Combined bacterium system for efficiently synthesizing resveratrol and method for synthesizing resveratrol

Abstract

The invention relates to a combined bacterium system for efficiently synthesizing resveratrol and a method for synthesizing resveratrol, and belongs to the technical field of bioengineering and biosynthesis. The preparation method comprises the following steps: respectively connecting 4CL, STS and FCS with scaffold proteins, constructing recombinant expression vectors for expressing 4CL-SpyTag, STS-SpyCatcher and FCS-SpyTag, respectively introducing the recombinant expression vectors into escherichia coli, carrying out expression to obtain a compound enzyme connected with the scaffold proteins, and then carrying out self-assembly to obtain a multienzyme complex 4CL-SpyTag-STS-SpyCatcher and FCS-SpyTag-STS-SpyCatcher; the constructed recombinant engineering bacterium or enzyme complex is added into p-coumaric acid synthesized from agricultural wastes for further synthesis of resveratrol; the multi-enzyme complex constructed by the invention and the new method for synthesizing the resveratrol not only provide a new high-valued utilization approach for the lignin, but also realize efficient and high-yield synthesis of the resveratrol.

Description

Technical Field

[0001] The invention relates to a combined bacterial system for efficiently synthesizing resveratrol and a method for synthesizing resveratrol, belonging to the technical field of bioengineering and biosynthesis. Background Art

[0002] Resveratrol (trans-3,5,4'-trihydroxystyrene) is a natural styrene polyphenol compound that is usually found in higher plants under natural conditions. As one of the most widely studied styrene compounds, resveratrol is widely used in the food, pharmaceutical and cosmetic industries. Currently, the extraction method of resveratrol is limited to extraction from specific plants. Not only is the extraction efficiency low and it is easy to cause organic solvent residues, but it also consumes a lot of natural resources and is extremely costly.

[0003] In recent years, research on resveratrol has focused on biosynthesis, among which the most important biosynthetic pathway is the tyrosine and phenylalanine pathway, which is mainly catalyzed by the cascade enzyme reaction performed by two family genes, 4-coumarate: coenzyme A ligase (4CL) and stilbene synthase (STS). First, p-coumaric acid is activated by connecting to coenzyme A (CoA) through 4CL to generate coumaroyl-CoA, and then Stilbenesynthase (STS) is used to further catalyze the production of resveratrol. Researchers usually use tyrosine or phenylalanine as the raw material to synthesize the intermediate product p-coumaric acid to further produce resveratrol. In order to obtain higher resveratrol yields, excessive addition of tyrosine, phenylalanine and p-coumaric acid, which are resveratrol precursors, seems to be the only way.

[0004] How to transform the two cascade enzymes and choose a more suitable method to obtain resveratrol has become a problem that needs to be solved urgently. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a combined bacterial system for synthesizing resveratrol and a method for synthesizing resveratrol. The present invention takes value creation as the direction, explores a new way to synthesize resveratrol, constructs a multi-enzyme complex system, and provides a method for producing resveratrol using cheap biomass material lignin.

[0006] In order to achieve the purpose of the above invention, the present invention adopts the following technical solutions:

[0007] The invention first provides a combined bacterial system for efficiently synthesizing resveratrol. The combined bacterial system comprises a recombinant engineering bacterium capable of expressing 4-coumaric acid: coenzyme A ligase (4CL), a recombinant engineering bacterium capable of expressing feruloyl-CoA synthetase (FCS) and a recombinant engineering bacterium capable of expressing stilbene synthase (STS); wherein the 3' ends of the 4CL and the FCS are connected with a scaffold protein SpyTag-linker fragment, the 3' end of the STS is connected with a scaffold protein SpyCatcher-GS linker fragment, and 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher are obtained after connecting the scaffold proteins.

[0008] Furthermore, the amino acid sequences of the 4CL, FCS and STS are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, the amino acid sequences of the SpyTag and SpyCatcher are shown in SEQ ID No: 13 and SEQ ID No: 14, and the amino acid sequences of 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher obtained after connecting the scaffold proteins are shown in SEQ ID No: 4, SEQ ID No: 5 and SEQ ID No: 6.

[0009] Furthermore, the combined bacterial system includes the following combined bacterial systems: (1) a combined bacterial system comprising a recombinant engineered bacterium capable of expressing 4-coumaric acid:CoA ligase (4CL) and a recombinant engineered bacterium capable of expressing stilbene synthase (STS); (2) a combined bacterial system comprising a recombinant engineered bacterium capable of expressing feruloyl-CoA synthetase (FCS) and a recombinant engineered bacterium capable of expressing stilbene synthase (STS).

[0010] The present invention also provides a multi-enzyme complex expressed and self-assembled by the combined bacterial system.

[0011] Furthermore, the multi-enzyme complex is obtained by self-assembly after the combined bacterial system expresses the corresponding enzymes; that is, the recombinant engineering bacteria that can express 4-coumaric acid: Coenzyme A ligase (4CL) or the recombinant engineering bacteria that can express feruloyl-CoA synthetase (FCS) are mixed with the recombinant engineering bacteria that can express stilbene synthase (STS), and the corresponding enzymes are expressed and then self-assembled.

[0012] According to an embodiment of the present invention, the multi-enzyme complex includes 4CL-SpyTag-STS-SpyCatcher and FCS-SpyTag-STS-SpyCatcher.

[0013] The present invention further provides application of the combined bacterial system or the multi-enzyme complex system in biosynthesis of resveratrol.

[0014] The present invention also provides a method for efficiently synthesizing resveratrol, the method comprising the following steps:

[0015] Agricultural waste biomass is used as raw material and treated with an alkaline solution to obtain p-coumaric acid; the combined bacterial system or the multi-enzyme complex is added to an initial reaction system containing the p-coumaric acid, glycerol and antibiotics to react, IPTG is added to induce bacterial growth during the reaction, and p-coumaric acid and glycerol are added in batch intermittent feeding to obtain resveratrol; wherein the p-coumaric acid is added by dissolving the p-coumaric acid in DMSO and adding it intermittently.

[0016] In the above method, the alkaline solution is a mixture of KOH solution and urea solution, wherein the volume ratio of KOH solution to urea solution is 1:1, and the concentration of KOH in the alkaline solution is 0.2-2.0 mol / L; the reaction temperature of the alkaline solution treatment is 4-60°C, and the solid-liquid ratio (S / L) of the agricultural waste biomass and the alkaline solution added is 1g:10-200mL.

[0017] More preferably, the concentration of KOH in the alkaline solution is 0.6 mol / L, the reaction temperature of the alkaline solution treatment is 50°C, and the solid-to-liquid ratio (S / L) of the agricultural waste biomass and the alkaline solution added is 1 g:30 mL.

[0018] Furthermore, in the above method, the concentration of p-coumaric acid in the initial reaction system is 1-3 mM, and the concentration of glycerol is 0.4%; more preferably, the concentration of p-coumaric acid in the initial reaction system is 2 mM.

[0019] Furthermore, in the above method, the antibiotics include kanamycin, chloramphenicol, and ampicillin; chloramphenicol and ampicillin are added to the combined bacterial system of the recombinant engineered bacteria capable of expressing 4-coumaric acid: Coenzyme A ligase (4CL) and the recombinant engineered bacteria capable of expressing stilbene synthase (STS); kanamycin and ampicillin are added to the combined bacterial system of the recombinant engineered bacteria capable of expressing feruloyl-CoA synthetase (FCS) and the recombinant engineered bacteria capable of expressing stilbene synthase (STS).

[0020] More preferably, 30 mg / L chloramphenicol and 50 mg / L ampicillin are added to the combined bacterial system of the recombinant engineering bacteria capable of expressing 4-coumaric acid: CoA ligase (4CL) and the recombinant engineering bacteria capable of expressing stilbene synthase (STS); 30 mg / L kanamycin and 50 mg / L ampicillin are added to the combined bacterial system of the recombinant engineering bacteria capable of expressing feruloyl-CoA synthetase (FCS) and the recombinant engineering bacteria capable of expressing stilbene synthase (STS).

[0021] Furthermore, in the above method, the recombinant engineered bacteria are added in the form of a bacterial solution, the initial OD value of the recombinant engineered bacteria solution is 0.6-0.8, and the amount of the recombinant engineered bacteria solution added is 4% of the total volume of the initial reaction system in terms of volume percentage;

[0022] Furthermore, in the above method, during the reaction, the reaction temperature is 15-45°C, the pH value of the reaction system is 4-12; the concentration of IPTG after being added to the reaction system is 0.1-1mM, and the concentration of DMSO after being added to the reaction system is 0-20%; and the intermittent feeding of p-coumaric acid and glycerol is added once every 8h.

[0023] Furthermore, in the above method, during the reaction, the reaction temperature is 30°C, the pH value of the reaction system is 8-9, the concentration of IPTG after being added to the reaction system is 0.17-0.3 mM, and the concentration of DMSO after being added to the reaction system is 5%; the intermittent feeding of p-coumaric acid and glycerol is respectively added at 0, 8, 16, 24, 32, 40, and 48 hours.

[0024] The present invention explores the application effect of the cascade enzyme system in the preparation of resveratrol through experiments. In the present invention, the system 4CL-STS composed of 4-coumaric acid: coenzyme A ligase (4CL) and stilbene synthase (STS) and the system FCS-STS composed of feruloyl-CoA synthetase (FCS) and stilbene synthase (STS) can both convert p-coumaric acid into resveratrol. The multi-enzyme composite system constructed by the present invention has two groups: 4CL-SpyTag-STS-SpyCatcher and FCS-SpyTag-STS-SpyCatcher, which can significantly improve the stability of the cascade reaction and significantly increase the yield of resveratrol.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) In the present invention, p-coumaric acid is obtained from a cheaper biomass as a key intermediate in the biosynthesis pathway of resveratrol, and more valuable resveratrol is synthesized. The raw material lignin used in the present invention is the most abundant sustainable source of renewable aromatic hydrocarbons and is a key precursor of p-coumaric acid. Replacing tyrosine and phenylalanine with lignin can greatly reduce costs and alleviate environmental pressure. At the same time, the present invention combines a multi-enzyme complex synthesis system to further optimize the fermentation conditions, so that the yield of resveratrol can reach 1.23g / L, which is 15 times the yield of resveratrol synthesized using glucose as a raw material in the prior art.

[0027] The present invention discloses a method for preparing p-coumaric acid from lignin, which comprises treating lignin with a UK solution prepared by KOH and urea to prepare p-coumaric acid. Compared with the method for preparing p-coumaric acid from lignin by treating lignin with NaOH in the prior art, the method provided by the present invention greatly increases the yield of p-coumaric acid.

[0028] (2) The present invention solves the problem that in the cascade enzyme reaction synthesis pathway of resveratrol, each enzyme exists in a discrete and free-floating state, resulting in low catalytic activity and poor stability, and a direct or indirect lack of synergy. The present invention adopts SpyTag / SpyCatcher orthogonal protein scaffold self-assembly technology to construct the enzymes involved in the cascade reaction into a multi-enzyme complex. The multi-enzyme complex is self-assembled by post-translational modification in the sequential biosynthetic cascade process through protein-protein interaction. From the perspective of cascade catalysis, the assembly of this enzyme significantly increases the overall reaction rate because the proximity of the enzyme active sites is conducive to the effective transfer of intermediates. It has been experimentally verified that the resveratrol yield obtained by the FCS-TC-STS system in 48 hours can reach 346.7 mg / L, and the resveratrol yield of the 4CL-TC-STS system is 173.2 mg / L, which is much higher than that of the single enzyme system. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The schematic flow chart of the synthesis of resveratrol according to the present invention is shown in FIG.

[0030] Figure 2 This is a map of plasmid 4CL (pACYCDuet-1) constructed in Example 1.

[0031] Figure 3 This is a map of the plasmid 4CL-SpyTag (pACYCDuet-1) constructed in Example 1.

[0032] Figure 4 This is a map of the plasmid FCS (pET-28a(+)) constructed in Example 1.

[0033] Figure 5 This is a map of the plasmid FCS-SpyTag (pET-28a(+)) constructed in Example 1.

[0034] Figure 6 This is a map of the plasmid STS (pETDuet-1) constructed in Example 1.

[0035] Figure 7 This is a map of the plasmid STS-SpyCatcher (pET-28a(+)) constructed in Example 1.

[0036] Figure 8Schematic diagrams of connecting scaffold proteins and self-assembly, wherein Figure A is a schematic diagram of 4CL, FCS and STS connecting scaffold proteins; Figure B is a schematic diagram of 4CL-SpyTag and STS-SpyCatcher, FCS-SpyTag and STS-SpyCatcher self-assembly to form a multi-enzyme complex.

[0037] Fig. 9 SDS-PAGE electrophoresis diagrams of 4CL, FCS, STS, 4CL-SpyTag (indicated as 4CLT in the figure), FCS-SpyTag (indicated as FCST in the figure), STS-SpyCatcher (indicated as STSC in the figure), 4CL-SpyTag-STS-SpyCatcher (indicated as 4CLT-STSC in the figure) and FCS-SpyTag-STS-SpyCatcher (indicated as FCST-STSC in the figure).

[0038] Fig.10 This is the HPLC standard curve for the depolymerization of lignin raw materials into p-coumaric acid by high performance liquid chromatography. The horizontal axis in the figure is the amount of p-coumaric acid, the vertical axis is the area of ​​the HPLC peak, the black dotted line is the standard curve of the p-coumaric acid standard, and the red line is the linear fitting of the p-coumaric acid sample.

[0039] Fig.11 HPLC spectrum of the reaction of several materials to produce p-coumaric acid by high performance liquid chromatography (A) and the relationship between different lignin raw materials and p-coumaric acid yield in the p-coumaric acid synthesis reaction (B); in the figure, Corncod: corn cob; Sorghum Straw: sorghum straw; Peanut Shell: peanut shell.

[0040] Fig.12 This is the relationship between the amount of NaOH solution added and the yield of p-coumaric acid in the NaOH solution method for producing p-coumaric acid (A) and the relationship between the amount of KOH solution added and the yield of p-coumaric acid in the UK solution method for producing p-coumaric acid (B).

[0041] Fig.13 The relationship between the solid-liquid ratio and the p-coumaric acid yield in the p-coumaric acid synthesis reaction (A), and the relationship between the reaction temperature and the p-coumaric acid conversion rate (B).

[0042] Fig.14 The results of multi-enzyme self-assembly of 4CL-SpyTag-STS-SpyCatcher (represented as 4CLT-STSC in the figure) and FCS-SpyTag-STS-SpyCatcher (represented as FCST-TSTC in the figure) under different pH conditions.

[0043] Fig.15This is a comparison chart of the reaction curve and the standard curve.

[0044] Fig.16 HPLC spectra of the synthesis of resveratrol by catalyzing p-coumaric acid using high performance liquid chromatography; Figure A is the reaction curve of the four systems for 24 hours, Figure B is the reaction curve of the four systems for 36 hours, and Figure C is the reaction curve of the four systems for 48 hours.

[0045] Fig.17 This is a graph showing the relationship between the resveratrol yield under different temperature (A), pH (B), IPTG addition amount (C), and DMSO addition amount (D) during the reaction of p-coumaric acid to synthesize resveratrol catalyzed by four complex enzyme systems.

[0046] Fig.18 This is the response surface plot of DMSO addition amount and pH (A), IPTG addition amount and temperature (B), IPTG addition amount and DMSO addition amount (C), DMSO addition amount and temperature (D), IPTG addition amount and pH value (E), and pH value and temperature (F) during the reaction of resveratrol synthesized from p-coumaric acid catalyzed by the FCST-STSC system. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0048] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0049] The formula of the modified M9 medium involved in the embodiments of the present invention is as follows: 5g / L yeast powder, 6.8g / LNa2HPO4, 3.0g / L KH2PO4, 0.5g / L NaCl, 1.0g / L NH4Cl, 5g / L CaCO3, additional metal elements (58mg / L MgSO4, 54mg / L FeCl3, 4mg / L ZnCl2, 4mg / L CoCl2, 4mg / L NaMoO4, 2mg / L CuCl2, 1mg / L H2BO4) and complex vitamins (340mg / L vitamin B1, 0.84mg / L vitamin B2, 0.084mg / L folic acid, 12.2mg / L fat-soluble vitamins, 2.8mg / L vitamin B6, 0.12mg / L vitamin B7, 10.8mg / L vitamin B5) are added to provide nutrition.

[0050] The schematic flow chart of the synthesis of resveratrol according to the present invention is as follows: Figure 1 shown.

[0051] Example 1: Preparation of 4CL, FCS, STS, 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher

[0052] Based on the amino acid sequence of 4-coumaric acid: CoA ligase from Arabidopsis thaliana published by NCBI (NCBI Reference Sequence ID: NP_175579.1), the amino acid sequence shown in SEQ ID No: 1 was obtained by codon optimization, which is referred to as 4CL.

[0053] SEQ ID No: 1 is as follows:

[0054] GSMEEDYKMAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIFQNISEF

[0055] ATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFVLSFLAASF

[0056] RGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIVCIDDNESVPIPEG

[0057] CLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGE

[0058] NPNLYFHSDDVILCVLPMFHIYALNSIMLCGLRVGAAILIMPKFEINLLLELIQRCKVTVAPM

[0059] VPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDAVNAKFPNAKLGQGYGMTEAGPV

[0060] LAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGDSLSRNQPGEICIRGHQIMKGYLNN

[0061] PAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEALLIGHPDITDV

[0062] AVVAMKEEAAGEVPVAFVVKSKDSELSEDDVKQFVSKQVVFYKRINKVFFTESIPKAPSGKI

[0063] LRKDLRAKLANGL

[0064] Based on the amino acid sequence of feruloyl-CoA synthetase from Pseudomonas published by NCBI (NCBI Reference Sequence ID: WP_049587830.1), the amino acid sequence of feruloyl-CoA synthetase shown in SEQ ID No: 2 was obtained by codon optimization, abbreviated as FCS.

[0065] SEQ ID No: 2 is as follows:

[0066] EFMNNEARSGSTDPGQRPRYRQVAIGHPQVQVSHVDDVLRMQPVEPLAPLPARLLERL

[0067] VHWAQVRPDTTFIAARQADGAWRSISYVQMLADVRTIAANLLGLGLSAERPLALLSGNDIE

[0068] HLQIALGAMYAGIAYCPVSPAYALLSQDFAKLRHVCEVLTPGVVFVSDSQPFQRAFEAVLDD

[0069] SVGVISVRGQVAGRPHISFDSLLQPGDLAAAADAAFAATGPDTIAKFLFTSGSTKLPKAVITTQ

[0070] RMLCANQQMLLQTFPTFAEEPPVLVDWLPWNHTTFGGSHNLGIVLYNGGSFYLDAGKPTPQ

[0071] GFAETLRNLREISPTAYLTVPKGWEELVKALEQDPALREVFFARIKLFFFAAAGLSQSVWDR

[0072] LDRIAEQHCGERIRMMAGLGMTEASPSCTFTTGPLSMAGYVGLPAPGCEVKLVPVGDKLE

[0073] ARFRGPHIMPGYWRSPQQTAEAFDEEGFYCSGDALKLADARQPELGLMFDGRIAEDFKLSS

[0074] GVFVSVGPLRNRAVLEGSPYVQDIVVTAPDRECLGLLVFPRLPECRRLAGLAEDASDARVL

[0075] ANDTVRSWFADWLERLNRDAQGNASRIEWLSLLAEPPSIDAGEITDKGSINQRAVLQRRAA

[0076] QVEALYRGEDPDALHAKVRP

[0077] Based on the amino acid sequence of stilbene synthase from Huadong grape published by NCBI (NCBI Reference Sequence ID: AFN21523.1), the amino acid sequence of stilbene synthase shown in SEQ ID No: 3 was obtained by codon optimization, abbreviated as STS.

[0078] SEQ ID No: 3 is as follows:

[0079] MASVEEFRNAQRAKGPATILAIGTATPDHCVYQSDYADYYFRVTKSEHMTELKKKFNR

[0080] ICDKSMIKKRYIHLTEEMLEEHPNIGAYMAPSLNIRQEIITAEVPKLGKEAALKALKEWGQP

[0081] KSKITHLVFCTTSGVEMPGADYKLANLLGLETSVRRVMLYHQGCYAGGTVLRTAKDLAEN

[0082] NAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSSAVIVGSDPDVSIERPLFQLVSAA

[0083] QTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEKCLTQAFDPLGISDWNSLFWIAHPGG

[0084] PAILDAVEAKLNLEKKKLEATRHVLSEYGNMSSACVLFILDEMRKKSLKGEKATTGEGLDW

[0085] GVLFGFGPGLTIETVVLHSIPTVTN

[0086] The 3' end of the optimized gene 4CL was inserted into the SpyTag-linker fragment: HMAHIVMVDAYKPTKGGSELAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAG AGAGPEGGGS (SEQ ID No: 13) to obtain the amino acid sequence shown in SEQ ID No: 4, which was named 4CL-SpyTag, abbreviated as 4CLT.

[0087] SEQ ID No: 4 is as follows:

[0088] MHMAHIVMVDAYKPTKGGSELAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGP

[0089] EGAGAGAGPEGGGSGSMEEDYKMAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHL

[0090] SLHDYIFQNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNC

[0091] PEFVLSFLAASFRGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIV

[0092] CIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVMLTHKGL

[0093] VTSVAQQVDGENPNLYFHSDDVILCVLPMFHIYALNSIMLCGLRVGAAILIMPKFEINLLLEL

[0094] IQRCKVTVAPMVPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDAVNAKFPNAKLGQ

[0095] GYGMTEAGPVLAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGDSLSRNQPGEICIRG

[0096] HQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEA

[0097] LLIGHPDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDVKQFVSKQVVFYKRINKVFF

[0098] TESIPKAPSGKILRKDLRAKLANGL

[0099] The 3' end of the optimized gene FCS was inserted into the SpyTag-linker fragment to obtain the amino acid sequence shown in SEQ ID No: 5, which was named FCS-SpyTag, abbreviated as FCST.

[0100] SEQ ID No: 5 is as follows:

[0101] MHMAHIVMVDAYKPTKGGSELAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGP

[0102] EGAGAGAGPEGGGSEFMNNEARSGSTDPGQRPRYRQVAIGHPQVQVSHVDDVLRMQPVE

[0103] PLAPLPARLLERLVHWAQVRPDTTFIAARQADGAWRSISYVQMLADVRTIAANLLGLGLSA

[0104] ERPLALLSGNDIEHLQIALGAMYAGIAYCPVSPAYALLSQDFAKLRHVCEVLTPGVVFVSDS

[0105] QPFQRAFEAVLDDSVGVISVRGQVAGRPHISFDSLLQPGDLAAADAAFAATGPDTIAKFLFT

[0106] SGSTKLPKAVITTQRMLCANQQMLLQTFPTFAEEPPVLVDWLPWNHTTFGGSHNLGIVLYNG

[0107] GSFYLDAGKPTPQGFAETLRNLREISPTAYLTVPKGWEELVKALEQDPALREVFFARIKLFFF

[0108] AAAGLSQSVWDRLDRIAEQHCGERIRMMAGLGMTEASPSCTTFTTGPLSMAGYVGLPAPGC

[0109] EVKLVPVGDKLEARFRGPHIMPGYWRSPQQTAEAFDEEGFYCSGDALKLADARQPELGLM

[0110] FDGRIAEDFKLSSGVFVSVGPLRNRAVLEGSPYVQDIVVTAPDRECLGLLVFPRLPECRRLA

[0111] GLAEDASDARVLANDTVRSWFADWLERLNRDAQGNASRIEWLSLLAEPPSIDAGEITDKGS

[0112] INQRAVLQRRAAQVEALYRGEDPDALHAKVRP

[0113] The 3' end of the optimized gene STS was inserted into the SpyCatcher-GS linker fragment: HMAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTW ISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGS GGGGS (SEQ ID No: 14), and the amino acid sequence shown in SEQ ID No: 6 was obtained, which was named STS-SpyCatcher, abbreviated as STSC.

[0114] SEQ ID No: 6 is as follows:

[0115] HMAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTI

[0116] STWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGG

[0117] GGSGGGGSMASVEEFRNAQRAKGPATILAIGTATPDHCVYQSDYADYYFRVTKSEHMTELK

[0118] KKFNRICDKSMIKKRYIHLTEEMLEEHPNIGAYMAPSLNIRQEIITAEVPKLGKEAALKALKE

[0119] WGQPKSKITHLVFCTTSGVEMPGADYKLANLLGLETSVRRVMLYHQGCYAGGTVLRTAKD

[0120] LAENNAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSSAVIVGSDPDVSIERPLFQL

[0121] VSAAQTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEKCLTQAFDPLGISDWNSLFWIA

[0122] HPGGPAILDAVEAKLNLEKKKLEATRHVLSEYGNMSSACVLFILDEMRKKSLKGEKATTGE

[0123] GLDWGVLFGFGPGLTIETVVLHSIPTVTN

[0124] The nucleotide sequences of 4CL and 4CL-SpyTag (as shown in SEQ ID No: 7 and SEQ ID No: 10) were synthesized and ligated to the pACYCDuet-1 vector to obtain the recombinant vectors pACYCDuet-1-4CL and pACYCDuet-1-4CL-SpyTag; the nucleotide sequences of FCS and FCS-SpyTag (as shown in SEQ ID No: 8 and SEQ ID No: 11) were synthesized and ligated to the pET-28a(+) vector to obtain the recombinant vectors pET-28a(+)-FCS and pET-28a(+)-FCS-SpyTag; the nucleotide sequences of STS and STS-SpyCatcher (as shown in SEQ ID No: 9 and SEQ ID No: 12) were synthesized and ligated to the pETDuet-1 vector to obtain the recombinant vectors pETDuet-1-STS and pET-28a(+)-STS-SpyCatcher.

[0125] SEQ ID No: 7:

[0126]

[0127] SEQ ID No:8:

[0128]

[0129] SEQ ID No:9:

[0130]

[0131] SEQ ID No:10:

[0132]

[0133] SEQ ID No:11:

[0134]

[0135] SEQ ID No:12:

[0136]

[0137] The recombinant vector map constructed by the present invention is as follows Figure 2-7 As shown. In the process of constructing the recombinant vector, the primers used to amplify the 4CL, FCS and STS gene fragments are as follows:

[0138] Table 1. Primers used to amplify 4CL, FCS and STS gene fragments

[0139]

[0140] The underlined parts in Table 1 represent specific sequences recognized by restriction endonucleases, indicating the recognition sites of restriction endonucleases such as BamHI (GGATCC) and XhoI (CTCGAG). These enzymes cut DNA at specific sequences, facilitating genetic manipulations such as cloning and DNA analysis.

[0141] The recombinant vectors constructed in this example were expressed in E. coli DH5α and sequenced to confirm the fidelity of the inserted fragments in the vectors pACYCDuet-1, pET-28a(+) and pETDuet-1.

[0142] The constructed recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) respectively. The recombinant engineered bacteria obtained after transformation were cultured in 5 mL LB medium, and appropriate antibiotics (50 mg / L chloramphenicol, 50 mg / L kanamycin and 100 mg / L ampicillin) were added. The culture was shaken well and cultured at 220 rpm and 37 ° C for 12 h. The initial culture was inoculated into 50 mL of new LB medium and cultured at 37 ° C and 220 rpm until OD 600 After culturing at 30°C for 18 hours, the recombinant bacteria were harvested by centrifugation. The harvested recombinant bacteria were resuspended in a mixed solution containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl and 20 mM imidazole buffer, and lysed by homogenizer at 4°C to obtain a crude enzyme extract.

[0143] Six enzyme proteins (such as Figure 8As shown, the crude enzyme extracts of the six enzyme proteins (including the enzyme proteins before and after the connection with the scaffold protein) were centrifuged at 12,000rpm for 20min, and the supernatant was loaded on a Ni-NTA agarose column (GE Healthcare, Chicago, IL, USA) for purification. The recombinant enzyme protein was eluted with 150mM imidazole, and the high-purity purified protein was collected. The final protein yields were measured as follows: the yields of 4CL, FCS and STS purified proteins were 57.31±2.3mg / L, 62.70±3.2mg / L and 59.48±1.2mg / L (LB medium), respectively. The yields of 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher purified proteins connected with the scaffold protein were 72.34±4.7mg / L, 93.90±3.2mg / L and 89.46±3.7mg / L (LB medium), respectively.

[0144] The enzyme protein before and after connecting to the scaffold protein was verified by SDS-PAGE gel electrophoresis. The results are as follows Fig. 9 As shown, the six groups of genes after codon optimization were overexpressed in Escherichia coli BL21 (DE3) cells, and the molecular weights of the isolated proteins were FCS: 68.83 KDa, 4CL: 62.00 KDa, STS: 42.82 KDa; FCS-SpyTag (FCST): 74.66 KDa, 4CL-SpyTag (4CLT): 67.84 KDa, STS-SpyCatcher (STSC): 56.05 KDa.

[0145] The strains used in the present invention are described as follows:

[0146] Table 2. Strains used

[0147] Bacteria illustrate E. coli DH5α For plasmid amplification and gene cloning BL21(DE3) For protein expression BL21-4CL E. coli BL21 carrying plasmid pACYCDuet-1-4CL or pACYCDuet-1-4CL-SpyTag BL21-FCS E. coli BL21 carrying plasmid pET-28a(+)-FCS or pET-28a(+)-FCS-SpyTag BL21-STS E. coli BL21 carrying plasmid pETDuet-1-STS or pET-28a(+)-STS-SpyCatcher

[0148] Example 2: Obtaining p-coumaric acid, a precursor for resveratrol production, from lignin raw materials

[0149] As a key intermediate in the synthesis of resveratrol, p-coumaric acid can be obtained from agricultural waste. This example selected several important agricultural waste biomass materials: corn straw, sorghum straw, corn cob and peanut shell.

[0150] Reaction steps: Taking corn cob material as an example, the corn cob residue is crushed and placed in a 60°C drying oven for 7 days. Then, after grinding, pass through a 40-mesh sieve and place in a drying oven for use. For each experiment, 2.5g of corn cob powder is weighed and placed in a conical flask, and UK solution (the volume ratio of KOH solution to urea solution is 1:1) is added according to the solid-liquid ratio S / L (g / mL) = 1:30, wherein the concentration of KOH in the reaction system is 1 mol / L. In addition, NaOH (1 mol / L) solution is also used as a control in this embodiment, and is also configured according to S / L (g / mL) = 1:30. Place the conical flask in a 50°C water bath shaker and react at 160 rpm for 1 to 10 hours. During the reaction, take 1 mL of sample at reaction times of 1, 2, 4, 6, 8, and 10 h, add H2SO4 to adjust the pH to neutral, and add pure water to make up to 1.5 mL. After making up to the volume, take 0.5 mL and add methanol to make up to 1 mL. Finally, test the formation of the product by HPLC.

[0151] HPLC detection: After each sampling, methanol was added to the fixed volume and filtered with a 0.22 μm syringe filter. High performance liquid chromatography system (Waters 2695, Waters 2489 UV / visible light detector), the parameters of the high performance liquid chromatography system are as follows:

[0152] Chromatographic column: Thermo Hypersil 176BDS C18 column (150×46mm, 5μm); mobile phase: methanol: water: glacial acetic acid (30:69.3:0.7); flow rate: 1.0mL / min; detection wavelength: 238nm; column temperature: 30℃; injection volume: 20μL; running time: 15min.

[0153] The product obtained by the above method was detected by HPLC and compared with the p-coumaric acid standard to obtain a standard curve as shown in Fig.10 shown.

[0154] The same method was used to synthesize p-coumaric acid, a precursor of resveratrol, using sorghum straw, corncob and peanut shell as raw materials. The product was detected by HPLC. The HPLC results are as follows: Fig.11 As shown in Figure A, it can be seen that after alkali treatment, the three lignin raw materials, corn cobs, sorghum straw and peanut shells, all showed an identifiable peak at about 8.5 minutes. The comparison with the p-coumaric acid standard confirmed that the signal came from p-coumaric acid. In addition, a second identifiable peak appeared at 9.5 minutes, which was confirmed to be ferulic acid by comparison with the ferulic acid standard. After the same treatment conditions, different p-coumaric acid yields were obtained for several materials. Among them, the corn cob material obtained the highest p-coumaric acid yield after UK alkali treatment, which can reach 19.73 mg / g.

[0155] Using corn cobs as raw materials, this example further investigates the effects of different amounts of NaOH alkaline solution added (0.2-2.0 mol / L), UK solution added (measured by the concentration of KOH in the reaction system, the concentration of KOH in the reaction system is 0.2-2.0 mol / L), temperature (4-60°C), and solid-liquid ratio (1:10 g-1:200 mL) on the synthesis yield of p-coumaric acid.

[0156] Fig.11 The results of B showed that the best lignin raw material for p-coumaric acid production was corn cob material.

[0157] Combination Fig.12 A and Fig.12 B The results show that using UK solution ( Fig.12 The yield of p-coumaric acid synthesized by the KOH concentration in B was significantly higher than that of the traditional NaOH solution treatment. Fig.12 B shows that under the four different UK solution concentration treatment conditions, the extraction amount of p-coumaric acid has a similar change trend. With the increase of alkali concentration, the output of p-coumaric acid gradually increases. In the first 2 hours of reaction, p-coumaric acid has a rapid accumulation; while the accumulation of p-coumaric acid in the subsequent stage only increased by 12.3%. After 8 hours of treatment, the accumulation of p-coumaric acid has a downward trend. This is due to its antioxidant properties. If it is left for too long, it will be oxidized and it will be detrimental to its accumulation. Therefore, the optimal UK solution dosage for p-coumaric acid production is when the concentration of KOH in the reaction system is 1.0 mol / L after addition, and the reaction time is 4h.

[0158] Fig.13 A shows that the optimal solid-liquid ratio in the synthesis of p-coumaric acid using UK solution is 1g:30mL, Fig.13 B shows that the optimal reaction temperature condition during the synthesis process is 50°C.

[0159] In summary, corn cob material is the best lignin raw material for the production of p-coumaric acid. The best conditions for the production of p-coumaric acid are: treatment with UK solution containing 1.0 mol / L KOH for 4 h, the best temperature is 50 ° C, and the solid-liquid ratio is 1 g: 30 mL. Finally, 48.76 ± 5.3 mg / g of p-coumaric acid can be obtained.

[0160] Example 3: Construction of multi-enzyme complex and characterization of reaction system

[0161] (1) Construction of multi-enzyme complex:

[0162] The constructed 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher connected with the scaffold protein SpyTag or SpyCatcher were combined into two reaction systems: 4CL-SpyTag-STS-SpyCatcher (4CLT-STSC) and FCS-SpyTag-STS-SpyCatcher (FCST-STSC) two combined enzyme systems. In order to test the self-assembly effect of the two systems, this example conducted an in vitro self-assembly experiment on the two systems.

[0163] Reaction steps: add 200 μL of Tris-HCl (50 mM) buffer with different pH values ​​to a 10 mL shaking tube, take 0.2 mg of purified 4CL-SpyTag and STS-SpyCatcher proteins and mix them, use the same Tris-HCl buffer to make the volume 500 μL, place in a constant temperature shaker, and react at 30 ° C, 200 rpm for 2 hours. In addition, the self-assembly reaction was carried out using Tris-HCl buffer with different pH values, and the self-assembly results were analyzed by 12% SDS-PAGE gel electrophoresis.

[0164] The results of SDS-PAGE gel electrophoresis are as follows Fig.14 As shown in the figure, the results show that a higher molecular weight protein band can be observed between the matching Catcher and Tag pairs. The corresponding molecular weight of SDS gel electrophoresis is consistent with the molecular weight of the complex. As a convenient indicator of the formation of a covalent complex, it shows that the multi-enzyme complex 4CL-SpyTag-STS-SpyCatcher (represented as 4CLT-STSC in the figure) was successfully synthesized. In the same way, the purified FCS-SpyTag and STS-SpyCatcher were mixed. The results in the figure show that the multi-enzyme complex FCS-SpyTag-STS-SpyCatcher (represented as FCST-STSC in the figure) was successfully synthesized.

[0165] Furthermore, in order to clarify the reaction conditions of self-assembly, this embodiment carried out self-assembly experiments at different pH values. Fig.14 The results show that when the pH value is 8, complete self-assembly is achieved, so pH 8 is the best condition for self-assembly. This discovery provides an important reference for the subsequent determination of the production conditions of resveratrol.

[0166] (2) Characterization of resveratrol production by multi-enzyme complex system:

[0167] Characterization of resveratrol production by FCST-STSC and 4CLT-STSC systems:

[0168] In order to determine whether the constructed multi-enzyme complex can effectively make the enzymes in the cascade reaction pathway more stable and improve the reaction efficiency, the recombinant engineered bacteria expressing FCS, STS and 4CL were mixed to obtain a mixed bacteria system expressing FCS and STS and a mixed bacteria system expressing 4CL and STS. The enzymes obtained from these two mixed bacteria systems were in a free state and served as the control groups, respectively recorded as FCS-STS and 4CL-STS. The multi-enzyme complex system in this embodiment is obtained by mixing recombinant engineered bacteria expressing 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher to obtain a mixed bacterial system of recombinant engineered bacteria of 4CL-SpyTag and STS-SpyCatcher and a mixed bacterial system of recombinant engineered bacteria of FCS-SpyTag and STS-SpyCatcher, respectively expressing the corresponding enzymes, and self-assembling in vitro to obtain two combined enzyme systems of 4CL-SpyTag-STS-SpyCatcher (4CLT-STSC) and FCS-SpyTag-STS-SpyCatcher (FCST-STSC). The two mixed bacterial systems are recorded as BL21-4CLT-TSTC (recorded as 4CLT-TSTC in this embodiment) and BL21-FCST-TSTC (recorded as FCST-TSTC) as experimental groups, with a total of four systems.

[0169] Reaction steps: the colonies obtained from the bacterial culture of each group of mixed bacterial systems were placed in 5 mL of LB medium, and the corresponding antibiotics were added according to different systems (30 mg / L kanamycin and 50 mg / L ampicillin were added to the FCS-STS and FCST-STSC systems; 30 mg / L chloramphenicol and 50 mg / L ampicillin were added to the 4CL-STS and 4CLT-STSC systems), and cultured overnight at 37°C as the starting culture; then 500 μL of the starting culture was inoculated into 50 mL of LB medium and cultured again at 37°C and 220 rpm; when OD 600 When it reaches 0.8-1.0, IPTG (the concentration in the reaction system after addition is 0.5mM) is added to induce gene expression, the temperature is adjusted to 30°C, and the culture is continued for 6 hours. The cells are then collected by centrifugation and resuspended in 25mL of modified M9 medium. Then, the culture is shaken at 30°C and 220rpm, and 500μL of 20% (v / v) glycerol and 2mM p-coumaric acid are supplemented every 8 hours. Samples are obtained at 0, 8, 16, 24, 32, 40, and 48h for subsequent sample analysis, and the reaction is terminated at 60h.

[0170] HPLC detection: Take 2 mL of the sample taken at each time point, vortex extract 3 to 5 times with an equal volume of ethyl acetate, separate the upper organic layer, and evaporate to dryness. Dissolve the dried residue in 10 mL of methanol and filter with a 0.22 μm syringe filter. The parameters of the HPLC system are as follows:

[0171] Chromatographic column: C18 chromatographic column (4.6mm×150mm, 5μm); mobile phase: 30% acetonitrile (mobile phase A) and 70% 0.2% phosphoric acid aqueous solution (mobile phase B); flow rate: 1.0mL / min; detection wavelength: 303nm; column temperature: 25℃; injection volume: 20μL; running time: 15min; elution gradient: using binary gradient, the methods are: 20% (4min), 20-50% (11min), 50% (3min), 50-100% (8min), 100% (5min), 100-20% (2min), 20% (2min).

[0172] According to the test results of the samples, compared with the peak points of the standard, it was verified that the peak times of p-coumaric acid and resveratrol were 4min and 7min respectively ( Fig.15 ), HPLC results are as follows Fig.16 As shown, the systems described in this example can all successfully produce resveratrol, and the systems in the two experimental groups have obtained higher yields of resveratrol than the systems in the two control groups.

[0173] After analysis, the newly synthesized product corresponds to the molecular weight of resveratrol, indicating that the four systems can efficiently catalyze the conversion of p-coumaric acid into resveratrol. Among them, the FCST-STSC system first produces resveratrol, and the resveratrol yield obtained after 48 hours is the highest, reaching 346.7 mg / L; the resveratrol yield of the 4CLT-STSC system is second, about 173.2 mg / L; the FCS-STS system equipped with only two enzymes reaches 118.4 mg / L, and the 4CL-STS system has the lowest yield, only 58.9 mg / L, close to the basic level. Comparison of the control group system with the experimental group system shows that the enzyme connected to the scaffold protein and self-assembled into a multi-enzyme complex for the synthesis of resveratrol can increase the yield of resveratrol by about 290%. The step of p-coumaryl-CoA produced by FCS instead of 4CL in the conversion of p-coumaric acid significantly improves the reaction efficiency, increasing the yield of resveratrol by about 100%.

[0174] Example 4: Optimization of resveratrol production system conditions

[0175] From the results of Example 3, it can be found that the multi-enzyme complex constructed by the present invention can significantly improve the production efficiency of resveratrol by replacing the cascade enzyme reaction; the conversion efficiency from p-coumaric acid to resveratrol can be further improved, and this example provides the best conditions for improving the protein expression level by optimizing the expression conditions. Therefore, in order to increase the yield of resveratrol, this example optimizes the synthesis conditions, adopts the control variable method and batch feeding strategy, and can further determine the optimal system for resveratrol synthesis.

[0176] This example investigates the effects of different pH values ​​(4.0-12.0), temperatures (15-45° C.), DMSO concentrations (0-20%, v / v), and IPTG concentrations (0.1-1 mM) in the reaction system on the conversion rate of resveratrol synthesis.

[0177] Fig.17 The results showed that the optimal conditions for resveratrol conversion were a temperature of 30°C and an optimal pH of 8.0. Fig.17 It can be seen that the conversion rate of resveratrol reached a peak when the IPTG addition amount was 0.2 mM, and decreased slightly when it was higher than 0.5 mM. Fig.17 In the experiment, the addition of DMSO can effectively increase the solubility of resveratrol in the reaction system. When the content of DMSO increases from 0% to 5%, the conversion of resveratrol only increases slightly; but when the concentration of DMSO is greater than 10%, the conversion rate of resveratrol decreases; at a concentration of 20%, only less than 0.4mM resveratrol is produced, indicating that the addition of a small amount of DMSO has little effect on the synthesis of resveratrol. Among them, under the influence of different condition variables, the FCST-STSC reaction system obtained the highest resveratrol yield. Under the same conditions, the 4CLT-STSC system has the second highest yield, and the free mixed bacteria system FCS-STS and 4CL-STS without a protein scaffold are the lowest.

[0178] In summary, the best resveratrol reaction system was determined: the FCST-STSC system was used; the best initial reaction conditions were: 30°C, pH 8.0, 5% DMSO, and 0.2 mM IPTG.

[0179] In order to further determine the optimal reaction conditions and obtain a higher yield of resveratrol, response surface analysis was performed under the previously obtained optimized conditions to determine the best combination of process parameters. Six groups of response surfaces were obtained by testing the FCST-STSC system ( Fig.18 ), the temperature range used is 15-45°C, the pH of the reaction system is 4-12, the amount of IPTG added in the reaction system is 0.1-1 mM, and the amount of DMSO is 0-20%.

[0180] The analysis revealed the optimal production conditions for the shake flask production stage of resveratrol synthesis from p-coumaric acid: the FCST-STSC system was used, and the batch feeding strategy was used: p-coumaric acid dissolved in 8.11% (v / v) DMSO was used as the raw material, p-coumaric acid and glycerol were added at 0, 8, 16, 24, 32, 40, and 48 hours, and the concentration of p-coumaric acid in the reaction system was 2mM. The culture was carried out for 48 hours at a temperature of 29.77°C, a pH value of 8.67, and a concentration of 0.17mM IPTG in the system after addition.

[0181] The results of the optimal conditions and methods showed that 973.43 mg / L of resveratrol could be synthesized within 24 hours using the intermittent feeding method, and the yield gradually increased throughout the experiment. The final resveratrol yield could reach 1.23 g / L, which is 15 times the yield of resveratrol synthesized by the tyrosine pathway using glucose as raw material in the previous study (Park JY et al. Biosynthesis of resveratrol using metabolically engineered Escherichia coli [J]. Applied Biological Chemistry, 2021, 64 (1).). The molar conversion rate of resveratrol reached 77.36%, 18.76 mg of p-coumaric acid could be obtained per gram of corn cob material, and 115.09 mg of p-coumaric acid could be produced per gram of lignin. This is the first time that resveratrol has been synthesized from agricultural waste materials. Therefore, the method for preparing resveratrol using the FCST-STSC system with lignin as raw material provides a new way for efficiently and economically producing resveratrol, a very valuable compound, in the biocatalytic reaction described in the present invention.

[0182] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail in this specification with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A combined bacterial system for efficiently synthesizing resveratrol, characterized in that: The combined bacterial system comprises a recombinant engineering bacterium capable of expressing 4-coumaric acid:CoA ligase (4CL), a recombinant engineering bacterium capable of expressing feruloyl-CoA synthetase (FCS) and a recombinant engineering bacterium capable of expressing stilbene synthase (STS); wherein the 3' ends of the 4CL and FCS are connected with a scaffold protein SpyTag-linker fragment, and the 3' end of the STS is connected with a scaffold protein SpyCatcher-GS linker fragment.

2. The combined bacterial system according to claim 1, characterized in that: The amino acid sequences of the 4CL, FCS and STS are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, the amino acid sequences of the SpyTag and SpyCatcher are shown in SEQ ID No: 13 and SEQ ID No: 14, and the amino acid sequences of 4CL-SpyTag, FCS-SpyTag and STS-SpyCatcher obtained after connecting the scaffold proteins are shown in SEQ ID No: 4, SEQ ID No: 5 and SEQ ID No:

6.

3. The combined bacterial system according to claim 1, characterized in that: The combined bacterial system includes the following combined bacterial system: (1) A combined bacterial system comprising a recombinant engineered bacterium capable of expressing 4-coumaric acid:CoA ligase (4CL) and a recombinant engineered bacterium capable of expressing stilbene synthase (STS); (2) A combined bacterial system comprising a recombinant engineered bacterium capable of expressing feruloyl-CoA synthetase (FCS) and a recombinant engineered bacterium capable of expressing stilbene synthase (STS).

4. The multi-enzyme complex expressed and self-assembled by the combined bacterial system according to any one of claims 1 to 3.

5. The multi-enzyme complex according to claim 4, characterized in that The multi-enzyme complex is obtained by self-assembly after the combined bacterial system according to any one of claims 1 to 3 expresses corresponding enzymes.

6. Use of the combined bacterial system according to any one of claims 1 to 3 or the multi-enzyme complex system according to any one of claims 4 to 5 in the biosynthesis of resveratrol.

7. A method for efficiently synthesizing resveratrol, the method comprising the following steps: Agricultural waste biomass is used as raw material and treated with alkaline solution to obtain p-coumaric acid; the combined bacterial system according to claim 1 or 2 or the multi-enzyme complex according to claim 3 or 4 is added to an initial reaction system containing the p-coumaric acid, glycerol and antibiotics to react, IPTG is added to induce bacterial growth during the reaction, and p-coumaric acid and glycerol are added in batches by intermittent feeding to obtain resveratrol; wherein the p-coumaric acid is added by dissolving the p-coumaric acid in DMSO and adding it by intermittent feeding.

8. The method according to claim 7, characterized in that The alkaline solution is a mixture of KOH solution and urea solution, wherein the volume ratio of KOH solution to urea solution is 1:1, and the concentration of KOH in the alkaline solution is 0.2-2.0 mol / L; the reaction temperature of the alkaline solution treatment is 4-60°C, and the solid-liquid ratio (S / L) of the agricultural waste biomass and the alkaline solution added is 1g:10-200mL; preferably, the concentration of KOH in the alkaline solution is 0.6 mol / L, the reaction temperature of the alkaline solution treatment is 50°C, and the solid-liquid ratio (S / L) of the agricultural waste biomass and the alkaline solution added is 1g:30mL.

9. The method according to claim 7, characterized in that: The concentration of p-coumaric acid in the initial reaction system is 1-3 mM, the concentration of glycerol is 0.4%, and the antibiotics include kanamycin, chloramphenicol, and ampicillin, which are added differently according to different bacteria; preferably, the concentration of p-coumaric acid in the initial reaction system is 2 mM.

10. The method according to claim 7, characterized in that The recombinant engineered bacteria are added in the form of bacterial solution. The initial OD value of the recombinant engineered bacteria solution is 0.6-0.

8. The amount of the recombinant engineered bacteria solution added is 4% of the total volume of the initial reaction system in terms of volume percentage.

11. The method according to claim 7, characterized in that During the reaction, the reaction temperature is 15-45°C, the pH value of the reaction system is 4-12; the concentration of IPTG after being added to the reaction system is 0.1-1mM, and the concentration of DMSO after being added to the reaction system is 0-20%; and the intermittent feeding of p-coumaric acid and glycerol is added once every 8 hours.

12. The method according to claim 11, characterized in that During the reaction, the reaction temperature is 30° C., the pH value of the reaction system is 8-9, the concentration of IPTG after being added to the reaction system is 0.17-0.3 mM, and the concentration of DMSO after being added to the reaction system is 5%; the intermittent feeding of p-coumaric acid and glycerol is respectively added at 0, 8, 16, 24, 32, 40, and 48 hours.

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