Recombinant escherichia coli for improving gernial A titer by key enzymes ispA and GAS of protein scaffold cascade pathway and application

Abstract

The application discloses a kind of recombinant escherichia coli for improving gimalenol A titer by protein scaffold cascade pathway key enzyme IspA and GAS and application thereof, fusion protein is expressed in the recombinant escherichia coli, fusion protein is obtained by four peptide binding modules respectively with pathway key enzyme IspA and GAS cascade, four peptide binding modules are connected to the N terminal of IspA and the C terminal of GAS by connecting peptide, four peptide binding modules are SpyCatcher, SpyTag, SnoopCatcher and SnoopTag;Four peptide binding modules form stable enzyme cascade assembly through specific covalent action, to realize the spatial close-range cascade catalysis of IspA and GAS, improve the synthesis efficiency of gimalenol A, finally by Cope rearrangement gimalenol A is converted into β-elemene.The application uses glycerol as carbon source, realizes the efficient synthesis of gimalenol A by IPTG induction and in-situ extraction, and drives its Cope rearrangement to be converted into β-elemene under high temperature condition.The yield of β-elemene reaches 7.2g / L and 31.2g / L respectively by shake flask and fermenter verification, and the product yield and substrate utilization are improved.

Description

Technical Field

[0001] This invention relates to a recombinant Escherichia coli that enhances gemmaene A titer through key enzymes IspA and GAS in a protein scaffold cascade pathway, and its applications, belonging to the fields of bioengineering and biosynthesis technology. Background Technology

[0002] β-Elemene is a natural bioactive compound with a cyclic sesquiterpene structure, possessing unique antitumor, anti-inflammatory, and antioxidant activities, and has been widely used in pharmaceuticals, cosmetics, and agriculture. However, traditional plant extraction methods rely on endangered plant resources, resulting in low efficiency, high costs, and environmental unsustainability. While chemical synthesis bypasses the limitations of plant resources, it faces challenges including complex reaction pathways, poor stereoselectivity, dependence on toxic catalysts, and byproduct contamination. In contrast, microbial biosynthesis using synthetic biology strategies (such as E. coli) demonstrates superior advantages in terms of pathway streamlines, stereospecificity, and sustainability.

[0003] Gemaene A, a direct precursor of β-elemene, is a widely distributed sesquiterpene compound in nature. Under heat treatment conditions, gemmaene A readily undergoes Cope rearrangement to form β-elemene. The biosynthesis of sesquiterpenes uses isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) as basic structural elements, catalyzed by farnesenyl pyrophosphate synthase (FPPS) to generate the sesquiterpene precursor FPP. Using FPP as a substrate, gemmaene A is then catalyzed by gemmaene A synthase (GAS).

[0004] In engineered strains, due to the lack of natural regulatory networks (such as feedback inhibition and precursor allocation mechanisms), the introduction of xenogeneic or synthetically designed metabolic pathways often leads to the following problems:

[0005] (1) The key precursor farnesyl pyrophosphate (FPP) is competitively consumed, resulting in insufficient target flux;

[0006] (2) Additional substrate FPP needs to be added. Due to the difficulty and high price of FPP preparation, the difficulty and cost of production are significantly increased.

[0007] (3) Accumulation of unstable intermediates (such as IDI and FPP) causes host toxicity and inhibits growth;

[0008] (4) The mismatch between enzyme fluxes leads to metabolic flux imbalance and limited yield.

[0009] Artificial protein scaffold technology can significantly reduce intermediate loss, decrease toxicity accumulation, and improve metabolic flux efficiency by enabling spatial co-localization and cascade reactions of enzymes, thus providing a new strategy for the microbial synthesis of complex natural products. Summary of the Invention

[0010] To address the limitations of existing technologies, such as limited metabolic flux, accumulation of intermediate toxicity, and low co-catalytic efficiency, this invention provides a protein scaffold-mediated covalent cascade assembly system for key enzymes. This system enables the efficient synthesis of gemmaene A from a glycerol substrate in *E. coli*, followed by thermally driven Cope rearrangement to generate β-elemene. This invention overcomes the limitations of traditional free enzyme systems, achieving high-level synthesis of the target product. It eliminates the need for the expensive substrate FPP, achieves high β-elemene yield, and significantly reduces production costs.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0012] A recombinant *E. coli* strain that enhances gemmaene A titer via key enzymes IspA and GAS in a protein scaffold cascade pathway. The recombinant *E. coli* strain expresses a fusion protein, which is obtained by cascading four peptide-binding modules with IspA (farnesenyl pyrophosphate synthase, FPPS) and GAS, respectively. The four peptide-binding modules are connected to the N-terminus of IspA and the C-terminus of GAS via linker peptides, and are SpyCatcher, SpyTag, SnoopCatcher, and SnoopTag.

[0013] Four peptide-binding modules form a stable enzyme cascade assembly through specific covalent interactions, thereby enabling spatially close cascade catalysis of IspA and GAS, improving the synthesis efficiency of gemmaene A, and finally converting gemmaene A into β-elemene through Cope rearrangement.

[0014] The abbreviations for SpyCatcher, SpyTag, SnoopCatcher, and SnoopTag are all related to SpyC, SpyTag, SnoopTag, and SnoopT.

[0015] This invention employs four peptide-binding modules, SpyC, SpyT, SnoopC, and SnoopT, which are cascaded with IspA and GAS.

[0016] The amino acid sequences of IspA and GAS are shown in SEQ ID No:1 and SEQ ID No:2, respectively; the amino acid sequences of SpyC, SpyT, SnoopC, SnoopT and the linker peptide are shown in SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 and SEQ ID No:7, respectively.

[0017] The aforementioned fusion proteins are one of the following groups: SpyCatcher-IspA and GAS-SpyTag fusion protein, SpyTag-IspA and GAS-SpyCatcher fusion protein, SnoopCatcher-IspA and GAS-SnoopTag fusion protein, or SnoopTag-IspA and GAS-SnoopCatcher fusion protein.

[0018] To further increase the yield of β-elemene, the fusion protein is a SnoopTag-IspA and GAS-SnoopCatcher fusion protein. In SnoopTag-IspA, SnoopTag is fused to the N-terminus of IspA via a 15-amino acid linker peptide, and in GAS-SnoopCatcher, SnoopCatcher is fused to the C-terminus of GAS via a 15-amino acid linker peptide.

[0019] SnoopTag and SnoopCatcher form a stable enzyme cascade assembly through specific covalent interactions, thereby achieving spatially close cascade catalysis of IspA and GAS, improving the synthesis efficiency of gemmaene A, and finally converting gemmaene A into β-elemene through Cope rearrangement.

[0020] The above-mentioned recombinant Escherichia coli capable of synthesizing gemmaene A from glycerol via metabolic pathways, and the synthesis of β-elemene from glycerol:

[0021] (a) The recombinant Escherichia coli was cultured using glycerol as a carbon source;

[0022] (b) During the culture process, add 8-12% of the culture medium volume of isopropyl myristate to extract gemmaene A in situ;

[0023] (c) Gemmaene A undergoes Cope rearrangement driven by heating conditions at a temperature ≥250℃ to generate β-elemene.

[0024] This application uses glycerol as a carbon source to synthesize gemmaene A via a protein scaffold cascade of IspA and GAS.

[0025] Compared to free enzyme strains, enzyme cascade strains grow faster. Free enzyme strains require 6 hours to reach an OD600 of 6, while enzyme cascade strains only require 4 hours.

[0026] The method for preparing β-elemene using the above method is as follows: Recombinant Escherichia coli is induced to culture with IPTG at a final concentration of 0.4-0.6 mM (preferably 0.5 mM) using glycerol as a substrate, and 8-12% (preferably 10%) of the culture volume of isopropyl myristate is added for in-situ extraction to biosynthesize the β-elemene precursor gemmaene A. Gemaene A is then converted to β-elemene via Cope rearrangement through a high-temperature gas injection port at a temperature exceeding 250°C using a GC system.

[0027] In shake flask culture, the yield of β-elemene exceeded 7 g / L. In fermenter culture, the yield of β-elemene exceeded 30 g / L.

[0028] The above shake flask culture method: Recombinant Escherichia coli was fermented in TB medium supplemented with 85-95 g / L glycerol. When the OD600 reached 5-7, protein expression was induced with 0.4-0.6 mM IPTG. Gemmaene A was extracted in situ by adding 8-12% (v / v) isopropyl myristate as an organic phase extractant.

[0029] Further optimization of the above-mentioned shake-flask culture method: Recombinant *E. coli* was fermented in TB medium supplemented with 90 g / L glycerol. When the OD600 reached 6, protein expression was induced using 0.5 mM IPTG, and 10% (v / v) isopropyl myristate was added as the organic phase extractant for in-situ extraction of gemmaene A. After optimization, the yield of β-elemene in shake flasks was 7.2 g / L, and the highest bacterial concentration with an OD600 of 27.1 was achieved, representing increases of 260% and 67.3% respectively compared to the yield of the free enzyme strain (2 g / L) and the OD600 of 16.2. The optimal substrate culture conditions for the free enzyme strain were a glycerol concentration of 20 g / L and a peak bacterial concentration time of 48 h; beyond these concentrations and time points, the bacterial concentration began to decline. In contrast, the optimal glycerol concentration for the enzyme cascade strain of this application was 90 g / L, and the peak bacterial concentration time was 168 h.

[0030] The above fermenter culture method is as follows: recombinant Escherichia coli is fermented in AM medium with 20-30 g / L glycerol as the initial substrate. When the OD600 reaches 75-80, protein expression is induced with 0.4-0.6 mM IPTG, and 8-12% (v / v) isopropyl myristate is added as an organic phase extractant to extract gemmaene A in situ.

[0031] Further optimization involved fermenting recombinant Escherichia coli in AM medium supplemented with 25 g / L glycerol as the initial substrate. When the OD600 reached 78, protein expression was induced with 0.5 mM IPTG, and gemmaene A was extracted in situ using 10% (v / v) isopropyl myristate as the organic phase extractant.

[0032] To avoid metabolic inhibition caused by excessive substrate, fed-batch fermentation is employed in the fermenter. Dissolved oxygen and feeding are linked; when the initial substrate in the AM medium is depleted, the dissolved oxygen level in the culture system rises rapidly and exceeds 30%. At this point, an automatic feed solution containing 400-600 g / L glycerol is added to maintain substrate supply, ensuring the glycerol concentration in the culture system remains between 0.01-0.1 g / L. Simultaneously, 13% ammonia or 30% phosphoric acid is added to maintain the pH at 7.0.

[0033] Through batch-fed fermentation in a fermenter, the yield of β-elemene was 31.2 g / L, the glycerol conversion rate was 8.9%, and the OD600 was 286. Compared with the free enzyme strain, the conversion rate was increased by 122% and the OD600 was increased by 27.1%.

[0034] To further increase the yield of β-elemene, the pre-induction culture conditions for both shake flask culture and fermenter culture were: 250 rpm rotation speed and 37℃ temperature; the post-induction culture conditions were: 200 rpm rotation speed and 25℃ temperature.

[0035] This application introduces a SnoopTag / SnoopCatcher peptide-binding module between IspA and GAS to achieve spatial co-localization and covalent cascade assembly of key enzymes, thereby increasing metabolic flux and reducing intermediate loss. The fusion protein is introduced into *E. coli* BL21(DE3) via a plasmid system. Using glycerol as the carbon source, gemmaene A is synthesized efficiently through IPTG induction and in-situ extraction, and its Cope rearrangement is driven to convert it to β-elemene under high temperature conditions. Shake-flask and fermenter tests verified that the β-elemene yield reached 7.2 g / L and 31.2 g / L, respectively, more than four times that of the free enzyme system. This invention effectively solves the problems of metabolic flux imbalance and intermediate toxicity accumulation, significantly improving the yield of the target product and substrate utilization, and has the advantages of low cost, high efficiency, and broad industrial application prospects.

[0036] The protein scaffold used for the efficient production of β-elemene was constructed in an E. coli chassis. The E. coli used was BL21(DE3) carrying the pJBEI-6409-MVA-IDI-IspA and pETDuet-AvGAS plasmids (obtained by referring to: Yihua Lu, Xinyi Zhang, Fei Wang, Yu Zhang, and Xun Li. Optimization of GermacreneASynthase for Efficient Production of β-Elemene in Escherichia coli. ACSSustainable Chemistry & Engineering 2024, 12(24), 9018-9026. DOI:10.1021 / acssuschemeng.3c07658.).

[0037] Any techniques not mentioned in this invention are based on existing technologies.

[0038] This invention utilizes recombinant *E. coli* strains with enhanced gemmaene A titers via a protein scaffold cascade pathway. Through scaffold-mediated assembly of key enzymes IspA and GAS, and by dynamically regulating enzyme expression levels and employing a modular pathway design, the synergistic efficiency of IspA and GAS is improved. This maintains the concentration of toxic intermediates below the sub-inhibition threshold, reduces host stress, minimizes metabolic diversion and side effects, and enhances glycerol conversion and cell viability, ultimately significantly increasing β-elemene yield. Further, fed-batch fermentation in a fermenter resulted in a β-elemene yield of 31.2 g / L, more than four times that of the scaffold-free control group (7.74 g / L), with a glycerol conversion rate of 8.9% (an increase of 122%) and a final OD600 concentration increase of 27.1%. The method provided by this invention not only significantly increases β-elemene yield but also improves glycerol utilization, which helps reduce commercial costs and thus increases industrial value. Attached Figure Description

[0039] Figure 1 This is a schematic flowchart illustrating the metabolism of β-elemene for the production of this invention.

[0040] Figure 2 This invention utilizes a protein scaffold-mediated enzyme cascade.

[0041] Figure 3 This is a diagram showing the yield of the enzyme cascade mediated by the protein scaffold in this invention.

[0042] Figure 4 Characterization diagram of the successful construction of the multi-enzyme complex system.

[0043] Figure 5OD600 plot showing β-elemene yield and bacterial concentration under optimized conditions.

[0044] Figure 6 Figures showing the yield, glycerol concentration, and OD600 of β-elemene in a feed-fed fermenter. Detailed Implementation

[0045] To better understand the present invention, the following embodiments further illustrate its content, but the content of the present invention is not limited to the embodiments described below. In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. Processes involved in the embodiments that are not described in detail are those that can be understood and easily implemented by those skilled in the art based on product specifications or basic knowledge in the field, and therefore will not be described in detail.

[0046] A schematic flowchart of the production of β-elemene according to the present invention is shown below. Figure 1 As shown.

[0047] Example 1

[0048] Preparation of SpyC-IspA, GAS-SpyT, SpyT-IspA, GAS-SpyC, SnoopC-IspA, GAS-SnoopT, SnoopT-IspA and GAS-SnoopC:

[0049] SpyC was synthesized by Shanghai Sangon Biotech Co., Ltd., and SnoopC was synthesized by Jiutian Gene Biotechnology Co., Ltd. SpyT and SnoopT were obtained by reverse PCR using Takara's PrimeSTAR MAX high-fidelity enzyme with designed primers (amino acid sequence acquisition method refers to: Jiale Qu, Sheng Cao, Qixin Wei, Huawei Zhang, Rui Wang, Wei Kang, Tian Ma, Liang Zhang, Tiangang Liu, Shannon Wing-Ngor Au, Fei Sun, Jiang Xia. Synthetic Multienzyme Complexes, Catalytic Nanomachineries for Cascade Biosynthesis InVivo. ACS Nano 2019, 13(9), 9895-9906. DOI:10.1021 / acsnano.9b03631.). They were then ligated to the N-terminus of IspA and the C-terminus of GAS, respectively, using a linker. Primers, related reaction systems, and reaction procedures are shown in the table below.

[0050] Primer design

[0051]

[0052]

[0053] PrimeSTAR MAX high-fidelity enzyme reaction system

[0054]

[0055] PrimeSTAR MAX High-Fidelity Enzyme Reaction Program

[0056]

[0057] Phosphorylation reaction system

[0058]

[0059]

[0060] Phosphorylation reaction procedure

[0061]

[0062] Connecting reaction system

[0063]

[0064] The amino acid sequences of IspA and GAS are shown in SEQ ID No:1 and SEQ ID No:2, respectively, and the amino acid sequences of SpyC, SpyT, SnoopC, SnoopT and linker are shown in SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 and SEQ ID No:7, respectively.

[0065] The sequence of SEQ ID No:1 is as follows:

[0066] MDFPQQLEACVKQANQALSRFIAPLPFQNTPVVETMQYGALLGGKRLRPFLVYAT

[0067] GHMFGVSTNTLDAPAAAVECIHAYSLIHDDLPAMDDDDLRRGLPTCHVKFGEANAILA

[0068] GDALQTLAFSILSDADMPEVSDRDRISMISELASASGIAGMCGGQALDLDAEGKHVPLD

[0069] ALERIHRHKTGALIRAAVRLGALSAGDKGRRALPVLDKYAESIGLAFQVQDDILDVVG

[0070] DTATLGKRQGADQQLGKSTYPALLGLEQARKKARDLIDDARQSLKQLAEQSLDTSAL

[0071] EALADYIIQRNK

[0072] The sequence of SEQ ID No: 2 is as follows:

[0073] MGDIAGAAALARVRNASMEKFTFPNLYCPFPERKNPHSEWLQDYALQWVIRFKLIDSESLYQRFSKAKFYLLTAGAYPHCQLEELKIANDVISWLFIWDDQCDISDLGKKPELLKTWCNRFLEILNGAELTPDDLPLGFALRDIRNRIINRGGITFFHHFVRNFEDYFYGCIEEAHNRVNVSVPDVEAYIKIRSANAAAALCLNLIEFCDRVMIPYSLRNHETLKKLTQMTINILAWSNDIFSAPREIANGEVHNLVFVIHHHQKIPLEKAMLAAAAMHNHEVQKLVNLESKIPSFSAETDAEITKYISGLHAWIRGNLDWYAHSGRYQITEKLELLAS

[0074] The sequence of SEQ ID No: 3 is as follows:

[0075] GSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSS

[0076] GKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATK

[0077] GDAHIEF

[0078] The sequence of SEQ ID No: 4 is as follows:

[0079] AHIVMVDAYKPTK

[0080] The sequence of SEQ ID No: 5 is as follows:

[0081] ASKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRL FENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYITNEPIPPK

[0082] The sequence of SEQ ID No:6 is as follows:

[0083] KLGDIEFIKVNK

[0084] The sequence of SEQ ID No:7 is as follows:

[0085] GGGGSGGGGSGGGGS

[0086] The constructed plasmids were transformed into E. coli competent cells (top 10) using the heat shock method. Thaw 100 μL of pre-prepared competent cells on ice. Then, add approximately 1 ng of plasmid to the thawed competent cells, mix gently, and incubate on ice for 5 min. Next, place the centrifuge tube containing the competent cells in a 42°C water bath for 60 s, remove it, and immediately incubate on ice for 5 min. Then, add preheated SOC liquid medium (40°C) to the centrifuge tube, mix by inverting, and finally incubate the centrifuge tube at 37°C and 250 rpm for 60 min. After incubation, evenly spread the mixture onto plates containing 100 mg / L ampicillin or 33 mg / L chloramphenicol, and then incubate at 37°C for 12 h. Subsequently, send the plates to a biotechnology company for sequencing to confirm the fidelity of the insert fragments in the vectors pJBEI-6409-MVA-IDI-IspA and pETDuet-AvGAS.

[0087] Example 2

[0088] Comparison of β-elemene titers produced by four fusion protein combinations:

[0089] like Figure 2 As shown, the four fusion protein combinations are: (1) SnoopT-IspA and GAS-SnoopC (strain FS2); (2) SnoopC-IspA and GAS-SnoopT (strain FS3); (3) SpyT-IspA and GAS-SpyC (strain FS4); (4) SpyC-IspA and GAS-SpyT (strain FS5) and the free enzyme control group (strain FS1).

[0090] The following are the steps for verifying the production of β-elemene from four fusion proteins in E. coli (BL21):

[0091] (1) The recombinant expression vector successfully constructed in Example 1 was transformed into Escherichia coli BL21(DE3) by heat shock method (as described in Example 1) [Escherichia coli is BL21(DE3) carrying pJBEI-6409-MVA-IDI-IspA and pETDuet-AvGAS plasmids, obtained by referring to: Yihua Lu, Xinyi Zhang, Fei Wang, Yu Zhang, and Xun Li. Optimization of Germacrene A Synthase for Efficient Production of β-Elemene in Escherichia coli. ACS Sustainable Chemistry & Engineering 2024, 12(24), 9018-9026. DOI:10.1021 / acssuschemeng.3c07658.];

[0092] (2) Pick a single colony that grows normally on the plate and put it into a 5 mL test tube containing LB liquid medium containing 100 mg / L ampicillin and 33 mg / L chloramphenicol. Incubate at 37°C and 250 rpm for 12 h to obtain the initial culture.

[0093] (3) Take 1 mL of the initial culture and inoculate it into 50 mL of new TB liquid medium containing 100 mg / L ampicillin, 33 mg / L chloramphenicol and 20 g / L glycerol (that is, add 20 g / L glycerol to the TB liquid medium), and culture the bacterial solution at 37°C and 250 rpm in a shaker until the OD600 reaches 6.

[0094] (4) When OD600 is approximately 6, add 25 μL of IPTG (1M) as an inducer to a final concentration of 0.5 mM. Then add 10% isopropyl myristate to the culture system. Place the shake flask in a shaker at 25°C and 200 rpm. Take samples (500-700 μL) every 24 hours to detect the OD600 of the bacterial culture and the yield of β-elemene after Cope rearrangement transformation, as detailed below:

[0095] Every 24 hours, 600 μL of *E. coli* culture was sampled and centrifuged at 12000 × g for 5 minutes for phase separation. The upper organic phase containing gemmaene A (200 μL) was collected for analysis. During injection, the high-temperature GC inlet (300 °C) thermally triggered the Cope rearrangement of gemmaene A to β-elemene, thus achieving in-situ transformation before detection. β-elemene quantification was performed using an Agilent 7890A gas chromatograph equipped with a flame ionization detector (GC-FID) and a Cyclosil-B β-cyclodextrin chiral capillary column (30 m × 0.25 mm × 0.25 μm). The temperature program started at 50 °C (hold for 1 minute), increased to 180 °C (hold for 5 minutes) at a rate of 10 °C / min, and then increased to 250 °C (hold for 3 minutes) at a rate of 15 °C / min. The injector and detector temperatures were maintained at 300 °C, with a separation ratio of 10:1. High-purity nitrogen was used as the carrier gas at a flow rate of 1.2 mL / min.

[0096] Comparative analysis of β-elemene yield showed that the SnoopT-IspA and GAS-SnoopC combination (strain FS2) exhibited excellent catalytic performance, achieving a β-elemene yield of 2.6 g / L in shake flasks, significantly higher than other combinations. Figure 3 ).

[0097] Example 3

[0098] Characterization of β-elemene production from a multi-enzyme complex system:

[0099] To verify that the increase in β-elemene titer was indeed caused by the successful assembly of the protein scaffold-mediated dual-enzyme complex, the protein-protein interactions were characterized using transmission electron microscopy (TEM).

[0100] The strain FS2 obtained in Example 2 was cultured in 5 mL of LB medium in a shaker. Antibiotics (33 mg / L chloramphenicol and 100 mg / L ampicillin) were added, and the mixture was shaken well and cultured at 250 rpm and 37 °C for 12 h to obtain the initial culture. 1 mL of the initial culture was inoculated into 50 mL of fresh LB medium containing 100 mg / L ampicillin, 33 mg / L chloramphenicol, and 20 g / L glycerol. The culture was then incubated in a shaker at 37 °C and 250 rpm until the OD600 reached approximately 0.6. 0.5 mM IPTG was added to induce protein expression. After 12 h, the mixture was centrifuged at 8000 × g for 10 min, the supernatant was discarded, and the cell pellet was obtained. The pellet was washed twice with 10 mM PBS (pH 7.4) and then resuspended in 7 mL of lysis buffer (7.8 g / L NaH2PO4·2H2O, 17.54 g / L NaCl, 0.68 g / L imidazole, pH 8.0). The resuspended cells were sonicated at 300W for 12 min, and then centrifuged at 8000×g for 10 min at 4°C. The supernatant was collected for downstream applications.

[0101] Pre-equilibrate 2 mL of Ni-NTA resin with 10 mL of lysis buffer, and load the supernatant onto 2 mL of Ni-NTA resin. After loading, wash the column with 25 mL of wash buffer (7.8 g / L NaH2PO4·2H2O, 17.54 g / L NaCl, 1.36 g / L imidazole, pH 8.0) to remove non-specific proteins, and then elute the target protein with 15 mL of elution buffer (7.8 g / L NaH2PO4·2H2O, 17.54 g / L NaCl, 17 g / L imidazole, pH 8.0).

[0102] The eluted multienzyme complex was diluted to 0.0005 mg / mL in a buffer solution consisting of 20 mM sodium dihydrogen phosphate and 150 mM NaCl (pH 7.0). 10 μL of the diluted solution was plated onto a carbon-coated copper grid and subsequently stained with phosphotungstic acid. The sample was imaged using a JEM-1400 transmission electron microscope at an accelerating voltage of 200 kV. Figure 4 As shown, the free enzyme group exhibits a small, nearly spherical particle distribution, while the enzyme cascade complex exhibits a larger, irregularly shaped structure, supporting the successful assembly of the multi-enzyme complex.

[0103] Example 4

[0104] Optimization of β-elemene production system conditions:

[0105] The results of Example 2 show that the four multi-enzyme complexes constructed in this invention can significantly improve the production efficiency of β-elemene, especially the combination of IspA-SnoopT and SnoopC-GAS (strain FS2) which exhibits superior catalytic performance. Therefore, in order to further improve the yield of β-elemene, the production conditions of strain FS2 were optimized in this example.

[0106] First, the IPTG induction concentrations were screened in shake flasks (0.1, 0.25, 0.5, 0.75, and 1.0 mM), meaning only the IPTG concentration was changed while other conditions remained consistent with Example 2. Figure 5 The results shown in Figure a indicate that after 48 hours of induction, the yield of β-elemene induced by a final concentration of 0.5 mM IPTG reached a peak of 2.69 g / L, with an OD600 of 15.2. After screening, the induction concentration (0.5 mM) in Example 2 was found to be the optimal induction condition.

[0107] Next, the concentrations of the inducing bacteria (OD600) were screened by shaking the flasks (0.8, 2, 4, 6, and 8), i.e., only the concentration of the inducing bacteria was changed while other conditions remained consistent with Example 2. Figure 5 The results shown in b indicate that after 48 hours of induction, the yield of β-elemene reached a peak of 2.68 g / L when the inducing bacterial concentration OD600 was 6, and the OD600 reached 16.08. The optimal inducing bacterial concentration OD600 was consistent with that in Example 2.

[0108] Subsequently, the substrate glycerol concentration was optimized using gradients of 20, 30, 40, and 50 g / L, while other conditions remained consistent with Example 2. As the shake-flask fermentation time progressed, the yield of β-elemene and OD600 continuously increased, reaching the experimental upper limit of 50 g / L glycerol after 96 h of induction (β-elemene: 5.3 g / L; OD600: 23.9). Figure 5 c). Further exploration of glycerol concentrations (60, 70, 80, 90, and 100 g / L) and extended fermentation times (96–168 h) revealed that β-elemene yield reached a peak of 7.2 g / L (OD600: 27.1) after 168 h at 90 g / L glycerol, while 100 g / L glycerol led to a decrease in both yield and OD600. Figure 5 d).

[0109] Example 5:

[0110] Engineered strains produce β-elemene in a 1.3L bioreactor:

[0111] To further evaluate the application potential of the high-yield engineered strain, strain FS2 from Example 2 was subjected to fed-batch fermentation in a 1.3L bioreactor to verify its ability to produce β-elemene under scale-up conditions. The initial culture obtained in Example 2 was inoculated into shake flasks at 2% of the shake flask culture volume and cultured under constant conditions until the OD600 reached 15. Subsequently, the shake-flask bacterial culture was inoculated at 10% fermenter volume into AM medium containing 25 g / L glycerol as the initial substrate, 100 mg / L ampicillin, and 33 mg / L chloramphenicol (25 g / L glycerol, 15.7 g / L K2HPO4·H2O, 4.2 g / L KH2PO4, 2.0 g / L (NH4)2SO4, 1.2 g / L MgSO4·7H2O, 1.7 g / L citric acid, 8.4 mg / L EDTA, 4.5 mg / L thiamine·HCl, and 10 mL / L trace metal solution (1.5 g / L MnCl2·H2O, 1.3 g / L Zn(CH3COO)2·2H2O, 0.25 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.3 g / L MnCl2·H2O, 0.3 g / L HCl, 100 mg / L MgSO4·7H2O, 1.7 g / L citric acid, 8.4 mg / L EDTA, 4.5 mg / L thiamine·HCl, and 10 mL / L trace metal solution). Fermentation was carried out using H3BO4, 0.25 g / L Na2MoO4·2H2O, and 5 g / L Fe(III)citrate hydrate at a fermentation temperature of 37°C. The dissolved oxygen (DO) range was set to 25%-30% and linked to the fermentation speed. The DO decreased with increasing bacterial density. When the DO fell below 25%, the fermentation speed was increased (maximum speed 1000 rpm) to maintain the DO within the set range. When the OD600 reached approximately 78, protein expression was induced using 0.5 mM IPTG. Gemmaene A was extracted in situ using 10% (v / v) isopropyl myristate as an organic phase extractant at a fermentation temperature of 25°C. To avoid metabolic inhibition caused by excessive substrate, a dissolved oxygen controlled feeding strategy was adopted. When the dissolved oxygen level exceeded 30%, an automatic feed solution containing 500 g / L glycerol (500 g / L glycerol, 12 g / L MgSO4·7H2O, 10.7 g / L (NH4)2SO4, 13 mg / L EDTA, and 10 mL / L trace metal solution (1.5 g / L MnCl2·H2O, 1.3 g / L Zn(CH3COO)2·2H2O, 0.25 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.3 g / L H3BO4, 0.25 g / L Na2MoO4·2H2O, and 5 g / L Fe(III)citrate hydrate) was added to maintain substrate supply, thus keeping the glycerol concentration in the culture system at 0.01-0.1 g / L. Meanwhile, the pH was maintained at 7.0 by adding 13% ammonia or 30% phosphoric acid.

[0112] Ultimately, the enzyme cascade strain (FS2) achieved a β-elemene yield of 31.21 g / L, an OD600 of 286.20, and a glycerol conversion rate of 8.9%. Figure 6 The yield of the free enzyme strain was 7.74 g / L, the OD600 was 225, and the glycerol conversion rate was 4.0% (Reference: Yihua Lu, Xinyi Zhang, Fei Wang, Yu Zhang, and Xun Li. Optimization of Germacrene A Synthase for Efficient Production of β-Elemene in Escherichia coli. ACSSustainable Chemistry & Engineering 2024, 12(24), 9018-9026. DOI:10.1021 / acssuschemeng.3c07658.). The yield of the enzyme cascade strain in the 1.3L fermenter was 4.03 times that of the free enzyme system (7.74 g / L), and the glycerol conversion rate was 2.22 times that of the free enzyme system.

Claims

1. A recombinant *E. coli* strain that enhances gemmaene A titer via key enzymes IspA and GAS in a protein scaffold cascade pathway, characterized in that: The recombinant Escherichia coli expressed a fusion protein, which was obtained by cascading four peptide-binding modules with IspA and GAS respectively. The four peptide-binding modules were connected to the N-terminus of IspA and the C-terminus of GAS respectively through linking peptides. The four peptide-binding modules were SpyCatcher, SpyTag, SnoopCatcher and SnoopTag. Four peptide-binding modules form a stable enzyme cascade assembly through specific covalent interactions, thereby enabling spatially close cascade catalysis of IspA and GAS, improving the synthesis efficiency of gemmaene A, and finally converting gemmaene A into β-elemene through Cope rearrangement. The fusion protein is one of the following groups: SpyCatcher-IspA and GAS-SpyTag fusion protein, SpyTag-IspA and GAS-SpyCatcher fusion protein, SnoopCatcher-IspA and GAS-SnoopTag fusion protein, or SnoopTag-IspA and GAS-SnoopCatcher fusion protein. Using the shake flask culture method, the yield of β-elemene exceeded 7 g / L; using the fermenter culture method, the yield of β-elemene exceeded 30 g / L. The amino acid sequences of IspA and GAS are shown in SEQ ID No:1 and SEQ ID No:2, respectively; the amino acid sequences of SpyC, SpyT, SnoopC, SnoopT and the linker peptide are shown in SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 and SEQ ID No:7, respectively.

2. The application of the recombinant Escherichia coli according to claim 1, characterized in that: Synthesis of β-elemene starting from glycerol: (a) The recombinant Escherichia coli was cultured using glycerol as a carbon source; (b) During the culture process, add 8-12% of the culture medium volume of isopropyl myristate to extract gemmaene A in situ; (c) Gemmaene A undergoes Cope rearrangement driven by heating conditions at a temperature ≥250 °C to generate β-elemene.

3. The application according to claim 2, characterized in that: Recombinant Escherichia coli was cultured using glycerol as a substrate at a final concentration of 0.4-0.6 mM IPTG. Isopropyl myristate was added at 8-12% of the culture volume for in-situ extraction and biosynthesis of β-elemene precursor gemmaene A. Gemaene A was converted to β-elemene via Cope rearrangement through a high-temperature gas injection port at over 250 °C using a GC system.

4. The application according to claim 2 or 3, characterized in that: Shake flask culture method: Recombinant Escherichia coli was fermented in TB medium supplemented with 85-95 g / L glycerol. When the OD600 reached 5-7, protein expression was induced with 0.4-0.6 mM IPTG. Gemmaene A was extracted in situ by adding 8-12% (v / v) isopropyl myristate as an organic phase extractant. The fermenter culture method is as follows: recombinant Escherichia coli is fermented in AM medium supplemented with 20-30 g / L glycerol as the initial substrate. When the OD600 reaches 75-80, protein expression is induced with 0.4-0.6 mM IPTG, and 8-12% (v / v) isopropyl myristate is added as an organic phase extractant to extract gemmaene A in situ.

5. The application according to claim 4, characterized in that: Shake flask culture method: Recombinant Escherichia coli was fermented in TB medium supplemented with 90 g / L glycerol. When the OD600 reached 6, protein expression was induced with 0.5 mM IPTG. Gemmaene A was extracted in situ by adding 10% (v / v) isopropyl myristate as an organic phase extractant. The fermenter culture method adopts batch feeding fermentation. Through the linkage between dissolved oxygen and feeding, when the initial substrate in the AM medium is depleted, the dissolved oxygen in the culture system will rise rapidly and exceed 30%. At this time, the feed solution containing 400-600 g / L glycerol will be automatically added to maintain the substrate supply, so that the glycerol concentration in the culture system is maintained at 0.01-0.1 g / L. At the same time, the pH is maintained at 7.0 by adding 13% ammonia water or 30% phosphoric acid.

6. The application according to claim 4, characterized in that: For both shake flask and fermenter culture methods, the pre-induction culture conditions were: rotation speed 250 rpm, temperature 37 ℃; and the post-induction culture conditions were: rotation speed 200 rpm, temperature 25 ℃.

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