福英
胡诚
汪莉
许震
Original Assignee
中国科学院生物物理研究所
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 中国科学院生物物理研究所 filed Critical 中国科学院生物物理研究所
Publication of WO2020062977A1 publication Critical patent/WO2020062977A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the invention provides an artificial photosynthesis protein that can be genetically encoded and its application.
  • the genetically-encoded artificial photosynthetic protein provided by the present invention can simulate the absorption of light energy by a natural photosynthesis system and catalyze the reduction of carbon dioxide to carbon monoxide.
  • Photosynthesis is the most important process on earth, which converts solar energy into chemical energy and converts carbon dioxide (CO 2 ) into biomass 1-4 .
  • CO 2 carbon dioxide
  • researchers have great interest in how to improve the efficiency of photosynthesis and reuse light systems to promote challenging chemical transformations.
  • the present invention relates to one or more of the following:
  • An artificial photosynthesis protein comprising a fluorescent protein (FP) in which a chromophore amino acid residue is modified by a photosensitizer to be converted into a photosensitized protein (PSP).
  • FP fluorescent protein
  • PSP photosensitized protein
  • the fluorescent protein includes a dark blue fluorescent protein such as Sirius, a blue fluorescent protein such as EBFP, Azurite, EBFP2, TagBFP, a cyan fluorescent protein such as ECFP, Cerulean, CyPet, mTurquoise, mTFP1 (Teal), Midoriishi-Cyan, green fluorescent proteins such as GFP, UKG, EGFP, Emerald, Superfolder, yellow fluorescent proteins such as YFP, sfYFP, EYFP, Venus, Citrine, YPet, PhiYFP, orange fluorescent proteins such as mHoneydew, mBanana, mKO , MKO ⁇ , mOrange, mOrange2, red fluorescent proteins such as TagRFP, TagRFP, 158T, mRuby, mCherry, deep red fluorescent proteins such as Katushka, mKate, mKate2, mPlum, E2-Crimson, m
  • a dark blue fluorescent protein such as Sirius
  • chromophore amino acid residues include, for example, amino acid residues corresponding to positions 65, 66, and 67 of the green fluorescent protein GFP, and particularly correspond to Tyrosine residue at position 66 of the green fluorescent protein GFP.
  • the artificial photosynthetic protein according to any one of items 1-5 further comprising modification of amino acid residues other than chromophore residues, such as amino acid residue insertions, substitutions, and deletions.
  • b) has a sequence shown in any of the above with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher Amino acid sequence of amino acid identity;
  • c) has one or more sequences (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16 17, 18, 19, 20, 21, 22, 23, 24, 25 or more, preferably one or several, such as 1, 2, 3, 4, 5 or more) amino acid sequences with amino acid differences.
  • sequences e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16 17, 18, 19, 20, 21, 22, 23, 24, 25 or more, preferably one or several, such as 1, 2, 3, 4, 5 or more amino acid sequences with amino acid differences.
  • a photosensitive carbon dioxide reductase comprising the artificial photosynthetic protein according to any one of items 1-9 and a carbon dioxide reduction catalyst conjugated to the artificial photosynthetic protein.
  • An artificial photosynthesis system comprising the artificial photosynthesis protein according to any one of items 1-9 or the photosensitive carbon dioxide reductase according to any one of items 10-15, preferably the artificial photosynthesis system is Gene-coded artificial photosynthesis system.
  • a method for preparing an artificial photosynthetic protein comprising: modifying a chromophore amino acid residue of a fluorescent protein (FP) with a photosensitizer, thereby converting it into a photosensitized protein (PSP).
  • FP fluorescent protein
  • PSP photosensitized protein
  • a fusion protein comprising the artificial photosynthesis protein according to any one of 1-9.
  • a recombinant expression system comprising the nucleic acid molecule according to item 23.
  • a recombinant host cell comprising the expression system of item 24.
  • the photosensitizer is able to use light energy to turn a weak reducing agent into a strong reducing agent, so it is a key component in natural and artificial photosynthesis mechanisms.
  • the inventors started with the modification of light-sensitive proteins, overcame various restrictions in the prior art, and reasonably designed a genetically-encoded artificial photosensitizers protein (PSP) and a light-sensitive CO 2 reductase.
  • PSP genetically-encoded artificial photosensitizers protein
  • CO 2 reductase a light-sensitive CO 2 reductase
  • the light-sensitive CO 2 reductase mutates a specific site (for example, position 95) of PSP to cysteine, and then specifically conjugates a terpyridine nickel (II) complex (for example, N- ( 2,6,2-terpyridin-4-yl) -iodoacetamide is modified by adding nickel ion).
  • a terpyridine nickel (II) complex for example, N- ( 2,6,2-terpyridin-4-yl) -iodoacetamide is modified by adding nickel ion).
  • PSP2 95C terpyridine Ni (II) a protein conjugate having the activity of photocatalytic carbon dioxide to carbon monoxide , which is called photocatalytic carbon dioxide reductase (PSP2 95C terpyridine Ni (II), referred to as PSP2T for short).
  • PSP295C terpyridine Ni (II) a protein conjugate having the activity of photocatalytic carbon dioxide to carbon monoxide , which is called photocatalytic carbon dioxide reductase (PSP2 95C terpyridine Ni (II), referred to as PSP2T for short.
  • PSP2T The molecular weight of PSP2T is only 27 kD, which can be prepared by chemically modifying the genetically expressed PSP protein with a terpyridine nickel (II) complex in vitro.
  • PSP2T works in water / DMF solution and does not require precious metals.
  • the conversion quantum efficiency of CO 2 / CO is 2.6%, which is much higher than most photocatalysts using nanocrystals or small molecule photosensitizers under similar conditions.
  • the CO 2 / CO conversion has a quantum efficiency of 3 .
  • the inventors were inspired in the study of PSP2T and understood the essence of complex photosynthesis mechanisms: visible light absorption, generation of strong reducing power, and carbon dioxide reduction.
  • the inventors analyzed the crystal structure of PSP ⁇ , which provides the necessary atomic structure information for the use of PSP ⁇ to promote a new enzyme reaction. Importantly, the inventors have demonstrated through research that three variables important for PSP2T activity can be conveniently and independently optimized by mutagenesis, resulting in significantly improved carbon dioxide reduction activity.
  • the photochemical properties of the chromophore can be fine-tuned to make its photo-excited state sufficiently oxidizing, capable of oxidizing weak sacrificial reducing agents (SR), thereby generating strong reducing groups that can be used to promote the reduction of carbon dioxide reduction catalysts.
  • SR weak sacrificial reducing agents
  • fluorescent proteins include proteins that emit light upon encountering excitation light.
  • the fluorescent protein is not particularly limited.
  • the fluorescent protein may include a dark blue fluorescent protein such as Sirius, a blue fluorescent protein such as EBFP, Azurite, EBFP2, TagBFP, a cyan fluorescent protein such as ECFP, Cerulean, CyPet, mTurquoise, mTFP1 (Teal), Midoriishi-Cyan, green fluorescent proteins such as GFP, UKG, EGFP, Emerald, Superfolder, yellow fluorescent proteins such as YFP, sfYFP, EYFP, Venus, Citrine, YPet, PhiYFP, orange fluorescent proteins such as mHoneydew, mBanana, mKO , MKO ⁇ , mOrange, mOrange2, red fluorescent proteins such as TagRFP, TagRFP, 158T, mRuby, mCherry, deep red fluorescent proteins such as Kat
  • the fluorescent protein can be from any suitable source, such as corals such as Galaxea fascicularis, anemones, jellyfish, and the like.
  • green fluorescent protein GFP GenBank Accession No. AAA27722
  • mutant fluorescent proteins such as YFP, CFP, EGFP, EYFP, ECFP, and the like.
  • the GFP gene encoding sequences registered in GenBank also include AEVGFPAM62653, AVGFP1X83959, AVGFP2X83960, and AEVGFPL29345.
  • Appropriate modifications of fluorescent proteins other than photosensitizers can also be included in the invention.
  • the present invention may include modifications to the GFP chromophore residue tripeptide sequence at amino acids 65, 66, 67-serine, tyrosine, glycine, for example, modifications to the 65th serine residue may include Alanine, leucine, cysteine, valine, isoleucine, or threonine are substituted to obtain a protein with a red-shifted spectrum.
  • the chromophore residues of the fluorescent protein may include, for example, SHG (EBFP2), TWG (ECFP, mCerulean), AYG (mTFP1), TYG (mEGFP, mEmerald), GYG (EYFP, Mvenus, mCitrine, YPet) ), CYG (mKO), MYG (tdTomato, TagRFP, mCherry, mKate, mPlum), QYG (mRFP1), etc.
  • SHG EBFP2
  • TWG ECFP, mCerulean
  • AYG mTFP1
  • TYG mEGFP, mEmerald
  • GYG EYFP, Mvenus, mCitrine, YPet
  • CYG CYG
  • MYG tdTomato
  • TagRFP mCherry
  • mKate mPlum
  • QYG mRF
  • the invention includes, for example, amino acid residue modifications known in the art, such as modification of amino acid residue tyrosine (Y) at position 66, such as Y66H includes a blue-emitting fluorescent protein; V163A- S175G has heat resistance and enhanced fluorescence intensity; F64I, F64V, F64A, F64G, F64L have enhanced fluorescence intensity; F64L-Y66H-Y145F-L236R, F64L-Y66H-Y145F-V163A-S175G-L236R, Y66H-Y145F- V163A-S175G and F64L-Y66H-Y145F are related to photostable fluorescent proteins.
  • amino acid residue modifications known in the art such as modification of amino acid residue tyrosine (Y) at position 66, such as Y66H includes a blue-emitting fluorescent protein
  • V163A- S175G has heat resistance and enhanced fluorescence intensity
  • the invention also relates to a fluorescent protein with a molecular weight of only about 27 kD discovered by the inventors in a previous study, which also has the potential to be transformed into a photosynthetic protein similar to the natural light system.
  • a fluorescent protein with a molecular weight of only about 27 kD discovered by the inventors in a previous study, which also has the potential to be transformed into a photosynthetic protein similar to the natural light system.
  • the core problem in designing efficient carbon dioxide photoreductive proteins based on fluorescent protein mutants is how to extend the life of the reducing intermediate state generated by the excitation of its chromophore and reduce its reduction potential.
  • the inventors chose a tyrosine analog (BpA) with a benzophenone substituent to modify the chromophore.
  • BpA tyrosine analog
  • Benzophenone is a commonly used photosensitizer in organic photocatalysis. When it is irradiated with light of a certain wavelength, its excited state will cross between systems into a triplet state with a longer life. This triplet state in turn reacts with the sacrificial reducing agent to form a highly active free radical state, catalyzing the downstream redox reaction.
  • the newly generated fluorescent protein retained this characteristic.
  • Studies using transient absorption spectroscopy show that after being excited by light, the new chromophores inserted into BpA can be almost completely converted to the triplet state; in the presence of a sacrificial reducing agent (such as ascorbic acid), the triplet intermediate is equivalent to rapid oxidation Sacrifice the reducing agent, thereby generating a free radical state. This free radical is protected by the protein backbone, so it can exist stably for more than 10 minutes in the absence of oxygen.
  • a sacrificial reducing agent such as ascorbic acid
  • the inventors After obtaining a fluorescent protein that can be excited by light to generate a strong reducing activity, the inventors further applied chemical or biological methods to introduce a terpyridine nickel complex at a specific site on the outer surface of the PSP (this is a known small molecule carbon dioxide Reduction electrochemical catalyst).
  • This modified protein has the activity of reducing carbon dioxide to generate carbon monoxide under light conditions. Its 24-hour carbon monoxide conversion number is up to 120, and the photon quantum yield is 2.6%, which is higher than most of the reported carbon dioxide photoreduction catalysts. This illustrates the optimization of electron transfer and the improvement of activity based on the self-assembly properties of proteins.
  • the present invention provides a genetically-encoded artificial photosynthesis protein (PSP), which is extended by a genetic codon, in a superfolder yellow fluorescent protein (sfYFP, amino acid sequence such as SEQ ID ID NO: (Shown in 1)
  • PSP genetically-encoded artificial photosynthesis protein
  • sfYFP amino acid sequence such as SEQ ID ID NO: (Shown in 1)
  • the amino acid position 66 is obtained by mixing benzophenone-alanine (BpA).
  • the 66th tyrosine (Tyr, Y) of sfYFP was replaced by benzophenone-alanine (BpA), and this amino acid substitution was introduced by the genetic codon expansion method.
  • sfYFP (SEQ ID NO: 1) is an artificially synthesized protein whose amino acid sequence is 88% similar to the Mesorhizobium® loti sequence.
  • benzophenone is incorporated at the 66th amino acid position of the superfolder yellow fluorescent protein (sfYFP, amino acid sequence shown in SEQ ID NO: 1) by genetic codon expansion-
  • the artificial photosynthetic protein obtained from alanine (BpA) is shown in SEQ ID NO: 2 and named sfYFP-BpA66.
  • tyrosine (Tyr) at 203 in sfYFP was mutated to phenylalanine (Phe) by genetic codon mutation.
  • the double mutant sfYFP-BpA66-Phe203 was named PSP1. Its amino acid sequence is shown in SEQ ID NO: 4.
  • a triple mutant sfYFP in which tyrosine (Tyr) at 203 of sfYFP was mutated to aspartic acid (Asp) and histidine (His) at 148 was mutated to glutamic acid (Glu).
  • Glu glutamic acid
  • PSP2 can reversibly form PSP2 radicals (PSP2 ⁇ ) in the photochemical reaction.
  • PSP2 ⁇ PSP2 radicals
  • the reversible formation of PSP2 ⁇ indicates that although PSP2 ⁇ can react with oxygen (a common characteristic of almost all super-reducing free radicals), the reaction produces PSP2 without destroying the chromophore. Since various carbon dioxide reducing agents are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of the catalyst.
  • the decay lifetime of the PSP2 triplet excited state (PSP2 *, Fig. 4a / b) is 123 ⁇ s.
  • the reduction potential of PSP2 ⁇ is less than -1.14V.
  • the pKa of PSP2 ⁇ is 10.6.
  • PSP2 the 95th amino acid was mutated from glutamic acid (Glu) to cysteine (Cys, single letter symbol: C) to obtain a PSP2-95Cys mutant (also denoted as PSP2-95C), which The amino acid sequence is shown in SEQ ID NO: 8.
  • the mutant was finally conjugated in the presence of a divalent nickel ion after modification with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (the compound 7 synthesized in Example 1).
  • PSP2T1 has a higher carbon dioxide reducing activity, in which N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specifically conjugated at the 95th and a half positions of the PSP2-95C mutant On cystine residues.
  • PSP2T2 The final conjugate obtained in the presence of divalent nickel ions is named PSP2T2, which shows a significantly increased number of carbon monoxide conversions (TON) ( Figure 3c / d).
  • the inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3).
  • a terpyridine nickel (II) complex is conjugated to the cysteine at position 95.
  • the terpyridine nickel (II) complex is a known small molecule carbon dioxide reduction electrochemical catalyst.
  • BpA66 indicates that the 66th tyrosine of a superfolded yellow fluorescent protein (sfYFP) is mutated to BpA, or Tyr66BpA (that is, the number indicates the amino acid position of the mutation, and the left side of the number is the amino acid residue before the mutation Group, amino acid residues to the right of the number).
  • Phe203 indicates that the tyrosine (Tyr) at position 203 of the superfolded yellow fluorescent protein (sfYFP) is mutated to phenylalanine (Phe), and can also be expressed as Tyr203Phe.
  • 95Cys indicates that the 95th position of the superfolded yellow fluorescent protein (sfYFP) is mutated from glutamic acid (Glu) to cysteine (Cys, single letter symbol: C), and can also be expressed as Glu95Cys or 95C. Amino acid mutations at other positions are also expressed as described above.
  • the present invention provides a photosensitive carbon dioxide reductase (PSP2 terpyridine Ni (II), also known as PSP2-terpyridine nickel complex conjugate, referred to as PSP2T), which Gene-coded artificial photosynthesis protein (PSP) specific site (for example, position 95) is mutated to cysteine, and then N- (2,6,2-terpyridine-4- ) -Iodoacetamide, and in the presence of divalent nickel ions, the divalent nickel ions and N- (2,6,2-terpyridin-4-yl) -iodoethyl conjugated to cysteine Obtained by amide coordination.
  • PSP2 terpyridine Ni II
  • PSP2T Photosensitive carbon dioxide reductase
  • the conjugation of artificial photosynthetic protein (PSP) with terpyridine is achieved by bringing the corresponding artificial photosynthetic protein to N- (2,6,2-terpyridin-4-yl) -iodoacetamide (implementation The compound 7) synthesized in Example 1 was reacted. Specifically, N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specifically conjugated to a single cysteine residue introduced in the PSP.
  • cysteine is introduced at position 95 in the PSP protein, and N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specific to the cysteine Conjugated PSP-terpyridine nickel (II) complex conjugates obtained in the presence of divalent nickel ions have the activity to catalyze the photochemical reaction of carbon dioxide to form carbon monoxide.
  • N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specific to the cysteine Conjugated PSP-terpyridine nickel (II) complex conjugates obtained in the presence of divalent nickel ions have the activity to catalyze the photochemical reaction of carbon dioxide to form carbon monoxide.
  • the mutant PSP2-95C is modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- ( 2,6,2-terpyridin-4-yl) -iodoacetamide is conjugated to cysteine at position 95).
  • the final conjugate obtained in the presence of divalent nickel ion is named PSP2T1, which has a relatively High carbon dioxide reducing activity.
  • the mutant PSP2-95C93Y97Y was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (the compound 7 synthesized in Example 1), and PSP2T2 was obtained in the presence of divalent nickel ions. Significantly increased carbon monoxide conversion (TON) ( Figure 3c / d). The inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3).
  • the terpyridine modification of the mutant PSP2-95C or PSP2-95C93Y97Y can be achieved by the organism. For example, transfer the PSP2-95C or PSP2-95C93Y97Y expression vector into an appropriate host cell, and add an appropriate expression inducer and terpyridine (for example, N- (2,6,2-terpyridine-4) to the medium. -Yl) -iodoacetamide), after expression, appropriate divalent nickel ions are added to obtain the corresponding protein-terpyridine nickel (II) complex conjugate.
  • an appropriate expression inducer and terpyridine for example, N- (2,6,2-terpyridine-4
  • -Yl) -iodoacetamide for example, N- (2,6,2-terpyridine-4
  • appropriate divalent nickel ions are added to obtain the corresponding protein-terpyridine nickel (II) complex conjugate.
  • the present invention provides a method for photocatalytic reduction of carbon dioxide by using the photosensitive carbon dioxide reductase according to the second aspect of the present invention.
  • the method includes the following steps: adding the second aspect of the present invention to the reaction system.
  • the photo-sensitive carbon dioxide reductase for example, PSP2T1 or PSP2T2, preferably PSP2T2
  • the sacrificial reducing agent are irradiated with visible light to reduce carbon dioxide in the reaction system to carbon monoxide. Visible light irradiation can be performed using a xenon lamp that simulates the sunlight spectrum.
  • carbon dioxide in the reaction system includes a case where the reaction system includes related reactants capable of generating carbon dioxide.
  • the present invention also provides one or more of the following:
  • a genetically-encoded artificial photosynthesis protein obtained by incorporation of benzophenone-alanine BpA at the amino acid position 66 of the superfolded yellow fluorescent protein sfYFP through genetic codon expansion, and its amino acid sequence As shown in SEQ ID NO: 2.
  • the artificial photosynthesis protein according to item 1 further comprising a Tyr203Phe mutation, and the amino acid sequence is shown in SEQ ID NO: 4.
  • the artificial photosynthesis protein according to item 1 further comprising Tyr203Asp and His148Glu mutations, and the amino acid sequence is shown in SEQ ID NO: 6.
  • the artificial photosynthesis protein according to item 3 further comprising a Glu95Cys mutation, and the amino acid sequence is shown in SEQ ID NO: 8.
  • the artificial photosynthesis protein according to item 3 further comprising mutations of Glu95Cys, Val93Tyr and Thr97Tyr, and the amino acid sequence is shown in SEQ ID NO: 10.
  • a photosensitive carbon dioxide reductase which is a specific conjugate of the artificial photosynthetic protein of item 5 or 6 and a terpyridine nickel complex, wherein the conjugate is obtained by dissolving N- (2,6,2 -Terpyridin-4-yl) -iodoacetamide is specifically conjugated to the cysteine at position 95, and the divalent nickel ion is further conjugated to the cysteine in the presence of a divalent nickel ion.
  • N- (2,6,2-terpyridin-4-yl) -iodoacetamide is obtained by coordination.
  • the advantage of the present invention is that compared with semiconductor nanocrystals and small molecule photosensitizers, the PSP constructed by the present invention provides unique advantages, for example, it has higher compatibility with a wide range of biological systems, does not rely on precious metals, Switchable photochemical properties, and the ability to self-assemble into precise three-dimensional structures, can allow for modular expansion of its functions and accurate mechanism characterization. As a result, PSP can potentially sensitize a variety of challenging chemical transformations, involving diverse fields such as solar energy conversion, photobiology, environmental restoration, and industrial biology.
  • the photosynthetic protein synthesized by the present invention can be synthesized in the organism through genetic coding, can react with oxygen to form a free radical form reversibly without destroying the chromophore, and is a robust photochemical reaction catalyst.
  • the triplet excited state produced by light irradiation has a long decay life, which is more conducive to reducing carbon dioxide.
  • UV-Vis Ultraviolet-visible absorption
  • X-band electron spin resonance (X-band ESR) spectra of PSP2 (basically a horizontal line) and PSP2 ⁇ (a curve with peaks and troughs) using NADH as a reducing agent before and after 405 nm laser irradiation.
  • NADH is used as a sacrificial reducing agent because it has no background ESR signal.
  • FIG. 5 BpA structure; b, BpAChm structure, BpAChm simulates the chromophore structure of PSP.
  • c UV-Vis spectrum of BpA before and after 405nm laser irradiation; d, UV-Vis spectrum of BpAChm before and after 405nm laser irradiation; in both cases, in the presence of 10 mM dithionite Under conditions, no benzophenone radicals were observed after irradiating the sample with a 405 nm laser pen for more than 10 minutes.
  • Figure 6.a UV-Vis spectra of PSP1 and PSP2 after 405nm laser irradiation for 10 minutes; conditions: 50 ⁇ MPSP, 100 mM Tris-HCl pH 7.0 buffer, 100 mM Asc (ascorbate); b, at 100 mM Tris-HCl pH UV-Vis spectrum of PSP2 in 7.0 buffer, irradiated with 405nm laser pen for 10 minutes in the absence of Asc, or treated with 100mMAsc in the absence of laser irradiation; in both cases, there is no PSP2 spectrum changes were observed and no PSP2 ⁇ radical formation was obtained; c, UV-Vis spectra of PSP1 ⁇ at different pH.
  • Figure 7 Structure of benzophenone (BP), BP-COOH, BP neutral radical, BP anionic group, PSP1 chromophore, PSP2 chromophore, PSP2 neutral radical, PSP2 anionic group and BpAChm Naming.
  • BP benzophenone
  • BP-COOH BP neutral radical
  • BP anionic group PSP1 chromophore
  • PSP2 chromophore PSP2 neutral radical
  • PSP2 anionic group BpAChm Naming.
  • Circular dichroism (CD) spectra of PSP2 in different pH buffers Conditions: 10 ⁇ M PSP2 in 100 mM buffers with different pH values (that is, Tris-HCl (pH 6.0-8.0), glycine-NaOH (pH 9-10.6), Carbonate-NaOH (pH 11.4-11.8)) is placed on quartz In a cup (200 ⁇ L, 1 cm path), the spectrum was measured with a circular dichroism spectrometer at room temperature.
  • FIG. 9 The formation of PSP2 ⁇ is reversible. Conditions: 50 ⁇ M PSP2, 50 mM NADH, 100 mM Tris-HCl pH 7.0 buffer. For each photoperiod, the sample was first irradiated with a 405nm laser (100mW / cm 2 ) for 10 minutes, and then the absorbance at 525nm (which represents the formation of PSP2 ⁇ , corresponding to one of the three isoabsorbance points), and then Incubate for 20 minutes in the dark before the start of the next light cycle.
  • a 405nm laser 100mW / cm 2
  • the absorbance at 525nm which represents the formation of PSP2 ⁇ , corresponding to one of the three isoabsorbance points
  • Figure 14 Amino acid sequences of sfYFP (SEQ ID NO: 1) and various sfYFP variants constructed according to the present invention (SEQ ID NOs: 2, 4, 6, 8, 10), of which the "*" at position 66 of the variant Represents BpA.
  • SEQ ID NO: 1 amino acid sequence of superfolded yellow fluorescent protein (sfYFP)
  • SEQ ID NO: 3 Nucleotide sequence of sfYFP-BpA66 expressed in E. coli
  • SEQ ID NO: 5 Nucleotide sequence of sfYFP-BpA66-Phe203 (PSP1) expressed in E. coli
  • SEQ ID NO: 7 nucleotide sequence of sfYFP-BpA66-Asp203 Glu148 (PSP2) expressed in E. coli
  • SEQ ID NO: 12 Nucleotide sequence of superfolded yellow fluorescent protein (sfYFP) expressed in E. coli
  • the reagents, plasmids, etc. used in the examples can be purchased from commercially available channels.
  • PCR reagents T4 DNA ligase and restriction endonuclease were purchased from Fermentas.
  • Ni-NTA affinity purification reagents and purification columns were purchased from Qiagen.
  • the pEVOL-BpARS plasmid was purchased from Addgene (Plasmid # 31190). The primers and mutated genes used were synthesized by Sangon Biotech.
  • Protein purification was performed on the AKTA UPC 900FPLC system (GEhealthcare).
  • the absorption spectrum was recorded at room temperature using an ultraviolet-visible mass spectrometer (Agilent 8453, Agilent technologies, CA, USA). Fluorescence spectra were recorded on a microplate reader equipped with Varioskan Flash SkanIt software 2.4.3RE (Varioskan Flash, Thermo Fisher Scientific Inc).
  • the fluorescence attenuation measurement was performed with a time-correlated single photon counting (TCSPC) fluorescence spectrometer (FL900 Edinburgh instruments Ltd.). Instantaneous absorption spectra with nanosecond time resolution were detected using an Edinburgh LP980 spectrometer (Edinburgh Instruments Ltd.) set up with nanosecond flash photolysis.
  • TCSPC time-correlated single photon counting
  • Cyclic voltammetry (CV) measurements were performed using a CH instrument 600D electrochemical system (CH Instrument, China).
  • Gas chromatography (GC) was performed with an SRI Multi-Gas Analyzer (SRI Instruments, Model 8610C) equipped with a TCD and HID detector.
  • Circular dichroism (CD) spectra were recorded with a circular dichroism (Applied Photophysics Ltd, Chirascan Plus).
  • ESR spectra were recorded with a 154 Bruker EMX-plus X-band spectrometer (Institute of Chemistry, Chinese Academy of Sciences, Beijing).
  • Transmission electron microscope (TEM) pictures were taken with a JEM-2100F electron microscope (Analytical Center, Department of Chemistry, Tsinghua University).
  • the following compounds are synthesized according to the following reaction route, and are used to detect the reduction potential of free radicals formed by the genetically encoded artificial photosynthesis protein prepared in the present invention during photoreaction.
  • PE is petroleum ether and EA is ethyl acetate.
  • PE is petroleum ether and EA is ethyl acetate.
  • FP fluorescent protein
  • DNA amplification was performed using a DNA thermal cycler: initial denaturation (94 ° C, 1min); followed by 30 strand reaction cycles: 94 ° C 1min, 60 ° C 1min, 68 ° C 1min; and finally extension at 68 ° C for 10min.
  • a vector pET22b (+) containing a superfolder yellow fluorescent protein (sfYFP, amino acid sequence shown in SEQ ID NO: 1) coding sequence (SEQ ID NO: 12) was used as a template for generating different sfYFP mutants. All constructs and their mutagenesis were verified by DNA sequencing analysis.
  • the construct pET22b-sfYFP (where the sfYFP coding sequence was synthesized by Universal Biosystems (Anhui) Co., Ltd.) and the BpA tRNA synthetase plasmid pEVOL-BpARS (the plasmid was purchased from Addgene (Plasmid # 31190), please refer to reference 38 for the method of use -39) Co-transformed into E. coli BL21 (DE3) strain for unnatural amino acid incorporation. Specifically, the construct pET22b / sfYFP-TAG66 mutant and pEVOL-BpARS were co-transformed into the E. coli BL21 (DE3) strain.
  • the pEVOL-BpARS plasmid carries a BpA-selective Methanococcus jannaschii tyrosyl tRNA synthetase and a MjtRNA tyrosyl amber suppressor tRNA (MjtRNA Tyr CUA ), thereby allowing the first to the sfYFP mutant
  • MjtRNA Tyr CUA MjtRNA tyrosyl amber suppressor tRNA
  • the double mutant sfYFP -BpA66-Phe203 is named PSP1, and its amino acid sequence is shown in SEQ ID NO: 4.
  • a triple mutant sfYFP in which tyrosine (Tyr) at 203 of sfYFP was mutated to aspartic acid (Asp) and histidine (His) at 148 was mutated to glutamic acid (Glu).
  • Glu glutamic acid
  • PSP2 the 95th glutamic acid (Glu) was mutated to Cys (C) to obtain a PSP2-95C mutant, whose amino acid sequence is shown in SEQ ID NO: 8.
  • This mutant was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- (2,6,2-terpyridine-4) -Yl) -iodoacetamide is specifically conjugated to cysteine at position 95).
  • the final conjugate obtained in the presence of divalent nickel ions is named PSP2T1, and it was found that PSP2T1 has a higher carbon dioxide Reducing activity.
  • mutant PSP2-95C93Y97Y the amino acid sequence of which is shown in SEQ ID NO: 10.
  • This mutant was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- (2,6,2-terpyridine-4) -Yl) -iodoacetamide is specifically conjugated to cysteine at position 95), and the final conjugate obtained in the presence of a divalent nickel ion is named PSP2T2, which shows a significantly increased number of carbon monoxide conversions (TON) ( Figure 3c / d). The inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3).
  • a single colony that had been transformed with the corresponding recombinant expression vector was placed in LB supplemented with ampicillin (100 ⁇ g / mL, purchased from Sigma-Aldrich) and chloramphenicol (25 ⁇ g / mL, purchased from Sigma-Aldrich).
  • the medium (4 mL, purchased from Sigma-Aldrich) was grown overnight at 37 ° C. 1 mL of the overnight culture was inoculated into 100 mL of liquid LB medium supplemented with ampicillin (100 ⁇ g / mL) and chloramphenicol (25 ⁇ g / mL) and BpA (1 mM).
  • the cells were then cultured at 37 ° C to an OD 600 of 1.1, followed by addition of 0.02% arabinose (purchased from Sigma-Aldrich) and 1 mM isopropyl ⁇ -D-1-galactopyranoside (IPTG, purchased from Sigma- Aldrich) induces protein expression. Continue incubation for 4-12 hours, collect cells and freeze at -70 ° C, ready for protein purification.
  • arabinose purchased from Sigma-Aldrich
  • IPTG isopropyl ⁇ -D-1-galactopyranoside
  • lysis buffer 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 10 mM imidazole
  • lysis buffer 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 10 mM imidazole
  • the supernatant was loaded onto a Ni-NTA column (Histrap 5ml, GE Healthcare). Wash the column twice with 5 ml of washing buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 50 mM imidazole), and then elute with an elution buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 250 mM imidazole). Captured protein.
  • the digested protein was purified by Sephadex gel column chromatography (Superdex 75 10 / 300GL; GE Healthcare) into a buffer containing 20 mM HEPES-NaOH, pH 7.5, and concentrated to ⁇ 30 mg / mL, and examined by SDS-PAGE.
  • SDS-PAGE SDS-PAGE.
  • the diffraction data was collected by Shanghai Synchrotron Radiation Facility (SSRF) using beamlines B17U or BL18U. Data processing and reduction were performed using HKL2000 package.
  • SSRF Synchrotron Radiation Facility
  • CCP4 suite a macromolecular structure crystal structure analysis software, Collaborative Computational Science No.4 Software
  • Molrep performs molecular replacement to analyze the structure of PSP2 ⁇ (radical state).
  • UV-visible spectrophotometer was used to detect the UV-Vis spectra of various PSP mutant proteins at room temperature and determine the pKa of PSP2 ⁇ . UV-Vis spectroscopy and pKa data can prove that the PSP mutant protein is converted to a deprotonated state under alkaline conditions.
  • PSP2 ⁇ PSP2 radicals
  • 50 mM NADH 50 ⁇ M PSP2 protein in 100 mM Tris-HCl pH 7.0 buffer was irradiated with a 405 nm laser for 10 minutes, and then using an Agilent 8453 UV-visible spectrophotometer (quartz cup, 100 ⁇ L, 1 cm path) UV-Vis spectra were recorded at room temperature.
  • the sample was first irradiated with a 405nm laser (100mW / cm 2 ) for 10 minutes, and then the absorbance at 525nm (which represents the formation of PSP2 ⁇ , corresponding to one of the three isoabsorbance points), and then Incubate for 20 minutes in the dark before the start of the next light cycle.
  • the reversible formation of PSP2 ⁇ indicates that although PSP2 ⁇ can react with oxygen (a common characteristic of almost all super-reducing free radicals), the reaction returns to PSP2 without destroying the chromophore. Since various carbon dioxide reducing agents are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of the catalyst.
  • the reduction potential was measured by cyclic voltammetry (CV).
  • the reduction potential data can indirectly prove the reducing power of PSP2 radicals. Measurements were performed with CH Instruments 600D potentiostat (instrument purchased from Shanghai Chenhua Instrument Co., Ltd.).
  • a 2 mM BpAChm solution in a 0.1 M NBu 4 PF 6 DMF solution was placed in a 3-electrode chamber with an Au working electrode, an Ag / AgCl reference electrode, and a Pt auxiliary electrode at 4 ° C. Prior to measurement, the system was purged with Ar for 15 minutes.
  • the CV parameters are as follows: scan rate: 10mV / s; sample interval: 1mV; sensitivity: 10 ⁇ A / V; quiet time: 4s; temperature: 0 ° C.
  • Electron spin resonance (ESR) experiments were performed to further characterize the production of free radical PSP2 ⁇ .
  • ESR Electron spin resonance
  • 45 ⁇ M PSP2 in 100 mM Tris-HCl pH 8.0 buffer was propagated with 405 nm laser radiation 10, and the yield of PSP2 free radicals was quantitatively determined by UV-Vis spectroscopy as 12%.
  • the PSP2 protein solution appeared light yellow, and after the 405nm laser irradiation, the protein solution became dark red, indicating that free radicals were formed.
  • X-band ESR spectra were then recorded on a Bruker E500 spectrometer at room temperature for dark red light-treated protein samples.
  • ESR capture parameters are as follows: adjustment frequency: 30-100kHz; microwave power: 0.05-10mW; adjustment amplitude: 2G.
  • a nanosecond flash photolysis device using an Edinburgh LP980 spectrometer (Edinburgh Instruments Ltd.) combined with a compact Q-switched Nd: YAG laser (Q-smart 850, Quantel, France) measures instantaneous absorption spectra with nanosecond time resolution.
  • the probe is a 150W pulsed xenon arc lamp for kinetic and spectral measurements from a few nanoseconds to 1ms. Photolysis of the samples was achieved using a single-flash laser excitation at 355 nm (1 Hz, 10 mJ / pulse, 50 mm 2 spot area, fwhm ⁇ 7 ns).
  • the probe light comes from a 450W pulsed xenon lamp.
  • a single detector (PMT R928P) was used to record transient signals, an oscilloscope was used to record kinetic traces, and an ICCD detector was used to record time-resolved spectra. Data were analyzed using LP900 software. A sample having an absorbance of 0.3 OD at a wavelength of 355 nm was first degassed with Ar for about 10 minutes before measurement.
  • the attenuation curve is fitted to the following equation, and the obtained lifetime values are listed.
  • the constant rate is calculated as follows:
  • a monocysteine mutant of PSP2 (refer to Table 3, 50 ⁇ M) in a reaction buffer (150 mM Tris-HCl buffer pH 8.8, 30% DMF) at room temperature with 100 ⁇ M tris (2-carboxyethyl) Phosphine (TCEP) treatment for 5 minutes. Then, a labeling reaction was performed at room temperature for 12 hours by adding 250 ⁇ M N- (2,6,2-terpyridin-4-yl) -iodoacetamide (the compound 7 synthesized in the example). After terpyridine modification, unbound terpyridine molecules were removed by dialysis against 10 mM Tris-HCl pH 8 buffer (Amicon Ultra-0.5 Centrifugal Filter Unit 3K, at least 3 times). The obtained mutants were then analyzed by LC-MS.
  • Figure 11 shows the quantitative formation of the PSP2-95C-terpy complex.
  • Photocatalytic carbon dioxide reduction was performed in a glass headspace bottle (total volume 10 ml) sealed with a corrugated stopper.
  • the reaction solution volume is 200 ⁇ L: 100 mM Tris-HCl buffer (pH 8.0, 50% DMF, to increase 4- (2,3-dihydro-1H-benzo [d ] Imidazol-2-yl) benzene-1,2-diol (BIH) derivative solubility) terpyridine nickel complex modified 40 ⁇ M PSP2 monocysteine mutant protein (for example, PSP2T1 or PSP2T2) and 80 ⁇ M Ni (ClO 4 ) 2 , 100 mM NaHCO 3 , 100 mM 4- (2,3-dihydro-1H-benzo [d] imidazol-2-yl) benzene-1,2-diol (BIH) sacrificial reducing agent (SR) were added to the glass headspace vial together.
  • Tris-HCl buffer pH
  • the sample was bubbled with argon (Ar) for 10 minutes, and then irradiated with a 300W Xe lamp (MICROSOLAR300, Beijing Perfilai Technology Co., Ltd.) with an AM 1.5 filter to simulate the solar spectrum.
  • a cutoff filter (UVCUT400) is used to achieve visible light ( ⁇ > 400nm) irradiation.
  • Gas chromatography (GC) (SRI instruments, 8160C GC) was used to analyze the photocatalytic gas generation rate.
  • Photon hits / molecules / second were calculated according to the method of reference 42 (see pages 60-61 for details).
  • the photon flux or intensity I is calculated by:
  • I is the number of photons per unit surface area per second
  • E is the number of Einsteins per unit area per second
  • N A is the Avogadro constant with a value of 6.02 * 10 23 .
  • the structure is roughly square, and each side of the square is long It is illuminated by about 16 photons per second, but not all of these photons are absorbed.
  • the target size of the photosensitizer should be calculated according to the following equation:
  • ⁇ is the target size
  • ⁇ is the molar extinction coefficient
  • N A is the Avogadro constant
  • the extinction coefficient at the maximum absorption wavelength of 375 nm is 23 mM -1 cm -1 .
  • the average extinction coefficient in the absorption region (400 nm to 450 nm) is about 3.65 mM -1 cm -1 .
  • TON co represents the carbon monoxide conversion number
  • Hits is the Hits calculated in Section 3.3.
  • the carbon monoxide concentration (in ppm) detected by gas chromatography was converted to the total number of moles of carbon monoxide produced. Because it takes 2 electrons to generate one molecule of carbon monoxide, a factor of 2 is included in the calculation.
  • the photon wavelength (400-450 nm) was determined from the absorbance of the protein mutant containing BpA, and the flux was calculated.
  • the calculation is based on the number of photons absorbed by PSP2T2 after 3 hours photolysis, and the calculated quantum efficiency (QE) is 2.6%.
  • the quantum efficiency (QE) based on incident photons is calculated according to the method described in reference 43:
  • the incident photons number can be calculated from the incident photon flux 1.2 ⁇ 10 21 photons ⁇ cm -2 ⁇ h -1 (at 130mW cm -2 ), and in the case of the present inventors' study, the irradiation area It is 1 cm 2 . After 3 hours photolysis, 0.2 ⁇ mol of carbon monoxide (CO) was produced, and the total volume of the reaction system was 0.2 ml. Based on the total number of irradiated photons, the QE calculated for the PSP2T2 system was 0.0067%.
  • the present inventors In order to design a photocatalytic carbon dioxide reductase, the present inventors first converted a fluorescent protein (FP) 13-14 into a photosensitizer protein (PSP) ( Figure 1). In order to achieve this goal, the following conditions must be met: (1) the PSP must be able to effectively absorb visible light; (2) when absorbing photons, the PSP must be converted into a photoexcited state (PSP *) that can exist for a longer time, thereby promoting Electron transfer reaction, which leads to the formation of PSP radicals (PSP ⁇ ); (3) PSP ⁇ must be a strong reducing agent that can drive the reduction of carbon dioxide reduction catalysts. Because carbon dioxide is highly inert, the reduction of carbon dioxide reduction catalysts usually requires a high overpotential 5.
  • PSP photosensitizer protein
  • Benzophenone is known to nearly 100% quantum efficiency from the excited singlet state to the triplet state crossing lines, having a lifetime of the triplet state is 5 times the original 10, which allows the occurrence of sacrificial reducing agent (SR) reduction 17- 21 .
  • the present inventors envisage that in the sfYFP mutant containing BpA66, the tripeptide Gly65-BpA66-Gly67 can be spontaneously and catalytically converted to (E) -4- (4-benzoylbenzylidene) -1,2-di
  • the chromophore of methyl-1H-imidazol-5 (4H) -one (BpAChm, structure shown in Figure 1f) should cross to the triplet state with high quantum efficiency.
  • the inventors envisage that once the FP chromophore is photochemically reduced, a color change should be observed. To test this assumption, the inventors first incorporated BpA into position 66 of sfYFP, and irradiated the mutant protein with a 405 nm laser (100 mW / cm 2 ) in the presence of 10 mM sodium dithionite. Since the 203 amino acid residue of sfYFP is tyrosine, it undergoes a rapid electron transfer process with the PSP chromophore due to the formation of a pi-stack. Therefore, PSP cannot be detected after light irradiation. . This may be due to the electron transfer from Tyr203 to PSP * and the consequent rapid charge binding.
  • the inventors mutated Tyr203 to Phe.
  • the sfYFP-BpA66-Phe203 double mutant (PSP1, SEQ ID NO: 4) changed color from yellow to red 30 seconds after laser irradiation ( Figure 1b, Figure 5). Irradiation leads to the disappearance of 390nm peak at 555nm and 765nm with two new peaks appear, indicating that photochemical reduction reaction has taken place 22.
  • the resulting photochemical product is called PSP1 ⁇ .
  • the inventors investigated whether a weaker biologically related reducing agent can promote the photochemical reduction of PSP1 .
  • the inventors found that sodium ascorbate cannot drive the photochemical reduction of PSP1.
  • the inventors mutated residues Phe203 and His148, and the residues near the PSP1 chromophore were mutated to Aspartic acid, glutamic acid or lysine.
  • PCET 23-24 proton coupled electron transfer (PCET) 23-24 (proton coupled electron transfer (PCET) 23-24 avoids the generation of high-energy anionic group intermediates, reduces the PSP ⁇ state energy, and therefore increases the energy difference between PSP * and PSP ⁇ , which The electron transfer rate from SR to PSP * will be accelerated to form PSP ⁇ .
  • E ⁇ -1.46V cannot accurately represent the same reduction potential of PSP2.
  • the UV-Vis spectrum showed that even when an 80-fold excess of Eu (II) -DTPA was used as the reducing agent, no PSP2 ⁇ absorption peak appeared (Fig. 6d). Based on these data, the inventors can speculate that the reduction potential of PSP2 ⁇ is less than -1.14V.
  • PSP2 ⁇ In order to test whether PSP2 ⁇ is sensitive to oxygen, the present inventors first produced PSP2 ⁇ by light irradiation, and then incubated it in the presence of air in 100 mM Tris-HCl pH 7.0 buffer for 20 minutes. As shown in Fig. 9, PSP2 ⁇ reacts with oxygen to generate PSP2. This light cycle can be repeated multiple times. The reversible formation of PSP2 ⁇ indicates that the reaction of PSP2 ⁇ with oxygen will not irreversibly destroy the chromophore. Since many carbon dioxide reduction catalysts are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of PSP-based catalysts.
  • the dihedral angle between two benzene rings in benzophenone is 56 ° 26 , which indicates that the two benzene rings do not form a conjugated ⁇ -electron system.
  • PSP2 ⁇ free radical state
  • one benzene ring in BpA66 proceeds The apparent dihedral rotation (Figure 2e) reduces the dihedral angle between the two benzene rings by 29.1 ° ( Figure 2d), resulting in the formation of an extended conjugated ⁇ -electron system and a significantly red-shifted UV-Vis spectrum.
  • this is the first report on the crystal structure of protonated and neutral benzophenone radicals.
  • Detailed structural information about the super-reducing benzophenone radicals caged in the PSP2 ⁇ protein rigid structure cage provides the necessary understanding for using this powerful reagent to drive challenging enzyme reactions.
  • N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) was specifically conjugated to PSP2 (to obtain LC-
  • MS spectrum confirms that, see Figure 10, Compound 7 is specifically conjugated to the introduced cysteine).
  • the inventors determined the efficiency of these cysteine mutants to catalyze a photocatalytic carbon dioxide reduction reaction in the presence of divalent nickel ions.
  • the PSP2-95C mutant has a higher carbon dioxide reducing activity after being modified with a terpyridine nickel (II) complex, it is called PSP2T1.
  • PSP2T1 terpyridine nickel
  • Conditions in the absence of Ni (II), NaHCO 3 , BIH, BpA66 incorporation or PSP and N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) Below, no significant amount of carbon monoxide is generated, which indicates that all the above components are necessary for photocatalytic carbon dioxide reduction (Figure 3c).
  • TEM transmission electron microscopy
  • PSP2-95C93Y97Y SEQ ID NO: 10
  • the inventors envisage that the presence of two tyrosine residues at 95C covalently linked to the catalyst terpyridine nickel complex may serve as a local proton donor to promote proton-coupled electron transfer to the carbon dioxide substrate, which would reduce the energy for carbon dioxide reduction Obstacle 5 .
  • PSP2-95C93Y97Y was modified by the catalyst N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) and behaved in the presence of divalent nickel ions (ie, PSP2T2 was obtained).
  • the inventors measured the transient absorption spectrum of the transient species produced by photoexcitation.
  • PSP2 was irradiated with a 355 nm laser
  • the inventors observed a recovery of the absorption at the 380 nm ground state and the attenuation of a new peak at 430 nm, which indicates the formation of the PSP2 triplet excited state (PSP2 *, Figure 4a / b).
  • the decay lifetime of PSP2 * is 123 ⁇ s, which is about 10 5 times the lifetime of the singlet excited state of fluorescent proteins, and is such as tris (bipyridine) ruthenium (II) halide ([Ru (bpy) 3 ] 2+ ) ( ⁇ 0.5 ⁇ s )
  • the triplet excited state lifetime of the photosensitizer is more than 200 times 28 .
  • the long lifetime of PSP2 * is important to promote an efficient electron transfer reaction between the electron donor and PSP2.
  • the transient absorption spectrum shows that as the ascorbate concentration increases, the PSP2 * lifetime decreases and a new peak appears at 500-560nm.
  • C represents cysteine, and the number before C indicates the site of mutation to cysteine.
  • PSP2 photosensitizing protein
  • PSP2 can be regarded as a protein analogue of [Ru (bpy) 3 ] 2+ containing no precious metals. Since PSP2 is genetically encoded, it can be easily introduced into various organisms and co-localized with specific protein complexes. This is difficult to achieve with small molecule or nanocrystalline photosensitizers.
  • PSP Compared with semiconductor nanocrystals and small molecule photosensitizers, PSP offers unique advantages, such as higher compatibility with a wide range of biological systems, no dependence on precious metals, mutation-based switchable photochemical properties, and self-assembly The ability to create accurate three-dimensional structures, which can allow for modular extension of its functionality and accurate mechanism characterization. As a result, PSP can potentially sensitize a variety of challenging chemical transformations, involving diverse fields such as solar energy conversion, photobiology, environmental restoration, and industrial biology.
  • PSP2T simple design captures the essence of 3-4 complex natural photosynthesis mechanism provides a valuable model for the study of protein 29 in a variety of electronic mechanisms / proton transfer, and through rational design and directed evolution for the next generation
  • Photosensitizers such as the most famous [Ru (bpy) 3 ] 2+
  • Utilizing the extraordinary self-assembly capabilities of proteins 30-31 , diverse enzyme-catalyzed reactions, and the inventor's ability to rapidly improve the rational design of miniature proteins 32-40 the design of PSP will introduce new chemical reactions in biological systems Created multiple exciting opportunities.

Landscapes

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

Abstract

La présente invention concerne un gène codant pour une protéine de photosynthèse artificielle et une utilisation associée. Plus particulièrement, l'invention concerne une protéine de photosynthèse artificielle, la protéine comprenant une protéine fluorescente (FP), un résidu d'acide aminé chromophore étant modifié par un photosensibilisateur de façon à être converti en une protéine photosensibilisée (PSP). L'invention concerne une réductase de dioxyde de carbone photosensible, comprenant la protéine de photosynthèse artificielle et un catalyseur de réduction de dioxyde de carbone conjugué à la protéine de photosynthèse artificielle. La protéine de photosynthèse artificielle construite et la réductase de dioxyde de carbone photosensible sont plus compatibles avec une large gamme de systèmes biologiques, ne reposent pas sur des métaux précieux, peuvent potentiellement sensibiliser une pluralité de transformations chimiques difficiles, et se rapportent à divers domaines tels que la conversion d'énergie solaire, la photobiologie, la restauration environnementale et la biologie industrielle.
PCT/CN2019/093277 2018-09-30 2019-06-27 Gène codant une protéine de photosynthèse artificielle et utilisation associée WO2020062977A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811156095.0A CN110964088B (zh) 2018-09-30 2018-09-30 一种可基因编码的人工光合作用蛋白质及其应用
CN201811156095.0 2018-09-30

Publications (1)

Publication Number Publication Date
WO2020062977A1 true WO2020062977A1 (fr) 2020-04-02

Family

ID=69952730

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/093277 WO2020062977A1 (fr) 2018-09-30 2019-06-27 Gène codant une protéine de photosynthèse artificielle et utilisation associée

Country Status (2)

Country Link
CN (1) CN110964088B (fr)
WO (1) WO2020062977A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116023460B (zh) * 2022-10-28 2023-11-10 无锡佰翱得生物科学股份有限公司 一种StayGold黄色荧光蛋白及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1558222A (zh) * 2004-02-03 2004-12-29 复旦大学 生物光敏蛋白-纳米半导体复合光电极的制备方法
CN102692400A (zh) * 2012-05-28 2012-09-26 浙江农林大学 一种用于激活微生物体内光敏蛋白的装置及应用
WO2016019075A1 (fr) * 2014-07-29 2016-02-04 Circuit Therapeutics, Inc. Système et procédé pour thérapie optogénétique
CN105420203A (zh) * 2014-09-03 2016-03-23 中国科学院生物物理研究所 酪氨酸类似物翻译系统和基因编码的蛋白质光致电子转移荧光传感器蛋白家族

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106047846B (zh) * 2013-03-22 2020-02-04 中国科学院生物物理研究所 8-羟基喹啉丙氨酸翻译系统及其应用
CN106591343B (zh) * 2016-11-29 2020-02-18 湖北大学 一种超折叠绿色荧光蛋白介导异源蛋白在大肠杆菌中分泌表达方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1558222A (zh) * 2004-02-03 2004-12-29 复旦大学 生物光敏蛋白-纳米半导体复合光电极的制备方法
CN102692400A (zh) * 2012-05-28 2012-09-26 浙江农林大学 一种用于激活微生物体内光敏蛋白的装置及应用
WO2016019075A1 (fr) * 2014-07-29 2016-02-04 Circuit Therapeutics, Inc. Système et procédé pour thérapie optogénétique
CN105420203A (zh) * 2014-09-03 2016-03-23 中国科学院生物物理研究所 酪氨酸类似物翻译系统和基因编码的蛋白质光致电子转移荧光传感器蛋白家族

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAO, LIMEI ET AL.: "Research Progress on Red Fluorescent Protein", FOREIGN MEDICAL SCIENCES (SECTION OF PHARMACY), vol. 33, no. 2, 30 April 2006 (2006-04-30), pages 131 - 136 *
LI, JIASONG: "Crystal Structure of MjTyrRS Mutants and Genetic Incorporation of Unnatural Amino Acids for the Design of Fluorescent Proteins", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE), no. 6, 15 June 2018 (2018-06-15) *
LIU, X.H.: "A genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic C02-reducing enzyme", NATURE CHEMISTRY, vol. 10, 5 November 2018 (2018-11-05), pages 1201 - 1206, XP036642750 *
LIU, XIAOHONG ET AL.: "Gene Encoding of Unnatural Amino Acids Extended Fluorescent Protein Chromophores", THE 8TH NATIONAL CONFERENCE ON CHEMICAL BIOLOGY OF CHINA, 31 December 2013 (2013-12-31) *

Also Published As

Publication number Publication date
CN110964088B (zh) 2021-07-13
CN110964088A (zh) 2020-04-07

Similar Documents

Publication Publication Date Title
Liu et al. A genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic CO2-reducing enzyme
Okura Photosensitization of porphyrins and phthalocyanines
Filippini et al. Mapping the surface groups of amine-rich carbon dots enables covalent catalysis in aqueous media
Soltau et al. Aqueous light driven hydrogen production by a Ru–ferredoxin–Co biohybrid
Tong et al. Pore‐Environment‐Dependent Photoresponsive Oxidase‐Like Activity in Hydrogen‐Bonded Organic Frameworks
Niu et al. Halogen substituted A2B type Co (III) triarylcorroles: Synthesis, electronic structure and two step modulation of electrocatalyzed hydrogen evolution reactions
US20070227590A1 (en) Supramolecular Complex of Pyridylnaphthalenediimide with Zinc Porphyrin Dendrimer Having Multiplicity of Artificial Photosynthetic Reaction Center
Dime et al. Control over the oxidative reactivity of metalloporphyrins. Efficient electrosynthesis of meso, meso-linked zinc porphyrin dimer
Cai et al. “Off–on–off” type of selectively pH-sensing 8-hydroxyquinoline-substituted gallium (iii) corrole
Bano et al. Off-on detection of glutathione based on the nitrogen, sulfur codoped carbon quantum dots@ MnO2 nano-composite in human lung cancer cells and blood serum
Fukuzumi et al. Bioinspired artificial photosynthesis systems
Gao et al. Iron–iron hydrogenase active subunit covalently linking to organic chromophore for light-driven hydrogen evolution
Yu et al. Stable iso-bacteriochlorin mimics from porpholactone: Effect of a β-oxazolone moiety on the frontier π-molecular orbitals
Sugiura et al. Environment of TyrZ in Photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein
Kang et al. Rational design of a miniature photocatalytic CO2-reducing enzyme
David et al. N-Arylated bisferrocene pyrazole for the dual-mode detection of hydrogen peroxide: an AIE-active fluorescent “turn ON/OFF” and electrochemical non-enzymatic sensor
WO2020062977A1 (fr) Gène codant une protéine de photosynthèse artificielle et utilisation associée
Liu et al. A Mo2-ZnP molecular device that mimics photosystem I for solar-chemical energy conversion
Wu et al. Artificial photosynthetic assemblies constructed by the self-assembly of synthetic building blocks for enhanced photocatalytic hydrogen evolution
JP2018034152A (ja) 多電子酸化還元触媒
Liu et al. Synthesis of a green [60] fullerene derivative through cage-opening reactions
Zhang et al. Enzyme-Mimetic Photo-decarboxylation Based on Geometry-Dependent Supramolecular Association
Rand et al. The oxygen-independent coproporphyrinogen III oxidase HemN utilizes harderoporphyrinogen as a reaction intermediate during conversion of coproporphyrinogen III to protoporphyrinogen IX
Vargas et al. Synthesis of a highly emissive carboxylated pyrrolidine-fused chlorin for optical sensing of TATP vapours
Duan et al. Synthesis of tetra (trimethylammonio) phthalocyanato zinc tetraiodide,[ZnPc (NMe3) 4] I4, and a spectrometric investigation of its interaction with calf thymus DNA

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: 19865600

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19865600

Country of ref document: EP

Kind code of ref document: A1