Escherichia coli engineering bacteria for producing uroporphyrin and construction method and application thereof

Abstract

The present application relates to the technical field of fermentation engineering, and provides an engineered Escherichia coli for producing uroporphyrin as well as a construction method and application thereof.The present application uses Escherichia coli to express porphyrin synthase of thermophilic bacteria, performs whole-cell catalysis of 5-ALA to synthesize uroporphyrin under high-temperature conditions, and completely shields endogenous regulation; further, the Escherichia coli is wrapped by COF material to prevent the loss of enzymes in the cell recovery process; and a protein scaffold based on dockerin-cohesin is constructed to further improve the yield of uroporphyrin III.The present application creates a brand-new uroporphyrin high-temperature whole-cell catalysis process, realizes the efficient production of uroporphyrin, and provides a basis and idea for the industrial production of uroporphyrin.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and more specifically, to an engineered Escherichia coli strain for producing uroporphyrin, its construction method, and its application. Background Technology

[0002] Porphyrin compounds are cyclic conjugated systems consisting of four pyrrole rings linked by methylene groups, forming a large π-bond structure. This structure endows porphyrin compounds with superior photoelectric properties and catalytic performance (Hiroto S, 2017, Chemical Reviews, 117(4): 2910-3043). In nature, porphyrin compounds are indispensable for life activities, such as chlorophyll, heme, and vitamin B12. In addition, porphyrin compounds also have wide applications in the medical, agricultural, and food industries. Uroporphyrin is a porphyrin compound with a porphyrin cyclic planar structure, where each pyrrole ring is connected to a formic acid group and an acetic acid group. The synthetic pathway of uroporphyrin I is highly conserved in organisms. It involves the synthesis of bile pigmentogen from 5-aminolevulinic acid (5-ALA) catalyzed by bile pigmentogen synthase (PbgS), followed by the synthesis of hydroxymethylcholesterol under the catalysis of hydroxymethylcholesterol synthase (HmbS). Hydroxymethylcholesterol then spontaneously oxidizes to form uroporphyrin I. Uroporphyrin III is synthesized from hydroxymethylcholesterol in the next step catalyzed by uroporphyrin III synthase (UroS).

[0003] These two porphyrin compounds serve as precursors to downstream porphyrin compounds, such as chlorophyll, heme, and vitamin B12, and cannot accumulate naturally in organisms, resulting in few successful biomanufacturing cases. The synthesis of other porphyrin compounds, including uroporphyrin, through fermentation is extremely difficult. Because porphyrins play a crucial role in living organisms, complex regulatory systems have developed over millions of years of evolution to control their concentrations within reasonable ranges. Furthermore, porphyrins themselves possess certain photosensitizing activity, capable of generating singlet oxygen that kills cells. Currently reported methods for synthesizing uroporphyrin through E. coli fermentation have extremely low yields and efficiency, making them unsuitable for industrial applications.

[0004] Arab overexpressed key enzymes PbgS, HmbS, and UroS in uroporphyrin synthesis, achieving uroporphyrin I at 550 mg / L and uroporphyrin III at 350 mg / L (Arab B, 2024, Synthetic Biology and Engineering, 2(1):10002), with a production efficiency of 6.25 mg / L / h, which is extremely low. Arab subsequently optimized the vitamin C concentration to adjust the reducing state in the fermentation system, obtaining uroporphyrin I at 450 mg / L and uroporphyrin III at 1000 mg / L in 144 h (Arab B, 2025, Bioengineering, 12(1): 83), but the production efficiency only increased to 10 mg / L / h. The above fermentation-based uroporphyrin synthesis process suffers from long fermentation cycles and low yields. Summary of the Invention

[0005] The purpose of this invention is to provide an engineered Escherichia coli strain for producing uroporphyrin, its construction method, and its application.

[0006] This invention focuses on constructing a high-yield strategy for uroporphyrin based on whole-cell catalysis. To achieve the objectives of this invention, in a first aspect, this invention provides an engineered *E. coli* strain for producing uroporphyrin, which is an *E. coli* strain exogenously expressing porphyrin synthase derived from thermophilic bacteria.

[0007] Preferably, the porphyrin synthase is derived from *Bacillus thermoglucosidase* (…). Parageobacillus thermoglucosidasius ),like Parageobacillus thermoglucosidasius 11955.

[0008] Option A: The porphyrin synthase includes bile pigment synthase (PbgS) and hydroxymethylcholesterol synthase (HmbS), and the main product produced is uroporphyrin I.

[0009] Option B: The porphyrin synthase includes bile pigmentogen synthase (PbgS), hydroxymethylcholine synthase (HmbS), and uroporphyrin III synthase (UroS), and the main product produced is uroporphyrin III.

[0010] Furthermore, the bile pigment synthase (PbgS), hydroxymethylcholesterol synthase (HmbS), and uroporphyrin III synthase (UroS) are co-localized through a protein scaffold to form a multi-enzyme complex, thereby increasing the synthesis ratio of uroporphyrin III.

[0011] Furthermore, the protein scaffold is a scaffold constructed based on the dockerin-cohesin molecular module.

[0012] Furthermore, the bile pigment synthase is connected to... Archaeoglobus fulgidus The dockerin molecule fusion in the source dockerin-cohesin molecular module is used as a fusion unit ①; The hydroxymethylcholine synthase and Clostridium thermocellum The dockerin molecule in the source dockerin-cohesin molecular module is fused as a fusion unit ②; The uroporphyrin III synthase and Clostridium clariflavum The dockerin molecule in the source dockerin-cohesin molecular module is fused as a fusion unit ③; Finally, the cohesin molecules from the dockerin-cohesin molecular modules from the above three sources are sequentially linked together using a linker.

[0013] Preferably, the Linker is (GGGGS)3.

[0014] Preferably, the multi-enzyme complex comprises two repeating fusion units ③.

[0015] Secondly, the present invention provides a method for constructing the engineered strain of *Escherichia coli*, the method comprising: constructing the engineered strain of *Escherichia coli* (… Escherichia coli The porphyrin synthase derived from thermophilic bacteria is expressed exogenously. For example, the starting strain is Escherichia coli BL21.

[0016] Thirdly, the present invention provides the application of the engineered Escherichia coli in the fermentation production of uroporphyrin.

[0017] Fourthly, the present invention provides a method for producing uroporphyrin: Using the engineered Escherichia coli strain described in Scheme A, a catalytic reaction is carried out in a fermentation system containing 5-aminolevulinic acid at a preset temperature, and the main reaction product is uroporphyrin I.

[0018] Using the engineered Escherichia coli strain described in Scheme B, a catalytic reaction was carried out in a fermentation system containing 5-aminolevulinic acid at a preset temperature, and the main reaction product was uroporphyrin III.

[0019] Preferably, the preset temperature is 45℃-75℃, and more preferably 60℃.

[0020] Preferably, the catalytic reaction is carried out in a batch feeding manner to maintain the concentration of 5-aminolevulinic acid in the fermentation system at 5 g / L.

[0021] Furthermore, the pH of the fermentation system is controlled at 6.5-8, preferably 7.5.

[0022] Furthermore, a reducing agent is also added to the fermentation system; The reducing agent may be selected from at least one of vitamin C, mannitol, gluconate, dithiothreitol, etc., with dithiothreitol being preferred.

[0023] Preferably, the amount of reducing agent added to the fermentation system is 0.5 g / L.

[0024] Furthermore, prior to the catalytic reaction, the method includes a step of coating the engineered Escherichia coli with a covalent organic framework (COF).

[0025] In one specific embodiment of the present invention, the covalent organic framework material COF42 used is prepared by crosslinking 2,5-diethoxyterephthalohydrazide and 1,3,5-tricarboxyphenyl.

[0026] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention pioneers a novel high-temperature whole-cell catalytic process for uroporphyrin, achieving highly efficient production of uroporphyrin and providing a foundation and approach for its industrial production. Specifically, it includes: (i) To address the complex endogenous regulation of uroporphyrin, thermophilic porphyrin synthases were expressed in *E. coli* to synthesize uroporphyrin via whole-cell catalysis of 5-ALA under high-temperature conditions, completely shielding endogenous regulation. For example, PbgS and HmbS derived from thermophilic bacteria were expressed in *E. coli* BL21 to achieve uroporphyrin production from 5-ALA fermentation broth at 60℃, yielding uroporphyrin I at a yield of 5 g / L and a production efficiency of 0.4 g / L / h.

[0027] (ii) To address the issue of protein leakage during cell recovery in the catalytic process, covalent organic frameworks (COFs) are used to encapsulate cells, ensuring that the enzymes can be completely recovered by centrifugation. For example, COFs are used to encapsulate E. coli BL21 to prevent enzyme loss during cell recovery.

[0028] (III) To address the issue of spontaneous oxidation of hydroxymethylcholine, a protein scaffold was constructed to reduce the spontaneous oxidation reaction and increase the yield of uroporphyrin III. For example, a dockerin-cohesin-based protein scaffold was constructed, which increased the yield of uroporphyrin III to 3 g / L, achieving a production efficiency of 0.25 g / L / h.

[0029] (iv) The present invention further achieves synchronous control of substrate concentration and intermediate generation by dynamically adjusting the feeding rate and reaction temperature, which significantly improves the final yield and purity of uroporphyrin I. Attached Figure Description

[0030] Figure 1This is a preferred embodiment of the high-temperature whole-cell catalytic production of uroporphyrin I. a: Schematic diagram of the whole-cell catalytic unit used to synthesize porphyrin intermediates from 5-aminolevulinic acid fermentation broth. b: Change in uroporphyrin I conversion rate with 5-ALA fermentation broth concentration. c: Time-series curve of uroporphyrin I yield. d: Continuous and efficient synthesis of uroporphyrin I using a fed-batch whole-cell catalytic process.

[0031] Figure 2 The following are optimized high-temperature whole-cell catalytic conditions in a preferred embodiment of the present invention. a: Effect of pH on high-temperature catalytic efficiency. b: Effect of catalytic cycle on catalytic efficiency. c: Changes in singlet oxygen levels during the first cycle across batches. d: Effect of dithiothreitol on catalysis. e: Effects of vitamin C, mannitol, and gluconate on catalysis. f: SDS-PAGE gel image of the catalytic supernatant during catalysis. g: Electron micrograph of cells encapsulated by COF material. h: Effect of COF material encapsulation on catalysis. i: Whole-cell catalysis photograph in a 5 L tank.

[0032] Figure 3 This invention provides a preferred embodiment of high-temperature whole-cell catalytic production of uroporphyrin III. a: Pie chart and HPLC chromatogram of uroporphyrin I and uroporphyrin III production after the introduction of UroS enzyme. b: Schematic diagram of a multi-enzyme complex scaffold designed based on dockerin-cohesin domain interactions. PbgS is fused from... A. fulgidus The dockerin structure (gray), HmbS merges from C. thermocellum Dockerin (light green), UroS is integrated from C. clariflavum Dockerin (green). The corresponding cohesin domains are represented by their corresponding colors and are used to mediate specific binding. c: Different yields of uroporphyrin III can be obtained by regulating the cohesin scaffold containing different numbers of Y domains. d: Product composition analysis results of the optimized uroporphyrin III whole-cell catalytic system. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0034] This invention relates to the following gene: from *Bacillus thermoglucosinolates* (… Parageobacillus thermoglucosidasius )of PbgS , HmbS , UroS The nucleotide sequences of the genes are shown in SEQ ID NO:1-3.

[0035] Archaeoglobus fulgidus The nucleotide sequences of the gene encoding the anchoring protein dockerin and the gene encoding the adhesion protein cohesin in the dockerin-cohesin molecular module are shown in SEQ ID NO:4-5, respectively.

[0036] Clostridium thermocellum The nucleotide sequences of the gene encoding the anchoring protein dockerin and the gene encoding the adhesion protein cohesin in the dockerin-cohesin molecular module are shown in SEQ ID NO:6-7, respectively.

[0037] Clostridium clariflavum The nucleotide sequences of the gene encoding the anchoring protein dockerin and the gene encoding the adhesion protein cohesin in the dockerin-cohesin molecular module are shown in SEQ ID NO:8-9, respectively.

[0038] The 5-ALA fermentation broth used in the following examples can be prepared by referring to the literature Systematic development of a highly efficient cell factory for 5-aminolevulinic acid production, Trends in Biotechnology, https: / / doi.org / 10.1016 / j.tibtech.2024.06.004.

[0039] Example 1: High-Temperature Whole-Cell Catalytic Production of Uroporphyrin I 1. Construction of a whole-cell catalytic system 5-Aminolevulinic acid (5-ALA) can be obtained through fermentation. This invention further develops a whole-cell catalysis for converting 5-ALA fermentation broth into uroporphyrin I and uroporphyrin III.

[0040] This invention develops a novel biocatalytic system that utilizes... Parageobacillus thermoglucosidasius The thermostable enzyme 11955 is efficiently expressed in *E. coli* BL21, enabling whole-cell catalysis at high temperatures (60°C). Theoretically, at this temperature, the host *E. coli*'s endogenous enzymes and regulatory systems will be inactivated, with only the thermostable enzyme remaining active, thus achieving highly selective and efficient transformation. Furthermore, the increased cell membrane permeability under high temperature conditions promotes the efflux of uroporphyrin without a dedicated efflux pump, reducing feedback inhibition. Figure 1 a).

[0041] As a proof of concept, this invention will originate from P. thermoglucosidasiusPbgS and HmbS from sample 11955 were constructed into the pet28a-Duet vector (purchased from Beijing Qingke Biotechnology Co., Ltd.) via homologous recombination to obtain the plasmid pET28aDuet-hemBC, which was then transformed into... E. coli BL21-BC cells, used for uroporphyrin I synthesis, were obtained from BL21 (DE3). BL21-BC cells were seeded at 37°C and cultured in 250 ml Erlenmeyer flasks containing 50 ml of LB medium. The optical density (OD) of the culture medium was measured... 600 When the concentration reached 0.4, 0.2 mM isopropyl-β-D-thiogalactoside was added to induce protein expression, and the culture temperature was adjusted to 28 ℃ for 24 h. After protein expression, the bacterial cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then the BL21-BC bacterial suspension was resuspended in diluted 5-ALA fermentation broth. The optical density of the bacterial suspension was adjusted to OD. 600 =60, the final total system volume was 3 mL, and the 5-ALA concentrations in the fermentation broth were 20 g / L, 10 g / L, 5 g / L, and 1 g / L, respectively. The above bacterial suspensions were transferred to a shaker and subjected to biotransformation at 60℃ and 220 rpm for 24 h. The results showed that when the final 5-ALA concentration was 5 g / L, the conversion rate reached 92.3% ( Figure 1 b); When the 5-ALA concentration was increased to 10 g / L, the conversion efficiency significantly decreased to 75.3%, indicating that high substrate concentration has an inhibitory effect on downstream catalytic enzymes. Under the condition of 5 g / L, the yield of UPI (uroporphyrin I) was 3.65 g / L, the reaction time was 3 hours, and the corresponding production rate was 1.22 g / L / h. Figure 1 c). Based on this result, a substrate concentration of 5 g / L and a reaction time of 3 hours were determined to be the optimal conditions for scale-up experiments. Following the scale-up experiments, this invention designed a fed-batch whole-cell catalytic process to reduce the complexity of cell recovery operations. First, BL21-BC cells were added to a 5 L bioreactor until the final optical density OD was reached. 600 The cell system with a concentration of 60 g / L underwent a 3 L reaction (first round). After 3 hours, to maintain a substrate concentration of 5 g / L, 0.8 L of 5-ALA fermentation broth was added sequentially, and BL21-BC cells were added to maintain the optical density (OD). 600 = 60, to maintain a substrate concentration of 5 g / L (second round of reaction), 1.1 L of 5-ALA fermentation broth was added after 3 hours (BL21-BC cells were added to maintain optical density OD). 600= 60), and after 3 hours of reaction, the final reaction volume reached 4.9 L (third round of reaction). The above three rounds of reaction constitute one catalytic cycle. The conversion rates of the three rounds of reaction in the first catalytic cycle were 83%, 78%, and 58%, respectively, corresponding to a UPI concentration of 30.2 mmol ( Figure 1 d). Compared with the small-scale experiment, the reaction efficiency decreased, indicating that the reactor conditions need to be optimized.

[0042] 2. Optimization of whole-cell catalytic system This invention re-optimized the pH (adjusting pH = 6.5, 7, 7.5, 8) and reaction time (cycles of 1.5h, 2h, 2.5h, 3h) in a 5 L reactor, and the results showed that pH = 7.5 ( Figure 2 a) and under the condition of reaction time 2 hours ( Figure 2 b) The conversion rate was the highest, approaching the level of small-scale trials. Furthermore, detection using a singlet oxygen detection kit revealed that porphyrin accumulation induces singlet oxygen generation (…). Figure 2 c), leading to enzyme inactivation. To mitigate the catalytic degradation caused by ROS, this invention tested the effects of adding various reducing agents (0.5 g / L vitamin C, 0.5 g / L mannitol, 0.5 g / L gluconate, and 0.5 g / L dithiothreitol) on the catalytic system. The results showed that dithiothreitol was the most effective. Figure 2 (d and e), the conversion rate remained at 75.2% at the end of the first catalytic cycle. However, after centrifugation to recover the cells, the first cycle was repeated, and the catalytic efficiency of the second cycle was found to be significantly lower. Further research revealed that high-temperature catalysis easily caused leakage of cell contents. PbgS and HmbS were detected in the supernatant by SDS-PAGE, indicating that PbgS and HmbS leaked out during the catalytic process, leading to a decrease in catalytic efficiency after recovery. Figure 2 f).

[0043] To prevent enzyme leakage, this invention uses a covalent organic framework (COF) to encapsulate cells. 4.2 mg of 2,5-diethoxyterephthalohydrazide (DETH) and 1.6 mg of 1,3,5-triformylbenzene (TB) were dissolved separately in 500 μl of ultrapure water containing 10 μl of 6 M acetic acid, and then centrifuged in 2 ml centrifuge tubes. Subsequently, 1 ml of… E. coli BL21 suspension (OD) 600=30) was mixed with 500 μl of the above-treated DETH and TB solutions and reacted at 37 ℃ and 100 rpm for 30 min. After the reaction, the obtained COF-coated E. coli cells were collected by centrifugation at 10,000 rpm for 1 min and washed three times with distilled water to obtain purified COF-coated cells ( Figure 2 g). After encapsulating cells with COF, the conversion rates in the second and third catalytic cycles were 61% and 18%, respectively. Figure 2 h), higher than 54% and 15% for unencapsulated cells. The efficiency of the third catalytic cycle was too low, so only the first two catalytic cycles were considered.

[0044] The whole-cell catalytic unit constructed in this invention can achieve two rounds of catalytic cycling, ultimately yielding approximately 9.8 L of supernatant containing uroporphyrin I, with a uroporphyrin I concentration of 5.53 g / L. Figure 2 i).

[0045] Example 2: High-Temperature Whole-Cell Catalytic Production of Uroporphyrin III To expand the range of products produced by whole-cell catalysis, in E. coli BL21 is derived from plasmid overexpression P. thermoglucosidasius The pET28aDuet-hemBCD plasmid was obtained from PbgS, HmbS, and UroS at 11955, and subsequently chemically transformed and introduced. E. coli BL21-BCD strain was obtained from BL21. Using BL21-BCD as a whole-cell catalyst and an optimized single-batch catalytic process, 2.14 g / L of uroporphyrin III was obtained within 2 h. Figure 3 a) The yield of uroporphyrin III was 0.43 g / g, indicating that the catalytic reaction was not complete.

[0046] Analysis of the reaction mixture revealed that 1.42 g / L of uroporphyrin I accumulated during the whole-cell catalysis of uroporphyrin III. This byproduct formation primarily stems from the spontaneous oxidation of hydroxymethylcholine under oxygen exposure. To reduce the occurrence of such undesirable oxidation reactions, this invention proposes constructing an artificial multi-enzyme cascade system to directionally deliver intermediate metabolites to subsequent reactions, thereby reducing random diffusion and inhibiting spontaneous oxidation.

[0047] In the design of the protein scaffold, this invention selects proteins derived from... Archaeoglobus fulgidus , Clostridium thermocellum , Clostridium clariflavum and Clostridium cellulolyticum Four pairs of orthogonal dockerin-cohesin modules. PbgS and A. fulgidusDockerin fusion in the source dockerin-cohesin molecular module, HmbS and C. thermocellum Dockerin fusion in the source dockerin-cohesin molecular module, UroS and C. clariflavum The dockerin fusions in the source dockerin-cohesin molecular module were linked together by (GGGGS)3 to obtain the plasmid pET28aDuet-doc-hemBCD.

[0048] At the same time, the above A. fulgidus , C. thermocellum and C. clariflavum The three cohesin modules from different sources are connected sequentially, using the same (GGGGS)3 ( Figure 3 (b) Protein scaffolds ① (fusion unit ①) carrying PbgS, ② (fusion unit ②) carrying HmbS, and ③ (fusion unit ③) carrying UroS were obtained respectively. Then, the nucleic acid constructs encoding the above protein scaffolds ①, ②, and ③ were co-constructed into the pCDFDuet-coh-C.cla plasmid to obtain plasmid pCDFDuet-coh-C.cla-1.

[0049] Based on the pCDFDuet-coh-C.cla plasmid, the number of protein scaffolds ③ carrying UroS was further adjusted (corresponding to W for PbgS, X for HmbS, and Y for UroS, respectively, see [reference]). Figure 3 (b) Plasmids pCDFDuet-coh-C.cla-2 and pCDFDuet-coh-C.cla-3 were obtained, wherein pCDFDuet-coh-C.cla-2 contains two repeated protein scaffolds ③ and pCDFDuet-coh-C.cla-3 contains three repeated protein scaffolds ③.

[0050] Finally, the plasmids pET28aDuet-doc-hemBCD and pCDFDuet-coh-C.cla-1 were simultaneously introduced into the cell via chemical transformation. E. coli In BL21; or, simultaneously introduce plasmids pET28aDuet-doc-hemBCD and pCDFDuet-coh-C.cla-2. E. coli In BL21; or, simultaneously introduce plasmids pET28aDuet-doc-hemBCD and pCDFDuet-coh-C.cla-3. E. coli In BL21.

[0051] This invention allows for precise control of the stoichiometry of enzymes in each pathway within a cascade complex by adjusting the number of repetitions in the protein scaffold, thereby achieving co-localization of different enzymes. Results showed that three scaffolds with different Y-domain repetition numbers produced significantly different yields of uroporphyrin III. Figure 3 c). Among them, the highest UP III (uroporphyrin III) yield was obtained when Y=2, which was 3.04 g / L, accounting for 85% of the total porphyrins. Figure 3 d).

[0052] In summary, by combining thermostable multi-enzyme systems from different sources, the whole-cell catalysis established in this invention can efficiently synthesize uroporphyrin I and uroporphyrin III, fully demonstrating the wide applicability and high efficiency of the system.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An engineered Escherichia coli strain for producing uroporphyrin, characterized in that, It is an engineered Escherichia coli strain that exogenously expresses porphyrin synthase derived from thermophilic bacteria; Preferably, the porphyrin synthase is derived from *Bacillus thermoglucosidase* (…). Parageobacillus thermoglucosidasius ).

2. The engineered Escherichia coli strain according to claim 1, characterized in that, The porphyrin synthase includes bile pigment synthase and hydroxymethylcholine synthase, and the main product produced is uroporphyrin I.

3. The engineered Escherichia coli strain according to claim 1, characterized in that, The porphyrin synthase includes bile pigment synthase, hydroxymethylcholine synthase and uroporphyrin III synthase, and the main product produced is uroporphyrin III.

4. The engineered Escherichia coli strain according to claim 3, characterized in that, The bile pigment synthase, hydroxymethylcholesterol synthase, and uroporphyrin III synthase are co-localized through a protein scaffold to form a multi-enzyme complex. The protein scaffold is a scaffold constructed based on the dockerin-cohesin molecular module.

5. The engineered Escherichia coli strain according to claim 4, characterized in that, The bile pigment synthase is connected via a linker to... Archaeoglobus fulgidus The dockerin molecule fusion in the source dockerin-cohesin molecular module is used as a fusion unit ①; The hydroxymethylcholine synthase and Clostridium thermocellum The dockerin molecule in the source dockerin-cohesin molecular module is fused as a fusion unit ②; The uroporphyrin III synthase and Clostridium clariflavum The dockerin molecule in the source dockerin-cohesin molecular module is fused as a fusion unit ③; Finally, the cohesin molecules from the dockerin-cohesin molecular modules from the above three sources are sequentially linked together using a Linker. Preferably, the Linker is (GGGGS)3.

6. The engineered Escherichia coli strain according to claim 5, characterized in that, The multienzyme complex comprises two repeating fusion units ③.

7. The method for constructing the engineered Escherichia coli according to any one of claims 1-3, characterized in that, The method includes: in Escherichia coli (Escherichia coli) Escherichia coli The porphyrin synthase derived from thermophilic bacteria was expressed exogenously in the sample.

8. The use of the engineered Escherichia coli according to any one of claims 1-6 in the fermentation production of uroporphyrin.

9. A method for producing uroporphyrin, characterized in that, Using the engineered Escherichia coli strain according to any one of claims 1 or 2, a catalytic reaction is carried out in a fermentation system containing 5-aminolevulinic acid at a preset temperature, and the main reaction product is uroporphyrin I; Using the engineered Escherichia coli strain described in any one of claims 3-6, a catalytic reaction is carried out in a fermentation system containing 5-aminolevulinic acid at a preset temperature, and the main reaction product is uroporphyrin III; Preferably, the preset temperature is 45℃-75℃, more preferably 60℃; Preferably, the catalytic reaction is carried out in a batch feeding manner to maintain the concentration of 5-aminolevulinic acid in the fermentation system at 5 g / L.

10. The method according to claim 9, characterized in that, Before the catalytic reaction, the process also includes a step of coating the engineered Escherichia coli with a covalent organic framework material; Preferably, the covalent organic framework material is prepared by crosslinking 2,5-diethoxyterephthalohydrazide and 1,3,5-tricarboxyphenyl.

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