CN114686452B

CN114686452B - Artificial protein skeleton and application thereof

Info

Publication number: CN114686452B
Application number: CN202110465975.1A
Authority: CN
Inventors: 马田
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-12-31
Filing date: 2021-04-28
Publication date: 2024-06-18
Anticipated expiration: 2041-04-28
Also published as: CN114686452A

Abstract

The invention provides an artificial protein skeleton and application thereof. The invention first provides an artificial protein backbone peptide fragment molecule comprising a non-covalent linkage recognition peptide derived from a type I non-iterative polyketide synthase. The invention specifically provides peptide fragment molecules consisting of amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 12 and encoding nucleic acid molecules thereof. The peptide fragment molecule or the nucleic acid molecule can be applied to an artificial protein skeleton technology in a microorganism, is used for assembling cell membrane enzymes or is applied to assembling cytoplasmic enzymes and cell membrane enzymes, and can improve the synthesis efficiency; or the recognition characteristic is utilized to realize the specific recognition, connection and co-localization of the enzyme.

Description

Artificial protein skeleton and application thereof

Technical Field

The invention relates to an artificial protein skeleton technology, in particular to an artificial protein skeleton applicable to a microorganism, a preparation method and related applications thereof.

Background

When a microbial synthesis system is artificially constructed, multi-step catalytic reactions are mostly carried out for synthesizing a target product. Natural biocatalytic systems typically form physically, spatially ordered multi-enzyme complexes, enzyme molecule scaffolds, or reaction microdomains within cells, and this efficient organization leads to efficient catalytic capabilities. However, the artificially constructed microbial synthesis system does not have the high-efficiency organization, namely, the distribution of the catalytic enzymes participating in synthesis in cells is different, the most direct influence is that the mass transfer efficiency of intermediate metabolites is lower, so that the problems of low synthesis efficiency of target pathways, unbalanced metabolic flows, influence on cell growth and the like are caused, the problem that if the technology of spatially highly ordered cathepsin molecules can be developed for the biological manufacturing potential (Dahl R,Zhang F,Alonso-Gutierrez J,Baidoo E,Batth T,Redding-Johanson A,Petzold C,Mukhopadhyay A,Lee T,Adams P.Engineering dynamic pathway regulation using stress-response promoters.Nat Biotechnol,2013,31(11):1039-1046). of the microbial synthesis system, namely, the artificial protein skeleton technology, the intermediate diffusion is prevented, the transmission efficiency of the intermediate metabolites is improved, the flow direction loss of the intermediate metabolites to competing pathways is reduced, the degradation loss of unstable intermediate is reduced, the intermediate passing time is shortened, the adverse balance is avoided, and the problem that the synthesis efficiency of the microbial synthesis system is low is improved to a great extent.

The artificial protein skeleton technology has the characteristics of simplicity, convenience, easiness in operation and high efficiency, the total pathway flux can be increased and the metabolic load can be reduced through engineering between a simple polypeptide interaction domain and a specific ligand thereof, and based on the characteristics, researchers have developed a few artificial protein skeleton technologies for enzyme assembly at present. For example, ligands for animal cell signaling can specifically recognize with receptors, and by utilizing this property Dubber et al developed a scaffold for protein assembly in E.coli, designated GBD _xSH3_yPDZ_z, by assembling three enzymes of the mevalonate pathway from acetyl-CoA synthesis by protein-peptide interactions between the ligand and the receptor, a significant improvement in target product yield was achieved (Dueber J,Wu G,Malmirchegini G,Moon T,Petzold C,Ullal A,Prather K,Keasling J.Synthetic protein scaffolds provide modular control over metabolic flux.Nat Biotechnol,2009,27(8):753-759).

However, the existing protein skeleton technology has very limited types of assembled enzymes, and is mainly applied to shorter metabolic pathways, such as assembly of most two enzymes, assembly of more than three enzymes, and very few developed protein skeleton technologies, and the application scenario is not clear.

Disclosure of Invention

The invention aims at providing a novel technology of an artificial protein skeleton.

The invention aims at the defects of the prior art, and develops an artificial protein skeleton technology (DEBS-EAL, deoxyerythronolide synthetase-enzyme assembly line) (figure 1) applied to microorganisms, wherein non-covalent connecting peptides capable of mutually specific recognition among structural modules are based on erythromycin polyketide synthase structures, and as the core of the technology, the connecting peptides capable of mutually specific recognition can be assembled and expressed on catalytic enzymes to be assembled respectively and applied to cytoplasmic enzymes and membrane enzymes of astaxanthin metabolic pathway varieties respectively.

In particular, in one aspect, the invention provides an artificial protein backbone peptide fragment molecule comprising a non-covalently linked recognition peptide derived from a type I non-iterative polyketide synthase.

According to a specific embodiment of the invention, the artificial protein backbone peptide fragment molecule of the invention, wherein the type I non-iterative polyketide synthase comprises one or more of erythromycin polyketide synthase, rapamycin polyketide synthase, tacks Mo Siju ketone synthase, gold chain fungus polyketide synthase, avermectin polyketide synthase, spinosad polyketide synthase, epothilone polyketide synthase, salinomycin polyketide synthase. Preferably, the peptide fragment molecules may be derived from specific recognition peptides that are mutually recognizable between modules of type I non-iterative polyketide synthases.

According to some embodiments of the invention, the invention provides a DEBS-EAL artificial protein backbone sequence, wherein:

Based on the gene sequence of I-type non-iterative polyketide synthase-erythromycin polyketide synthase, two pairs of non-covalent connection gene sequences are respectively positioned at the C end (M2-C) of the module 2, the N end (M3-N) of the module 3, the C end (M4-C) of the module 4 and the N end (M5-N) of the module 5, and the two pairs are respectively and specifically identified and connected. The rapamycin polyketide synthase has 2 pairs of non-covalent linkage sequences, rapA-C, rapB-N and RapB-C, rapC-N respectively. The tacks Mo Siju ketone synthase non-covalent linkage sequence has 2 pairs, fkbB-C, fkbC-N and FkbC-C, fkbA-N respectively. The ligase peptide fragment molecules have amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 12.

In another aspect, the invention also provides a nucleic acid molecule encoding an artificial protein backbone peptide fragment molecule (ligase peptide fragment), which encodes the ligase peptide fragment molecule described above. In some embodiments of the invention, the nucleic acid molecule has the nucleotide sequence shown as SEQ ID NO. 13-SEQ ID NO. 24.

In another aspect, the invention also provides the use of the artificial protein backbone peptide fragment molecule or the nucleic acid molecule in the preparation of an artificial protein backbone, wherein the target protein is assembled by using a non-covalent linkage recognition peptide derived from type I non-iterative polyketide synthase as an artificial protein backbone. Preferably, the protein of interest is a non-polyketide synthase.

In another aspect, the invention also provides a construct comprising a nucleic acid molecule according to the invention.

According to a specific embodiment of the invention, the construct according to the invention further comprises a nucleic acid molecule of a metabolic pathway gene of interest.

According to a specific embodiment of the invention, the construct according to the invention may be a plasmid, a host cell or a microorganism.

In another aspect, the present invention also provides a method for synthesizing a metabolite of an organism using an artificial protein scaffold, comprising:

introducing a non-covalent linkage recognition peptide derived from a type I non-iterative polyketide synthase into a target product metabolic pathway enzyme to construct a microbial mutant strain containing an artificial protein backbone;

Fermenting and culturing the microorganism mutant strain to obtain the target metabolite.

According to a specific embodiment of the present invention, in the method for synthesizing a metabolic product of an organism using an artificial protein scaffold of the present invention, the microorganism may be any one or more of escherichia coli, saccharomyces cerevisiae, pichia pastoris, bacillus, aspergillus oryzae, aspergillus nidulans, aspergillus niger, neurospora crassa, alternaria alternata, or fusarium.

According to a specific embodiment of the present invention, in the method for synthesizing a metabolic product of an organism using an artificial protein backbone, the metabolic product may be any one or more of terpenoids, vitamins, polyketides, non-ribosomal peptides, or ribosomal peptides, and is not limited to astaxanthin.

According to a specific embodiment of the present invention, the type I non-iterative polyketide synthase comprises one or more of erythromycin polyketide synthase, rapamycin polyketide synthase, tacks Mo Siju ketone synthase, aurin polyketide synthase, avermectin polyketide synthase, spinosyn polyketide synthase, epothilone polyketide synthase, salinomycin polyketide synthase. Preferably, the non-covalently linked recognition peptide is derived from a specific recognition peptide that is mutually recognizable between the modules of a type I non-iterative polyketide synthase. In some embodiments of the invention, the non-covalently linked recognition peptide comprises one or more of the polypeptides consisting of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 12.

According to a specific embodiment of the present invention, in the method for synthesizing a metabolic product of an organism using an artificial protein scaffold of the present invention, the target metabolic pathway enzyme may be a cytoplasmic enzyme and/or a cell membrane enzyme.

According to some embodiments of the present invention, there is provided a method of constructing a DEBS-EAL artificial protein scaffold technology and introducing it into a cellular membrane enzyme of an astaxanthin synthesis pathway, comprising the steps of:

The M2-C connecting sequence is fused to the C end of the gene crtY, the M3-N and M4-C are respectively fused to the N end and the C end of the gene crtZ, and the M5-N connecting sequence is fused to the N end of the gene crtW, so that a bacterial mutant A2 simultaneously containing crtY _M2C、crtZ_M3N-M4C、crtW_M5N is constructed.

According to some embodiments of the present invention, the present invention provides a method for constructing a DEBS-EAL artificial protein scaffold technology, and introducing the same into astaxanthin cytoplasmic enzyme and cell membrane enzyme, comprising the steps of:

The M2-C connecting sequence is fused to the C end of the gene IDI, the M3-N and M4-C are respectively fused to the N end and the C end of the gene crtE, and the M5-N connecting sequence is fused to the N end of the gene crtB, so that a bacterial mutant A3 containing IDI _M2C、crtE_M3N-M4C、crtB_M5N at the same time is constructed.

According to some embodiments of the invention, the invention constructs a bacterial mutant A4 using the DEBS-EAL artificial protein backbone technique, comprising the steps of: a RapA-C linker was fused to the C-terminus of the gene IDI, rapB-N and RapB-C to the N-terminus and C-terminus, respectively, of the gene crtE, and a RapC-N linker was fused to the N-terminus of the gene crtB, to construct bacterial mutant A4 containing both Idi _RapA-C、crtE_{RapB-N-RapB-C} and crtB _RapC-N.

According to some embodiments of the invention, the invention constructs a bacterial mutant A5 using the DEBS-EAL artificial protein backbone technique, comprising the steps of: a FkbB-C linker was fused to the C-terminus of the gene IDI, fkbC-N and FkbC-C to the N-terminus and C-terminus, respectively, of the gene crtE, and a FkbA-N linker was fused to the N-terminus of the gene crtB, to construct bacterial mutant A5 containing both Idi _FkbB-C、crtE_{FkbC-N-FkbC-C} and crtB _FkbA-N.

According to some embodiments of the invention, the invention constructs a bacterial mutant A6 using the DEBS-EAL artificial protein backbone technique, comprising the steps of: the M2-C junction sequence is fused to the C-terminal of the gene IDI, and the M3-N is fused to the N-terminal of the gene crtE, so as to construct a bacterial mutant A6 containing IDI _M2C、crtE_M3N at the same time.

According to some embodiments of the invention, the invention constructs a bacterial mutant A7 using the DEBS-EAL artificial protein backbone technique, comprising the steps of: a RapA-C connecting sequence is fused to the C end of the gene IDI, rapB-N is fused to the N end of the gene crtE, and a bacterial mutant A7 containing Idi _RapA-C、crtE_RapB-N is constructed.

According to some embodiments of the invention, the invention constructs a bacterial mutant A8 using the DEBS-EAL artificial protein backbone technique, comprising the steps of: a FkbB-C connecting sequence is fused to the C end of the gene IDI, fkbC-N is fused to the N end of the gene crtE, and a bacterial mutant strain A8 containing Idi _FkbB-C、crtE_FkbC-N is constructed.

In some embodiments of the invention, artificial protein backbone techniques were developed starting from the structure of type I non-iterative polyketide synthase-erythromycin synthesized polyketide synthase. Currently known type I non-iterative polyketide synthases have more than three thousand protein structures, and the specific recognition peptides between the modules in these structures are also diverse, for example, the polyketide synthases (RAPS) of rapamycin can be divided into three modules, and the three modules (RapA and RapB, rapB and RapC) are assembled from two sets of mutually recognizable specific recognition peptides for the entire polyketide synthase multimodule. These specific recognition peptides are different from those in the polyketide synthase structure synthesized by erythromycin, but still have the function of specifically recognizing and assembling. Similar epothilones, salinomycin, etc. all have the same characteristics. The specific recognition peptides can be applied to self-assembly of other path enzymes other than polyketide synthases according to the method for constructing the artificial protein skeleton, namely, the inter-module recognition peptides with potential three thousands of I-type non-iterative polyketide synthases can be applied to assembly application of various enzymes.

The DEBS-EAL artificial protein skeleton technology can be applied to assembly between cell membrane enzymes, and can also be applied to assembly between cytoplasmic enzymes and cell membrane enzymes, so that the synthesis efficiency can be improved, and the later has very obvious improvement effect. According to some specific embodiments of the invention, the method comprises the steps of constructing an artificial protein skeleton expression vector carrying a target enzyme and a mutant strain, and fermenting, extracting, detecting and quantifying the obtained mutant strain to realize 45.8% improvement of the yield of astaxanthin assembled between cell membrane enzymes and 220.5% improvement of the yield of astaxanthin assembled between cell membrane enzymes and cytoplasmic enzymes.

According to specific embodiments of the present invention, the peptide fragment molecules of the present invention as artificial protein backbones may be used with one pair(s) of non-covalently linked recognition peptides derived from type I non-iterative polyketide synthases, or two or more pairs may be used simultaneously for tandem assembly of multiple proteins.

According to some embodiments of the present invention, the peptide fragment molecules as artificial protein scaffold of the present invention may be used with a pair (group) of non-covalently linked recognition peptides derived from non-iterative polyketide synthases of type I and introduced into astaxanthin synthesis pathway enzyme two-enzyme assembly comprising the steps of:

The M2-C junction sequence is fused to the C-terminal of the gene IDI, and the M3-N is fused to the N-terminal of the gene crtE, so as to construct a bacterial mutant A6 containing IDI _M2C、crtE_M3N at the same time.

A RapA-C connecting sequence is fused to the C end of the gene IDI, rapB-N is fused to the N end of the gene crtE, and a bacterial mutant A7 containing Idi _RapA-C、crtE_RapB-N is constructed.

A FkbB-C connecting sequence is fused to the C end of the gene IDI, fkbC-N is fused to the N end of the gene crtE, and a bacterial mutant strain A8 containing Idi _FkbB-C、crtE_FkbC-N is constructed.

According to a specific embodiment of the invention, the artificial protein backbone peptide fragment molecule pair of the invention has specific recognition properties, and can be used for specific recognition, connection and co-localization between enzymes. In some embodiments of the invention, the invention constructs co-localized expression strains in which the DEBS-EAL recognition peptide specifically recognizes the linkage.

In some more specific embodiments, the present invention converts plasmids pQC005 and pQC006 into competent cells of E.coli BL21 (DE 3) containing plasmid pMH1 to obtain E.coli mutant A9.

In other more specific embodiments, the present invention converts plasmids pQC007 and pQC008 to competent cells of E.coli BL21 (DE 3) containing plasmid pMH1 to obtain E.coli mutant A10.

In other more specific embodiments, the present invention converts plasmids pQC009 and pQC010 into competent cells of E.coli BL21 (DE 3) containing plasmid pMH1 to obtain E.coli mutant A11.

In some embodiments of the invention, the invention also provides related uses of the strain.

In summary, the invention provides an artificial protein skeleton capable of assembling a plurality of enzymes, and definitely provides that the technology has very remarkable effect of improving catalytic efficiency when applied to cytosolic enzymes and membrane enzymes, thousands of similar artificial protein skeletons are arranged behind the technology, so that the application environment, application range and optional use library of the artificial protein skeleton can be enriched and expanded, a new strategy and thought are provided for efficient synthesis of an artificial biosynthesis system, and a new break is provided for related application and development of multienzyme assembly. In addition, the artificial protein skeleton can be applied to other situations requiring specific recognition, connection and co-localization.

Drawings

Fig. 1 is a schematic view of technical features of the present invention.

FIGS. 2A to 2M are schematic diagrams showing the structures of plasmids constructed in the examples of the present invention.

FIG. 3 is a schematic diagram of an E.coli mutant constructed in the examples of the present invention.

FIGS. 4A-4B are graphs comparing astaxanthin production (mg/g DCW) as a fermentation product of E.coli mutant strains constructed in examples of the present invention.

FIG. 5 is a graph showing the results of a co-localized fluorescence microscope for protein-specific recognition ligation in an embodiment of the invention.

Detailed Description

The scheme of the present invention will be explained below in connection with specific examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Ligase peptide fragment molecules

In one aspect of the invention, the invention provides non-covalent linking peptides, i.e., ligase peptide fragment molecules, between the various modules of erythromycin polyketide synthase, rapamycin polyketide synthase, and tacks Mo Siju ketone synthase. According to an embodiment of the invention, these ligase peptide fragment molecules have the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 12.

Ligation gene nucleic acid molecules

In another aspect of the invention, the invention provides a connector gene nucleic acid molecule. According to an embodiment of the invention, the ligase nucleic acid molecule encodes the preceding ligase peptide fragment molecule. Thus, the nucleic acid molecules according to embodiments of the present invention are capable of efficiently encoding the ligase peptide fragments described above. According to an embodiment of the invention, the connector gene nucleic acid molecule has a nucleotide sequence shown as SEQ ID NO. 13-SEQ ID NO. 24.

Constructs

In yet another aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises a connector gene nucleic acid molecule as described above and a target metabolic pathway gene nucleic acid molecule. Thus, constructs according to embodiments of the invention may encode ligase peptide fragments and metabolic pathway enzymes by expressing nucleic acid molecules in order to obtain efficient synthesis of the compound of interest.

DEBS-EAL artificial protein skeleton and establishment method thereof

In yet another aspect of the present invention, an artificial protein scaffold technology based on erythromycin polyketide synthase, rapamycin polyketide synthase, and tacks Mo Siju ketone synthase structures and methods of establishing the same are presented. According to an embodiment of the invention, the method comprises: culturing the construct under conditions suitable for expression of the ligase peptide and the pathway enzyme to obtain a culture product; and extracting the target compound from the culture product. Thus, the target compound high-yield strain can be obtained by quantitatively analyzing the target compound.

Example 1 determination of DEBS-EAL Artificial protein backbone sequence

The gene sequence of erythromycin polyketide synthase is analyzed, two pairs of noncovalently connected gene sequences (Gokhale,R.S.Dissecting and exploiting intermodular communication inpolyketide synthases.[J].Science,1999,284(5413):482-485.). are defined through the interaction between a protein structure and a module, are respectively positioned at the C end (M2-C) of the module 2, the N end (M3-N) of the module 3, the C end (M4-C) of the module 4 and the N end (M5-N) of the module 5, and are specifically identified and connected in pairs. Similarly, the analysis defined 2 pairs of rapamycin polyketide synthase non-covalent attachment sequences, rapA-C, rapB-N and RapB-C, rapC-N, respectively; the tacks Mo Siju ketone synthase non-covalent linkage sequence has 2 pairs, fkbB-C, fkbC-N and FkbC-C, fkbA-N respectively. The ligase peptide fragment molecules have amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 12. The nucleic acid sequence of each connecting gene is shown in SEQ ID NO. 13-SEQ ID NO. 24.

M2-C amino acid sequence (SEQ ID NO: 1):

GTEVRGEAPSALAGLDALEGALPEVPATEREELVQRLERMLAALRPVAQAADASGTGANPSGDDLGEAGVDELLEALGRELDGD

M3-N amino acid sequence (SEQ ID NO: 2):

MTDSEKVAEYLRRATLDLRAARQRIRELESD

M4-C amino acid sequence (SEQ ID NO: 3):

FAASPAVDIGDRLDELEKALEALSAEDGHDDVGQRLESLLRRWNSRRADAPSTSAISEDASDDELFSMLDQPFGGGEDL

M5-N amino acid sequence (SEQ ID NO: 4):

MSGDNGMTEEKLRRYLKRTVTELDSVTARLREVEHRAGE

RapA-C amino acid sequence (SEQ ID NO: 5):

FAPAEPEPRLHEQELRRALAGISIDKFREAGVLDTLLRLAAMEGLAVPKPDSESDDEAFVDEMDADALIKHVLEEER

RapB-N amino acid sequence (SEQ ID NO: 6):

MREDQLLDALRKSVKENARLRKANTSLRAAMD

RapB-C amino acid sequence (SEQ ID NO: 7):

MNPRVQSTTLLAEIDRIEKMFTSVTFDDRQASAIKDRLSSVLNKWQRISSPEEVSTTALSSASASEILDFIDREFGDPTA

RapC-N amino acid sequence (SEQ ID NO: 8):

MPEQDKVVEYLRWATAELHTTRAKLEALAAANT

FkbB-C amino acid sequence (SEQ ID NO: 9):

LTLLAPDGNGTPVGGEPPAQVSVGAPATDSEVASPDDELIDDMDADALIAHVLKG

FkbC-N amino acid sequence (SEQ ID NO: 10):

MAENDLIEALRTSVKDNAQLRRENTALRAAAN

FkbC-C amino acid sequence (SEQ ID NO: 11):

DRIGELLSPDDPVTVVLAQLDRLEALVAGVDPGARQADAIGTRLDALLNRWRRETRPTTPAGVLSADATADEIFDLIDRELR

FkbA-N amino acid sequence (SEQ ID NO: 12):

MPEQDKTVEYLRWATTELQRTRAELAAHS

M2-C nucleic acid sequence (SEQ ID NO: 13):

ggtactgaagttagaggtgaagcaccatctgctttggcaggtttagatgctttggaaggtgcattaccagaagttccagctacagaaagagaagaattggttcaaagattggaaagaatgttagctgcattaagaccagttgctcaagctgcagatgcatctggtactggtgctaatccatcaggtgacgatttgggtgaagcaggtgttgatgaattgttagaagctttaggtagagaattggatggtgac

M3-N nucleic acid sequence (SEQ ID NO: 14):

atgactgattctgaaaaggttgcagaatatttgagaagagctacattggatttgagagctgcaagacaaagaattagagaattggaatcagat

M4-C nucleic acid sequence (SEQ ID NO: 15):

tttgctgcatctccagcagttgatattggtgacagattggatgaattggaaaaggctttggaagcattgtcagctgaagatggtcatgatgatgttggtcaaagattggaatctttgttaagaagatggaactctagaagagctgatgcaccatctacttcagcaatttctgaagatgcttcagatgatgaattgttttctatgttggatcaaagatttggtggtggtgaagatttg

M5-N nucleic acid sequence (SEQ ID NO: 16):

atgtcaggtgacaatggtatgactgaagaaaagttgagaagatatttgaagagaactgttacagaattggattctgttacagcaagattaagagaagttgaacatagagctggtgaa

RapA-C nucleic acid sequence (SEQ ID NO: 17):

ttcgcaccggcagaaccggaaccgcgcctgcatgaacaggaactgcgccgtgcactggccggtattagcattgataaatttcgtgaagcaggtgtgctggataccctgctgcgcctggcagcaatggaaggtctggcagtgccgaaaccggatagtgaaagtgatgatgaagcatttgtggatgaaatggatgccgatgcactgattaagcatgttctggaagaagaacgc

RapB-N nucleic acid sequence (SEQ ID NO: 18):

atgcgtgaagatcagctgctggatgccctgcgcaaaagcgtgaaagaaaatgcacgcctgcgtaaagccaataccagcctgcgtgcagcaatggat

RapB-C nucleic acid sequence (SEQ ID NO: 19):

atgaatccgcgcgttcagagcaccaccctgctggcagaaattgatcgcattgaaaaaatgtttaccagcgttacctttgatgatcgtcaggcaagcgccattaaggatcgtctgagcagcgtgctgaataagtggcagcgtattagcagcccggaagaagtgagcaccaccgcactgagcagtgcaagcgccagcgaaattctggattttattgatcgtgaatttggtgacccgaccgcc

RapC-N nucleic acid sequence (SEQ ID NO: 20):

atgccggaacaggataaagttgtggaatatctgcgttgggccaccgccgaactgcataccacccgcgccaaactggaagcactggcagccgcaaatacc

FkbB-C nucleic acid sequence (SEQ ID NO: 21):

ctgaccctgctggcaccggatggcaatggcaccccggttggtggtgaaccgccggcacaggtgagcgttggcgcacctgcaaccgatagcgaagttgcaagtccggatgatgaactgattgatgatatggatgcagatgcactgattgcacatgtgctgaaaggc

FkbC-N nucleic acid sequence (SEQ ID NO: 22):

atggcagaaaatgatctgattgaagccctgcgtaccagtgtgaaagataatgcccagctgcgccgtgaaaataccgccctgcgtgcagccgccaat

FkbC-C nucleic acid sequence (SEQ ID NO: 23):

gatcgcattggcgaactgctgagtccggatgacccggttaccgtggtgctggcacagctggatcgcctggaagcactggtggccggtgtggaccctggcgcacgtcaggctgatgccattggcacccgcctggatgccctgctgaatcgctggcgtcgcgaaacccgtccgaccaccccggcaggtgttctgagcgcagatgcaaccgccgatgaaatttttgatctgattgatcgcgaactgcgt

FkbA-N nucleic acid sequence (SEQ ID NO: 24):

atgccggaacaggataaaaccgttgaatatctgcgttgggcaaccaccgaactgcagcgcacccgtgccgaactggccgcacatagt

example 2 construction of an Artificial protein backbone expression vector carrying an enzyme of interest

All nucleic acid molecules in this example were obtained by PCR amplification using the primers shown in Table 1. The primers used were all synthesized by the same general method as those of the Optimus of the Chemie, inc., and the peptide sequences (M2C and M3N, M C and M5N, rapA-C and RapB-N, rapB-C and RapC-N, fkbB-C and FkbC-N, fkbC-C and FkbA-N) were all synthesized by the same general method of the Style, inc. (pERY. Sup.1 is a plasmid carrying the synthetic genes of M2C and M3N, pERY is a plasmid carrying the synthetic genes of M4C and M5N, pET28a-RAPS is a plasmid carrying the synthetic genes of RapA-C, rapB-N, rapB-C, rapC-N, pET28a-FK506 is a plasmid carrying the synthetic genes of FkbB-C, fkbC-N, fkbC-C, fkbA-N). The fluorescent protein genes used were all synthesized in general Biotechnology Inc. (pET 28a-EGFP is a plasmid carrying EGFP gene, pET28a-mCherry is a plasmid carrying mCherry gene). The DNA polymerase used was purchased from NEB (Q5 DNA polymerase, M0491L), the DNA purification kit was purchased from Omega Bio-Tek (Gel & PCR Clean Up kit D2000, omega Bio-Tek), the multichip cloning kit was purchased from Noruzan Biotechnology Co., ltd (Vazyme C115), the plasmid miniprep kit was purchased from Tiangen Biotechnology Co., ltd (DP 103, TIANGEN), the restriction enzyme was purchased from Thermo Scientific, and plasmid sequencing was accomplished in Optimu, inc.

TABLE 1

The specific construction method of the plasmid is as follows:

1) Construction of pYJ407 plasmid:

The M3N fragment was amplified from plasmid pERY using the pYJ405-1-F/405-1-R primer pair, designated F405-1; the CrtE gene fragment was amplified from plasmid pFZ153(Ma T,Zhou Y,Li X,et al.Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp.ATCC 55669for heterologous overproduction in Escherichia coli[J].Biotechnology Journal,2016,11(2):228-237) using pYJ405-2-F/405-2-R primer pair, designated F405-2; the M4C fragment was amplified from plasmid pERY using the pYJ405-3-F/405-3-R primer pair, designated F405-3; the pYJ405-4-F/405-4-R primer pair amplified the M5N fragment from plasmid pERY, designated F405-4; the pYJ405-5-F/405-5-R primer pair amplified CrtI +CrtB+Idi fragment from plasmid pFZ153, designated F405-5; the pYJ405-6-F/405-6-R primer pair amplified the M2C fragment from plasmid pERY, designated F405-6; the pYJ405-7-F/405-7-R primer pair amplified CrtY +CrtZ+ CrtW + vector fragment from plasmid pFZ153, designated F405-7. Subsequently, overlap PCR amplification was performed using the pYJ405-1-F/405-3-R primer pair to obtain the splice fragments F405-OL1 of F405-1, F405-2 and F405-3; and performing overlap PCR amplification by using a pYJ405-4-F/405-6-R primer pair to obtain spliced fragments F405-OL2 of F405-4, F405-5 and F405-6, and finally performing three-fragment splicing on F405-OL1, F405-OL2 and F405-7 by using a multi-fragment cloning kit to obtain the pYJ405 plasmid. The M3N+ CrtE +M4C fragment, designated F407-1, was amplified from plasmid pYJ405 using the pYJ405-2-F/407-1-R primer pair; the rtI fragment was amplified from plasmid pFZ153 using the 407-2-F/407-2-R primer pair, designated F407-2; M5N was amplified from plasmid pERY using the 407-3-F/407-3-R primer pair, designated F407-3; the rtB fragment was amplified from plasmid pYJ405 using the 407-4-F/1536-1-R primer pair, designated F407-4; the M2C+IDI+ CrtY +CrtZ fragment, designated F407-5, was amplified from plasmid pYJ405 using the 1536-2-F/1536-2-R primer pair; the CrtW + vector fragment, designated F407-6, was amplified from the pYJ405 plasmid using the 1536-3-F/pYJ405-1-R primer pair. Then, overlap PCR amplification was performed using the pYJ405-2-F/1536-1-R primer pair to obtain the splice fragments F407-OL of F407-1, F407-2, F407-3 and F407-4. Finally, F407-OL, F407-5 and F407-6 fragments were spliced using a multi-fragment cloning kit to obtain the pYJ407 plasmid (FIG. 2A).

2) Construction of pYJ408 plasmid:

the CrtE + CrtI +CrtB fragment, designated F408-1, was amplified from plasmid pFZ using the pYJ408-1-F/1536-1-R primer pair; the idi+ CrtY fragment, designated F408-2, was amplified from plasmid pFZ153 using the 1536-2-F/pYJ408-2-R primer pair; the M2C fragment was amplified from plasmid pERY using the pYJ408-3-F/pYJ408-3-R primer pair, designated F408-3; the M3N fragment was amplified from plasmid pYJ407 using the pYJ408-4-F/pYJ408-4-R primer pair, designated F408-4; the CrtZ fragment was amplified from plasmid pYJ407 using the pYJ408-5-F/pYJ408-5-R primer pair, designated F408-5; the M4C fragment was amplified from plasmid pYJ407 using the pYJ408-6-F/pYJ408-6-R primer pair, designated F408-6; the M5N fragment was amplified from plasmid pYJ407 using the pYJ408-7-F/pYJ408-7-R primer pair, designated F408-7; the vector fragment was amplified from plasmid pFZ using the pYJ408-8-F/pYJ408-8-R primer pair, designated F408-8. Then, performing overlap PCR amplification by using a pYJ408-1-F/pYJ408-2-R primer pair to obtain spliced fragments F408-OE of F408-1 and F408-2; the overlap PCR amplification was performed using the pYJ408-3-F/pYJ408-7-R primer pair to obtain the splice fragments F408-OL of F408-3, F408-4, F408-5, F408-6 and F408-7. Finally, the F408-OE, F408-OL, F408-8 fragments were spliced using a multi-fragment cloning kit to obtain the pYJ408 plasmid (FIG. 2B).

3) PXS33 construction of plasmid:

The RapB-N fragment was amplified from the pET28a-RAPS plasmid using pXS-1-F/R primer pair, designated 33F1; the CrtE-CrtI-CrtB-Idi fragment, designated 33F2, was amplified from the pFZ153 plasmid using the pXS-2-F/R primer pair; the RapA-C fragment was amplified from pET28a-RAPS plasmid using pXS-3-F/R primer pair, designated 33F3; the CrtY-CrtW-CrtZ fragment, designated 33F4, was amplified from the pFZ153 plasmid using pXS33-4-F and pLS12-11-R primer pairs; the plasmid vector fragment was amplified from the pFZ plasmid using the pLS12-12-F and pXS-5-R primer pairs, designated 33F5. Then performing overlap PCR amplification by using pXS-1-F/pXS-33-2-R primer pairs 33F1 and 33F2 to obtain a 33OE1 fragment; the 33OE2 fragment was obtained by performing overlap PCR amplification with pXS-3-F/pLS 12-11-R primer pair 33F3 and 33F 4. Finally, the 33OE1, 33OE2 and 33F5 fragments were cloned using the Gibson assembly method to obtain the pXS plasmid (FIG. 2C).

4) PXS35 construction of plasmid:

the FkbC-N fragment was amplified from pET28a-FK506 plasmid using pXS-1-F/R primer pair, designated 35F1; a FkbB-C fragment, designated 35F3, was amplified from the pET28a-FK506 plasmid using pXS-3-F/R primer pair. Then performing overlap PCR amplification by using pXS-1-F/pXS 33-2-R primer pairs 35F1 and 33F2 to obtain a 35OE1 fragment; the 35OE2 fragment was obtained by performing overlap PCR amplification with pXS-3-F/pLS 12-11-R primer pair 35F3 and 33F 4. Finally, 35OE1, 35OE2 and 33F5 fragments were cloned using the Gibson assembly method to obtain the pXS plasmid (FIG. 2D).

5) PXS36 construction of plasmid:

The RapB-N fragment was amplified from the pET28a-RAPS plasmid using pXS-1-F/R primer pair, designated 33F1; the RapA-C fragment was amplified from pET28a-RAPS plasmid using pXS-3-F/R primer pair, designated 33F3; the CrtY-CrtW-CrtZ fragment, designated 33F4, was amplified from the pFZ153 plasmid using pXS33-4-F and pLS12-11-R primer pairs; the plasmid vector fragment was amplified from the pFZ153 plasmid using the pLS12-12-F and pXS-5-R primer pairs, designated 33F5; the CrtE fragment was amplified from the pFZ153 plasmid using pXS-2-F/pXS 36-2-R primer pair, designated 36F2; the RapB-C fragment was amplified from the pET28a-RAPS plasmid using pXS-3-F/R primer pair, designated 36F3; the CrtI fragment was amplified from the pFZ153 plasmid using the pXS36-4-F/R primer pair, designated 36F4; the RapC-N fragment was amplified from the pET28a-RAPS plasmid using pXS-5-F/R primer pair, designated 36F5; the CrtB-CrtI fragment was amplified from the pFZ153 plasmid using pXS-6-F and pXS-33-2-R primer pairs, designated 36F6. Then performing overlap PCR amplification by using pXS-1-F/pXS-4-R primer pairs 33F1, 36F2, 36F3 and 36F4 to obtain 36OL1 fragment; the 33OL2 fragment was obtained by performing overlap PCR amplification with pXS-5-F/pLS 12-11-R primer pairs 36F5, 36F6, 33F3 and 33F 4. Finally, 36OL1, 36OL2 and 33F5 fragments were cloned using the Gibson assembly method to obtain pXS plasmid (FIG. 2E).

6) PXS38 plasmid construction:

The FkbC-N fragment was amplified from pET28a-FK506 plasmid using pXS-1-F/R primer pair, designated 35F1; the FkbB-C fragment was amplified from pET28a-FK506 plasmid using pXS-3-F/R primer pair, designated 35F3; the CrtY-CrtW-CrtZ fragment, designated 33F4, was amplified from the pFZ153 plasmid using pXS33-4-F and pLS12-11-R primer pairs; the plasmid vector fragment was amplified from the pFZ153 plasmid using the pLS12-12-F and pXS-5-R primer pairs, designated 33F5; the FkbC-C fragment was amplified from pET28a-FK506 plasmid using pXS-3-F/R primer pair, designated 38F3; the FkbA-N fragment was amplified from the pET28a-FK506 plasmid using pXS-5-F/R primer pair, designated 38F5. Then performing overlap PCR amplification with pXS-1-F/pXS 36-4-R primer pair 35F1, 36F2, 38F3, and 36F4 to obtain 38OL1 fragment; the overlapping PCR amplification was performed with pXS-5-F/pLS 12-11-R primer pair 38F5, 36F6, 35F3 and 33F4 to obtain the 38OL2 fragment. Finally, 38OL1, 38OL2 and 33F5 fragments were cloned using the Gibson assembly method to obtain pXS plasmid (FIG. 2F).

7) Construction of pLS22 plasmid:

The CrtE-CrtI-CrtB-Idi fragment, designated 22F1, was amplified from pFZ153 plasmid using the pYJ405-2-F/pYJ405-5-R primer pair; the M3N-CrtY-CrtZ-CrtW fragment, designated 22F2, was amplified from the pYJ407 plasmid using the pYJ405-6-F/pLS12-11-R primer pair; the vector was amplified from the pYJ407 plasmid using the pLS12-12-F/pYJ405-1-R primer pair, designated 22F3. Thereafter, 22F1, 22F2 and 22F3 fragments were cloned using the Gibson assembly method to obtain the pLS22 plasmid (fig. 2G).

8) Construction of pQC005 plasmid:

The M3N fragment was amplified from the pYJ407 plasmid using the F005-1F/F005-1R primer pair, designated F005-1; the CrtE fragment was amplified from pFZ153 plasmid using the F005-2F/F005-2R primer pair, designated F005-2; amplifying EGFP fragment from pET28a-EGFP plasmid using F005-3F/F005-3R primer pair, designated F005-3; the vector was amplified from pFZ153 plasmid using the F005-4F/F005-4R primer pair, designated F005-4. Thereafter, F005-1, F005-2, F005-3 and F005-4 fragments were cloned using Gibson assembly to obtain pQC005 plasmid (FIG. 2H).

9) Construction of pQC006 plasmid:

Amplifying mCherry fragment from pET28a-mCherry plasmid using F006-1F/F006-1R primer pair, designated F006-1; the M2C fragment was amplified from the pYJ407 plasmid using the F006-2F/F006-2R primer pair, designated F006-2; the vector was amplified from pFZ81 plasmid (Ma T,Zhou Y,Li X,et al.Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp.ATCC 55669for heterologous overproduction in Escherichia coli[J].Biotechnology Journal,2016,11(2):228-237) using the F006-3F/F006-3R primer pair, designated F006-3. Thereafter, the F006-1, F006-2 and F006-3 fragments were cloned using Gibson assembly to give the pQC006 plasmid (FIG. 2I).

10 PQC007 plasmid construction:

The M5N fragment was amplified from the pYJ407 plasmid using the F007-1F/F007-1R primer pair, designated F007-1; the vector was amplified from the pFZ plasmid using the F007-4F/F007-4R primer pair, designated F007-4. Thereafter, F007-1, F005-2, F005-3 and F007-4 fragments were cloned using Gibson assembly to obtain pQC007 plasmid (FIG. 2J).

11 PQC008 plasmid construction:

Amplifying mCherry fragment from pET28a-mCherry plasmid using F006-1F/F008-1R primer pair, designated F008-1; the M4C fragment was amplified from the pYJ407 plasmid using the F008-2F/F008-2R primer pair, designated F008-2; thereafter, the F008-1, F008-2 and F006-3 fragments were cloned using Gibson assembly to obtain pQC008 plasmid (FIG. 2K).

12 PQC009 plasmid construction:

The vector was amplified from pFZ.sup.153 plasmid using the F005-4F/F005-2R primer pair and designated F009-2. Thereafter, F005-3 and F009-2 fragments were cloned using Gibson assembly to obtain pQC009 plasmid (FIG. 2L).

13 PQC010 plasmid construction:

Amplifying mCherry fragment from pET28a-mCherry plasmid using F006-1F/F010-1R primer pair, designated F010-1; amplifying the vector from pFZ81 plasmid with F010-2F/F006-3R primer pair, designated as F010-2; thereafter, F010-1 and F010-2 fragments were cloned using Gibson assembly to obtain pQC010 plasmid (FIG. 2M).

EXAMPLE 3 construction of astaxanthin-synthesizing E.coli mutant

In order to develop the DEBS-EAL artificial protein skeleton technology and explore the applicability thereof, a series of E.coli mutant strains are designed and constructed in the embodiment. In the construction of the strain, plasmids containing different genes were transformed into E.coli by the method of chemical transformation of Ca ²⁺ (molecular cloning Experimental guidelines (third edition): preparation of competent cells of E.coli). First pFZ and pMH1 were transformed into E.coli BL21 (DE 3), competent cells of E.coli BL21 (DE 3) containing plasmids pFZ and pMH1 were prepared, then the target plasmid was transformed into the competent cells, LA plates containing kanamycin (50 mg/L), chloramphenicol (34 mg/L) and ampicillin (100 mg/L) were plated, and the plates were cultured overnight in a biochemical incubator at 37 ℃. Reagents such as CaCl ₂ and glycerol used for preparing competence in the embodiment are purchased from national drug group, and antibiotics are purchased from biological engineering Co.

Construction of A1 Strain: plasmid pFZ153 was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ81 and pMH1 to obtain E.coli mutant A1 (FIG. 3).

A2 strain construction: plasmid pYJ408 was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ and pMH1 to obtain E.coli mutant A2 (FIG. 3) containing both CrtY _M2C、CrtZ_M3N-M4C、CrtW_M5N.

A3 strain construction: plasmid pYJ407 was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ and pMH1 to obtain E.coli mutant containing both Idi _M2C、CrtE_M3N-M4C、CrtB_M5N (FIG. 3).

A4 strain construction: plasmid pXS was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ81 and pMH1 to obtain E.coli mutant A4 containing both Idi _RapA-C、CrtE_{RapB-N-RapB-C} and CrtB _RapC-N (FIG. 3).

A5 strain construction: plasmid pXS was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ81 and pMH1 to obtain E.coli mutant A5 containing both Idi _FkbB-C、CrtE_{FkbC-N-FkbC-C} and CrtB _FkbA-N (FIG. 3).

A6 strain construction: plasmid pLS22 was transformed into competent cells of escherichia coli BL21 (DE 3) containing plasmid pFZ81 and pMH1 to obtain escherichia coli mutant A6 (fig. 3) containing IDI _M2C、CrtE_M3N simultaneously.

Construction of A7 strain: plasmid pXS33 was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ81 and pMH1 to obtain E.coli mutant A7 (FIG. 3) containing both Idi _RapA-C、CrtE_RapB-N.

A8 strain construction: plasmid pXS was transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pFZ81 and pMH1 to obtain E.coli mutant A8 (FIG. 3) containing both Idi _FkbB-C、CrtE_FkbC-N.

EXAMPLE 4 fermentation of astaxanthin-synthesizing E.coli mutant

To evaluate the effect of the introduction of artificial protein backbone on the synthesis of astaxanthin metabolism, the strains A1, A2, A3, A4, A5, A6, A7, A8 constructed in example 3 were cultured, single colonies were picked up, inoculated into 10mL of LB medium containing kanamycin (50 mg/L), chloramphenicol (34 mg/L) and ampicillin (100 mg/L), and cultured overnight at 37℃at 220rpm to obtain seed solutions. The seed solution was transferred to 200mL of LB medium containing the corresponding antibiotic at an inoculum size of 1%, and cultured at 30℃and 200 rpm. When OD ₆₀₀ reached 0.7-0.9, 0.1mM IPTG (isopropyl-. Beta. -d-thiogalactoside) (available from Bio-engineering Co., ltd.) was added to induce expression.

Example 5 extraction, detection and quantification of astaxanthin

Methanol and acetone used in this example were purchased from national drug group, acetonitrile and trifluoroacetic acid were purchased from Thermo company, and astaxanthin standard (a 141428) was purchased from Shanghai aladine Biochemical technologies Co.

The fermentation broth (2 mL) sampled in example 4 was collected by centrifugation and astaxanthin was extracted by vortexing with 1:4 (v: v) methanol and acetone in a dark environment until the cells were colorless. The extract was centrifuged at 13,000rpm for 10min, and the supernatant was taken into a sample bottle and subjected to HPLC detection. Astaxanthin detection and quantification was performed by using an Agilent high performance liquid system (1260) and a column Agilent C18 (5 μm. Times.150 mm. Times.4.6 mm) was used. Samples were eluted using mobile phases A (H ₂ O,0.1% trifluoroacetic acid) and B (acetonitrile, 0.1% trifluoroacetic acid) at a flow rate of 1.0mL/min, according to the following gradient: 0min,50% b;5min,100% B;23min,100% B;24min,50% B;30min,50% B. The column temperature was 25℃and the detection wavelength was 474nm. Astaxanthin yield was quantitatively analyzed based on peak area. As a result of the detection, compared with the control strain A1 (5 mg/g DCW), the astaxanthin yield was increased in the mutant strain carrying both the cellular membrane enzyme (A2) and the cytoplasmic enzyme (A3, A4, A5 and A6), in which the astaxanthin yield was increased by 46% (7.3 mg/g DCW) in the strain A2, the astaxanthin yield was increased by 222% (16.1 mg/g DCW) in the strain A3, the astaxanthin yield was increased by 148% (12.4 mg/g DCW) in the strain A4, and the astaxanthin yield was increased by 127% (11.3 mg/g DCW) in the strain A5 (FIG. 4A). In addition, only a single pair of recognition peptides had an effect of increasing the yield when used, in which the astaxanthin yield of the A6 strain was increased by 139% (12.0 mg/g DCW), the astaxanthin yield of the A7 strain was increased by 114% (10.7 mg/g DCW), and the astaxanthin yield in the A8 strain was increased by 137% (11.9 mg/g DCW) (FIG. 4B).

EXAMPLE 6 DEBS Co-localized expression Strain construction with specific recognition of the ligation by the EAL recognition peptide

A series of E.coli mutants were constructed in this example. In the construction of the strain, plasmids containing different genes were transformed into E.coli by the method of chemical transformation of Ca ²⁺ (molecular cloning Experimental guidelines (third edition): preparation of competent cells of E.coli). First, pMH1 was transformed into E.coli BL21 (DE 3), competent cells of E.coli BL21 (DE 3) containing the plasmid pMH1 were prepared, then the objective plasmid was transformed into the competent cells, LA plates containing kanamycin (50 mg/L), chloramphenicol (34 mg/L) and ampicillin (100 mg/L) were plated, and the plates were cultured overnight in a biochemical incubator at 37 ℃. Reagents such as CaCl ₂ and glycerol used for preparing competence in the embodiment are purchased from national drug group, and antibiotics are purchased from biological engineering Co.

Construction of A9 Strain: plasmids pQC005 and pQC006 were transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pMH1 to obtain E.coli mutant A9 (FIG. 3).

A10 strain construction: plasmids pQC007 and pQC008 were transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pMH1 to obtain E.coli mutant A10 (FIG. 3).

Construction of A11 strain: plasmids pQC009 and pQC010 were transformed into competent cells of E.coli BL21 (DE 3) containing plasmid pMH1 to obtain E.coli mutant A11 (FIG. 3).

Example 7 DEBS Co-localized observations of specific recognition links of EAL recognition peptides

In this example, the microscope immersion oil was Nikon Immersion Oil F cc (MXA 22168), the glass wiping paper was KIMTECH SCIENCE KIMWIPES, and 24 x 50mm (80340-3610) microscope cover slip of Shitai standard grade was purchased from Easybio/Bai Aoyi Jie corporation, the microscope slide-Shitai standard grade was domestic, and a Nikon A1R laser confocal microscope was used for fluorescence observation.

The cultured strains A9, A10 and A11 are taken to be placed on a glass slide, and the glass slide is covered. A100X (oil immersed) objective lens is selected for fluorescence observation, the excitation wavelength and the emission wavelength of an EGFP channel are 488/509nm and the excitation wavelength and the emission wavelength of an mCheery channel are 587/610nm respectively. Confocal microscopy imaging results showed that in the strain expressing the fusion proteins Idi-mCherry-M2C/CrtE-EGFP-M3N, red fluorescence and green fluorescence were mainly concentrated at two levels of cells, achieving co-localization of specific recognition junctions (panel b in fig. 5), whereas in the Idi-mCherry/CrtE-EGFP control strain without recognition peptide, red fluorescence was dispersed in the cytoplasm (panel a in fig. 5). Meanwhile, the M4C-M5N recognition peptide also showed a two-stage aggregation of red fluorescence to cells in E.coli (Panel C in FIG. 5). Therefore, the M2C-M3N or M4C-M5N recognition peptide has the functions of specific recognition, connection and co-localization.

Sequence listing

<110> Shenzhen advanced technology research institute of China academy of sciences

<120> An artificial protein backbone and use thereof

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Ala Leu Glu Gly Ala Leu Pro Glu Val Pro Ala Thr Glu Arg Glu Glu

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Leu Val Gln Arg Leu Glu Arg Met Leu Ala Ala Leu Arg Pro Val Ala

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Gln Ala Ala Asp Ala Ser Gly Thr Gly Ala Asn Pro Ser Gly Asp Asp

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Leu Gly Glu Ala Gly Val Asp Glu Leu Leu Glu Ala Leu Gly Arg Glu

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Leu Asp Gly Asp

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Asp Leu Arg Ala Ala Arg Gln Arg Ile Arg Glu Leu Glu Ser Asp

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Glu Lys Ala Leu Glu Ala Leu Ser Ala Glu Asp Gly His Asp Asp Val

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Gly Gln Arg Leu Glu Ser Leu Leu Arg Arg Trp Asn Ser Arg Arg Ala

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Asp Ala Pro Ser Thr Ser Ala Ile Ser Glu Asp Ala Ser Asp Asp Glu

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Ala Ile Lys Asp Arg Leu Ser Ser Val Leu Asn Lys Trp Gln Arg Ile

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Ser Ser Pro Glu Glu Val Ser Thr Thr Ala Leu Ser Ser Ala Ser Ala

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Ser Glu Ile Leu Asp Phe Ile Asp Arg Glu Phe Gly Asp Pro Thr Ala

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Leu Ala Gln Leu Asp Arg Leu Glu Ala Leu Val Ala Gly Val Asp Pro

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Asn Arg Trp Arg Arg Glu Thr Arg Pro Thr Thr Pro Ala Gly Val Leu

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Ser Ala Asp Ala Thr Ala Asp Glu Ile Phe Asp Leu Ile Asp Arg Glu

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ttagctgcat taagaccagt tgctcaagct gcagatgcat ctggtactgg tgctaatcca 180

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agaagatgga actctagaag agctgatgca ccatctactt cagcaatttc tgaagatgct 180

tcagatgatg aattgttttc tatgttggat caaagatttg gtggtggtga agatttg 237

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<213> M5-N nucleic acid

<400> 16

atgtcaggtg acaatggtat gactgaagaa aagttgagaa gatatttgaa gagaactgtt 60

acagaattgg attctgttac agcaagatta agagaagttg aacatagagc tggtgaa 117

<210> 17

<211> 231

<212> DNA

<213> RapA-C nucleic acids

<400> 17

ttcgcaccgg cagaaccgga accgcgcctg catgaacagg aactgcgccg tgcactggcc 60

ggtattagca ttgataaatt tcgtgaagca ggtgtgctgg ataccctgct gcgcctggca 120

gcaatggaag gtctggcagt gccgaaaccg gatagtgaaa gtgatgatga agcatttgtg 180

gatgaaatgg atgccgatgc actgattaag catgttctgg aagaagaacg c 231

<210> 18

<211> 96

<212> DNA

<213> RapB-N nucleic acids

<400> 18

atgcgtgaag atcagctgct ggatgccctg cgcaaaagcg tgaaagaaaa tgcacgcctg 60

cgtaaagcca ataccagcct gcgtgcagca atggat 96

<210> 19

<211> 240

<212> DNA

<213> RapB-C nucleic acids

<400> 19

atgaatccgc gcgttcagag caccaccctg ctggcagaaa ttgatcgcat tgaaaaaatg 60

tttaccagcg ttacctttga tgatcgtcag gcaagcgcca ttaaggatcg tctgagcagc 120

gtgctgaata agtggcagcg tattagcagc ccggaagaag tgagcaccac cgcactgagc 180

agtgcaagcg ccagcgaaat tctggatttt attgatcgtg aatttggtga cccgaccgcc 240

<210> 20

<211> 99

<212> DNA

<213> RapC-N nucleic acids

<400> 20

atgccggaac aggataaagt tgtggaatat ctgcgttggg ccaccgccga actgcatacc 60

acccgcgcca aactggaagc actggcagcc gcaaatacc 99

<210> 21

<211> 165

<212> DNA

<213> FkbB-C nucleic acids

<400> 21

ctgaccctgc tggcaccgga tggcaatggc accccggttg gtggtgaacc gccggcacag 60

gtgagcgttg gcgcacctgc aaccgatagc gaagttgcaa gtccggatga tgaactgatt 120

gatgatatgg atgcagatgc actgattgca catgtgctga aaggc 165

<210> 22

<211> 96

<212> DNA

<213> FkbC-N nucleic acids

<400> 22

atggcagaaa atgatctgat tgaagccctg cgtaccagtg tgaaagataa tgcccagctg 60

cgccgtgaaa ataccgccct gcgtgcagcc gccaat 96

<210> 23

<211> 246

<212> DNA

<213> FkbC-C nucleic acids

<400> 23

gatcgcattg gcgaactgct gagtccggat gacccggtta ccgtggtgct ggcacagctg 60

gatcgcctgg aagcactggt ggccggtgtg gaccctggcg cacgtcaggc tgatgccatt 120

ggcacccgcc tggatgccct gctgaatcgc tggcgtcgcg aaacccgtcc gaccaccccg 180

gcaggtgttc tgagcgcaga tgcaaccgcc gatgaaattt ttgatctgat tgatcgcgaa 240

ctgcgt 246

<210> 24

<211> 87

<212> DNA

<213> FkbA-N nucleic acids

<400> 24

atgccggaac aggataaaac cgttgaatat ctgcgttggg caaccaccga actgcagcgc 60

acccgtgccg aactggccgc acatagt 87

<210> 25

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 25

ctttaagaag gagatatacc atgactgatt ctgaaaaggt tgcaga 46

<210> 26

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 26

tgttttttcg cacacacggt atctgattcc aattctctaa ttctt 45

<210> 27

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 27

ttagagaatt ggaatcagat accgtgtgtg cgaaaaaaca tgtgc 45

<210> 28

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 28

actgctggag atgcagcaaa cgacaccgct gccagttttt tatcg 45

<210> 29

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 29

aaaaactggc agcggtgtcg tttgctgcat ctccagcagt tgata 45

<210> 30

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 30

tgtcacctga catggttaat tcctccttta caaatcttca ccaccaccaa atg 53

<210> 31

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 31

tgaagatttg taaaggagga attaaccatg tcaggtgaca atggtatgac tg 52

<210> 32

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 32

ccaatcaccg tggtcggttt ttcaccagct ctatgttcaa cttctc 46

<210> 33

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 33

ttgaacatag agctggtgaa aaaccgacca cggtgattgg tgctg 45

<210> 34

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 34

tcacctctaa cttcagtacc tttaagctgg gtaaatgcag ataatc 46

<210> 35

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 35

ctgcatttac ccagcttaaa ggtactgaag ttagaggtga agcac 45

<210> 36

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 36

cataccgcgg catagtgtaa tcctccttta gtcaccatcc aattctctac ct 52

<210> 37

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 37

ggatggtgac taaaggagga ttacactatg ccgcggtatg atctgattct gg 52

<210> 38

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 38

accttttcag aatcagtcat ggtatatctc cttcttaaag ttaaac 46

<210> 39

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 39

gaattcacta gttacagcgg acgttgccac agatgggc 38

<210> 40

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 40

aacgtccgct gtaactagtg aattcga 27

<210> 41

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 41

cgctccacgc tccgctgatc tctggcagcg 30

<210> 42

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 42

cagagatcag cggagcgtgg agcgtgacgc 30

<210> 43

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 43

cctttacaaa tcttcaccac caccaaatct ttgatccaac atag 44

<210> 44

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 44

tcaaagattt ggtggtggtg aagatttgta aaggaggaat taaccatgaa accg 54

<210> 45

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 45

cagtcatacc attgtcacct gacatgagta ttacctcctt taaatc 46

<210> 46

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 46

gatttaaagg aggtaatact catgtcaggt gacaatggta tgactg 46

<210> 47

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 47

gattcagcag ggacggatta ttttcaccag ctctatgttc aac 43

<210> 48

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 48

ttgaacatag agctggtgaa aataatccgt ccctgctgaa tc 42

<210> 49

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 49

gatataccat gaccgtgtgt gcgaaaaaac atgtgcatct g 41

<210> 50

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 50

cacctctaac ttcagtacct tgcatcgcct gttgacggtg 40

<210> 51

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 51

caccgtcaac aggcgatgca aggtactgaa gttagaggtg 40

<210> 52

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 52

cagaatcagt catatagtaa tcctccttca gtcaccatcc 40

<210> 53

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 53

ggatggtgac tgaaggagga ttactatatg actgattctg 40

<210> 54

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 54

cagggcattc caaatccaca aatctgattc caattctc 38

<210> 55

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 55

gagaattgga atcagatttg tggatttgga atgccctg 38

<210> 56

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 56

ctgctggaga tgcagcaaac ttcccgggtg gcgcgtcacg 40

<210> 57

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 57

cgtgacgcgc cacccgggaa gtttgctgca tctccagcag 40

<210> 58

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 58

ccattgtcac ctgacattac tcattcctcc tttacaaatc ttcaccacc 49

<210> 59

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 59

ggtggtgaag atttgtaaag gaggaatgag taatgtcagg tgacaatgg 49

<210> 60

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 60

gctctgccac tgcggcggtt tcaccagctc tatgttcaac 40

<210> 61

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 61

gttgaacata gagctggtga aaccgccgca gtggcagagc 40

<210> 62

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 62

cagatgcaca tgttttttcg cacacacggt catggtatat c 41

<210> 63

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 63

ctttaagaag gagatatacc atgcgtgaag atcagctgct ggatgc 46

<210> 64

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 64

catggtatat ctccttctta aag 23

<210> 65

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 65

accgtgtgtg cgaaaaaaca tg 22

<210> 66

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 66

catgtttttt cgcacacacg gtatccattg ctgcacgcag gctgg 45

<210> 67

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 67

gattatctgc atttacccag cttaaattcg caccggcaga accggaac 48

<210> 68

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 68

tttaagctgg gtaaatgcag ataatc 26

<210> 69

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 69

taaaggagga ttacactatg ccg 23

<210> 70

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 70

cggcatagtg taatcctcct ttagcgttct tcttccagaa catgc 45

<210> 71

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 71

ctttaagaag gagatatacc atggcagaaa atgatctgat tgaag 45

<210> 72

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 72

catgtttttt cgcacacacg gtattggcgg ctgcacgcag ggc 43

<210> 73

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 73

gattatctgc atttacccag cttaaactga ccctgctggc accggatg 48

<210> 74

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 74

cggcatagtg taatcctcct ttagcctttc agcacatgtg caatc 45

<210> 75

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 75

cgataaaaaa ctggcagcgg tgtcgatgaa tccgcgcgtt cagagcac 48

<210> 76

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 76

cgacaccgct gccagttttt tatcg 25

<210> 77

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 77

taaaggagga attaaccatg aaacc 25

<210> 78

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 78

ggtttcatgg ttaattcctc ctttaggcgg tcgggtcacc aaattcacg 49

<210> 79

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 79

gatttaaagg aggtaatact catgccggaa caggataaag ttgtgg 46

<210> 80

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 80

catgagtatt acctccttta aatc 24

<210> 81

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 81

aataatccgt ccctgctgaa tc 22

<210> 82

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 82

gattcagcag ggacggatta ttggtatttg cggctgccag tgc 43

<210> 83

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 83

cgataaaaaa ctggcagcgg tgtcggatcg cattggcgaa ctgctgag 48

<210> 84

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 84

ggtttcatgg ttaattcctc ctttaacgca gttcgcgatc aatcaga 47

<210> 85

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 85

gatttaaagg aggtaatact catgccggaa caggataaaa ccgttg 46

<210> 86

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 86

gattcagcag ggacggatta ttactatgtg cggccagttc ggcac 45

<210> 87

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 87

gccaatccgg atatagttcc tc 22

<210> 88

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 88

gaggaactat atccggattg gc 22

<210> 89

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 89

gatataccat gactgattct gaaaaggttg cag 33

<210> 90

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 90

catgtttttt cgcacacacg gtgctacccc caccgccatc tgattccaat tctctaattc 60

<210> 91

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 91

accgtgtgtg cgaaaaaaca tg 22

<210> 92

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 92

cctcccgaca ccgctgccag ttttttatcg 30

<210> 93

<211> 76

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 93

cgataaaaaa ctggcagcgg tgtcgggagg tggaggttca ggtggaggtg gatctatggt 60

gtctaaaggt gaagag 76

<210> 94

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 94

ggttaattcc tcctttactt gtacaactca tccatacc 38

<210> 95

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 95

caagtaaagg aggaattaac catgaaaccg 30

<210> 96

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 96

cagaatcagt catggtatat ctccttctta aag 33

<210> 97

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 97

ctgcatttac ccagcttaaa ggaggtggag gttcaggtgg aggtggatct atggtgtcta 60

aaggtgaaga gg 72

<210> 98

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 98

gcttcacctc taacttcagt accgctaccc ccaccgccct tgtacaactc atccatacc 59

<210> 99

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 99

ggtactgaag ttagaggtga agc 23

<210> 100

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 100

ctatagggcg aattggagct cttagtcacc atccaattct ctacc 45

<210> 101

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 101

taagagctcc aattcgccct atag 24

<210> 102

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 102

tttaagctgg gtaaatgcag ataatc 26

<210> 103

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 103

ctttaagaag gagatatacc atgtcaggtg acaatggtat gactg 45

<210> 104

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 104

catgtttttt cgcacacacg gtgctacccc caccgccttc accagctcta tgttcaac 58

<210> 105

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 105

catggtatat ctccttctta aag 23

<210> 106

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 106

caactgctgg agatgcagca aagctacccc caccgccctt gtacaactca tccatacc 58

<210> 107

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 107

gctttgctgc atctccagca gttg 24

<210> 108

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 108

ctatagggcg aattggagct cttacaaatc ttcaccacca ccaaatc 47

<210> 109

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 109

ctatagggcg aattggagct cttacttgta caactcatcc atacc 45

<210> 110

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 110

taagagctcc aattcgccct atag 24

Claims

1. A construct comprising a junction gene nucleic acid molecule and a target product metabolic pathway gene nucleic acid molecule encoding a ligase peptide and a metabolic pathway enzyme by expressing the nucleic acid molecule;

wherein the ligase peptide fragment is derived from a non-covalent linkage recognition peptide of a type I non-iterative polyketide synthase;

The metabolic pathway enzyme is a cell membrane enzyme or a combination enzyme of a cytoplasmic enzyme and a cell membrane enzyme;

the ligase peptide fragment is used as a protein skeleton peptide fragment molecule for the tandem assembly of metabolic pathway enzymes, and the ligase peptide fragment and the metabolic pathway enzymes are selected from one of the following combination modes:

The ligase peptide fragment is divided into two pairs: M2-C and M3-N, M-C and M5-N, wherein the metabolic pathway enzymes are lycopene cyclase crtY, beta-carotene hydroxylase crtZ and beta-carotene ketolase crtW; or alternatively

The ligase peptide fragment is divided into two pairs: M2-C and M3-N, M-C and M5-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE and phytoene synthetase crtB; or alternatively

The ligase peptide fragment is divided into two pairs: rapA-C and RapB-N, rapB-C and RapC-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE, phytoene synthetase crtB; or alternatively

The ligase peptide fragment is divided into two pairs: fkbB-C and FkbC-N, fkbC-C and FkbA-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE, phytoene synthetase crtB; or alternatively

The ligase peptide fragment is a pair of: M2-C and M3-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI and geranylgeranyl pyrophosphoric acid synthetase crtE; or alternatively

The ligase peptide fragment is a pair of: rapA-C and RapB-N, wherein the metabolic pathway enzymes are isoprene pyrophosphate isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE; or alternatively

The ligase peptide fragment is a pair of: fkbB-C and FkbC-N, wherein the metabolic pathway enzymes are isoprene pyrophosphate isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE;

Wherein the amino acid sequence of M2-C is shown as SEQ ID NO.1, the amino acid sequence of M3-N is shown as SEQ ID NO.2, the amino acid sequence of M4-C is shown as SEQ ID NO.3, the amino acid sequence of M5-N is shown as SEQ ID NO.4, the amino acid sequence of RapA-C is shown as SEQ ID NO.5, the amino acid sequence of RapB-N is shown as SEQ ID NO.6, the amino acid sequence of RapB-C is shown as SEQ ID NO.7, the amino acid sequence of RapC-N is shown as SEQ ID NO.8, the amino acid sequence of FkbB-C is shown as SEQ ID NO.9, the amino acid sequence of FkbC-N is shown as SEQ ID NO.10, the amino acid sequence of FkbC-C is shown as SEQ ID NO. 11, and the amino acid sequence of FkbA-N is shown as SEQ ID NO. 12.

2. The construct of claim 1, which is a plasmid, host cell or microorganism.

3. A method for synthesizing a metabolite of an organism using an artificial protein scaffold, comprising:

Introducing a non-covalent linkage recognition peptide derived from a type I non-iterative polyketide synthase into a target product metabolic pathway enzyme to construct a microbial mutant strain containing an artificial protein backbone; wherein the metabolic pathway enzyme is a cell membrane enzyme or a combination enzyme of a cytoplasmic enzyme and a cell membrane enzyme;

Fermenting and culturing the microorganism mutant strain to obtain a target metabolite;

Wherein the non-covalent linkage recognition peptide derived from the type I non-iterative polyketide synthase is used as a protein backbone peptide fragment molecule for tandem assembly of metabolic pathway enzymes selected from one of the following combinations:

The non-covalent linking recognition peptides are in two pairs: M2-C and M3-N, M-C and M5-N, wherein the metabolic pathway enzymes are lycopene cyclase crtY, beta-carotene hydroxylase crtZ and beta-carotene ketolase crtW; or alternatively

The non-covalent linking recognition peptides are in two pairs: M2-C and M3-N, M-C and M5-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE and phytoene synthetase crtB; or alternatively

The non-covalent linking recognition peptides are in two pairs: rapA-C and RapB-N, rapB-C and RapC-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE, phytoene synthetase crtB; or alternatively

The non-covalent linking recognition peptides are in two pairs: fkbB-C and FkbC-N, fkbC-C and FkbA-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE, phytoene synthetase crtB; or alternatively

The non-covalent linkage recognition peptides are a pair of: M2-C and M3-N, wherein the metabolic pathway enzymes are isoprene pyrophosphoric acid isomerase IDI and geranylgeranyl pyrophosphoric acid synthetase crtE; or alternatively

The non-covalent linkage recognition peptides are a pair of: rapA-C and RapB-N, wherein the metabolic pathway enzymes are isoprene pyrophosphate isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE; or alternatively

The non-covalent linkage recognition peptides are a pair of: fkbB-C and FkbC-N, wherein the metabolic pathway enzymes are isoprene pyrophosphate isomerase IDI, geranylgeranyl pyrophosphate synthetase crtE;

Wherein the amino acid sequence of M2-C is shown as SEQ ID NO. 1, the amino acid sequence of M3-N is shown as SEQ ID NO. 2, the amino acid sequence of M4-C is shown as SEQ ID NO. 3, the amino acid sequence of M5-N is shown as SEQ ID NO. 4, the amino acid sequence of RapA-C is shown as SEQ ID NO. 5, the amino acid sequence of RapB-N is shown as SEQ ID NO. 6, the amino acid sequence of RapB-C is shown as SEQ ID NO. 7, the amino acid sequence of RapC-N is shown as SEQ ID NO. 8, the amino acid sequence of FkbB-C is shown as SEQ ID NO. 9, the amino acid sequence of FkbC-N is shown as SEQ ID NO. 10, the amino acid sequence of FkbC-C is shown as SEQ ID NO. 11, and the amino acid sequence of FkbA-N is shown as SEQ ID NO. 12.

4. The method of claim 3, wherein the microorganism is any one or more of escherichia coli, saccharomyces cerevisiae, pichia pastoris, bacillus, aspergillus oryzae, aspergillus nidulans, aspergillus niger, neurospora crassa, alternaria alternata, or fusarium.

5. The method of claim 3 or 4, wherein the target metabolite is astaxanthin.