Multienzyme complex assembly system and genetically engineered bacterium for producing l-cysteine at high yield

Abstract

Provided is a multienzyme complex assembly system for producing L-cysteine at high yield. A reasonable metabolic modification target is determined by means of screening of critical pathway enzymes CysE, CysM, NrdH, and CysK in a L-cysteine synthesis pathway and L-cysteine transport-related channel proteins EamA and EamB, two affinity short peptides RIDD and RIAD are respectively fusion-expressed with the critical pathway enzymes and the channel proteins, and by means of combination optimization, the assembly of the critical pathway enzymes and the channel proteins is implemented, thereby accelerating the reaction rate of the L-cysteine synthesis pathway, improving the metabolic flux, and enhancing the degree of aggregation of products in the vicinity of a cell membrane. On the basis of the construction and application strategy of the multienzyme complex assembly system, further provided are a genetically engineered bacterium for producing L-cysteine at high yield and a construction method therefor, implementing efficient synthesis of L-cysteine.

Description

A multi-enzyme complex assembly system and genetically engineered bacteria for high-yield L-cysteine Technical Field

[0001] The invention belongs to the field of metabolic engineering, and particularly relates to a multi-enzyme complex assembly system and genetically engineered bacteria for high-yield L-cysteine. Background Art

[0002] As an important organic sulfur source in organisms, L-cysteine ​​plays a variety of key roles in metabolism and antioxidant defense networks. In proteins, the sulfhydryl groups of two cysteines can covalently bond together to form a disulfide bond, which stabilizes the three-dimensional structure of the protein molecule and ensures the correct folding of the protein molecule, especially in structural proteins such as keratin and bone and connective tissue proteins. At the same time, it is also an important cofactor in many enzyme-catalyzed reactions and plays a very important role in the physiological activities of cells. L-cysteine ​​and its derivatives have been widely used in the pharmaceutical industry (antipyretic, analgesic, anti-inflammatory), cosmetics industry (anti-oxidation, anti-radiation, anti-aging), food industry (bread additives, flavoring agents, antioxidants, preservatives) and animal husbandry industry (promoting the growth of poultry and livestock and shortening the time of stocking).

[0003] In the biotechnological production of L-cysteine, fermentation expression is mainly carried out through genetically engineered bacteria using Corynebacterium glutamicum or Escherichia coli as host bacteria; however, the yield of genetically engineered bacteria using Bacillus glutamicum as the host bacteria is relatively low, about 950 mg / L, so Escherichia coli is the preferred host for microbial fermentation production of L-cysteine.

[0004] In Escherichia coli, the synthetic pathway of L-cysteine ​​is mainly divided into carbon metabolism pathway and two sulfur metabolism pathways.In carbon metabolism, as a key enzyme in L-cysteine ​​synthesis, serine acetyltransferase (also referred to as SAT) CysE and acetyl-CoA jointly catalyze the precursor L-serine of L-cysteine ​​to be O-acetylserine (OAS).In addition, serine acetyltransferase CysE and O-acetylserine sulfhydrylase A (OASS-A, encoded by gene cysK) can also form cysteine ​​synthase complex (CSC).In two sulfur assimilation pathways, there are two kinds of key enzymes, namely OASS-A and O-acetylserine sulfhydrylase B (OASS-B, encoded by gene cysM). OASS-A catalyzes the conversion of OAS and sulfide (S2-) into the product L-cysteine ​​in the sulfate pathway; OASS-B catalyzes the conversion of OAS and thiosulfate into SSC in the thiosulfate pathway. The intermediate product SSC is then converted into L-cysteine ​​and sulfite under the catalysis of glutaredoxin NrdH. Sulfite can also continue to enter the sulfate pathway as a sulfur source.

[0005] Metabolic pathways are often catalyzed by multiple enzymes. In natural metabolic pathways, the timing and location of enzyme activity, as well as the concentrations of metabolites, are tightly regulated. Cells are highly dynamic and complex metabolic systems, where most enzymes function not in isolation but instead form supramolecular complexes. These complexes optimize substrate access, preventing the loss of intermediates and enhancing the controllability and efficiency of catalytic reactions. In recent years, strategies such as compartmentalization and intermediate channeling have been proposed. These strategies artificially immobilize enzymes on scaffolds or confine them to specific regions, co-localizing related enzymes in key metabolic pathways to construct substrate channels, thereby increasing metabolic rates and target product conversion efficiency. These strategies also allow for spatial control of enzyme position and distance, thereby enhancing the stability and catalytic activity of multi-enzyme assembly systems. Currently, relatively mature bioscaffolding technologies, such as DNA scaffolds, RNA scaffolds, and protein scaffolds, have been developed. These macromolecular self-assembly systems have been successfully applied to multi-enzyme complexes in metabolic pathways and have successfully improved metabolic efficiency. Among them, a pair of artificial protein scaffolds, RIAD-RIDD, was designed as a protein skeleton system due to its high specificity of binding to the ligand and receptor and nanomolar high affinity. The RIDD peptide is composed of the dimerization and docking (D / D) domains at the N-termini of the two regulatory subunits (R subunits) of the adenylate cyclase-dependent protein kinase (PKA). The RIAD peptide is derived from the 14-18 residue amphipathic helix on the anchoring domain of the A kinase anchor protein (AKAP). The RIAD and RIDD peptides form a stable dimeric α-helical docking domain through protein-protein interactions. The RIAD and RIDD peptide pairs have the following characteristics: (1) They are very small, 44 and 18 amino acids respectively, which minimizes the interference with the structure, position and activity of the enzyme when used as tags for fusion; (2) The peptide pair has high affinity, and the K between the RIDD dimer and the RIAD peptide is 0. D =1.2nM, which can ensure the integrity of the intracellular enzyme complex; (3) RIDD and RIAD have a 2:1 binding stoichiometric ratio, which can obtain a branched structure.

[0006] Therefore, systematic research on the key enzymes and channel proteins in the L-cysteine ​​synthesis pathway and the construction of a multi-enzyme complex assembly system may become a potential method to increase L-cysteine ​​production, which is of great significance for the industrial production of L-cysteine.

[0007] Summary of the Invention

[0008] To address the above problems, the present invention designed and constructed a scaffold-free multi-enzyme complex assembly system based on the affinity short peptide RIAD-RIDD, which assembles important pathway enzymes and channel proteins in the cysteine ​​synthesis pathway, thereby accelerating the reaction rate of the L-cysteine ​​synthesis pathway, improving metabolic flux, and enhancing the aggregation of products near the cell membrane, thereby obtaining genetically engineered Escherichia coli bacteria with high L-cysteine ​​production.

[0009] In a first aspect, the present invention provides a multi-enzyme complex assembly system for high-yield L-cysteine, which is constructed by the following method: a short peptide RIAD and a short peptide RIDD are fused and expressed with at least one L-cysteine ​​channel protein and at least one L-cysteine ​​key pathway enzyme, respectively, so that the L-cysteine ​​channel protein connected to the short peptide RIAD and the L-cysteine ​​key pathway enzyme connected to the short peptide RIDD are combined via RIAD-RIDD to obtain a multi-enzyme complex assembly system for high-yield L-cysteine.

[0010] Specifically, the L-cysteine ​​channel proteins include EamA and EamB.

[0011] Specifically, the L-cysteine ​​key pathway enzymes include CysM, NrdH, and CysK.

[0012] In a second aspect, the present invention provides a genetically engineered bacterium that produces high L-cysteine ​​yield, which is constructed by the following method:

[0013] The strain E. coli W3110 EYC was used as the starting strain to construct the above-mentioned multi-enzyme complex assembly system for high-yield L-cysteine, and the overexpressed cysE f The plasmid of the gene is used to obtain the genetically engineered bacteria with high L-cysteine ​​production.

[0014] The present invention constructs a multi-enzyme complex assembly system and an optimal high-yield L-cysteine ​​genetic engineering strain E. coli W3110 EYC::eamA through the following design ideas AD ::cysM DD ::nrdH DD / pTrc99a-cysE f :

[0015] (1) A laboratory-preserved engineered strain E. coli W3110 EYC for producing cysteine ​​was selected as the starting strain (deposited in China Center for Type Culture Collection, with a collection number of CCTCC NO: M 20191026, which has been published in CN 111019877A); the strain contained a serine acetyltransferase mutant CysE f coding genes.

[0016] (2) In the strains obtained in (1), different expression plasmids pTrc99a-cysE were constructed. f 、pTrc99a-cysE f -eamA, pTrc99a-cysE f -eamA AD 、pTrc99a-cysE f -eamB, pTrc99a-cysE f -eamB AD , studied the effect of short peptide tag RIAD fused to the C-terminus of channel proteins EamA and EamB on the fermentation production of L-cysteine.

[0017] (3) In the strain obtained in (1), the CRISPR / Cas9 gene editing technology was used to fuse the short peptide RIDD to the C-termini of the key enzymes CysE, CysM, NrdH, and CysK in the L-cysteine ​​synthesis pathway to verify the effect of the addition of the short peptide RIDD on L-cysteine ​​production and bacterial growth.

[0018] (4) Based on the experimental results of (2) to (3), in the strain obtained in (1), the short peptide RIAD was fused to the C-terminus of the channel proteins EamA and EamB, and the short peptide RIDD was fused to the C-terminus of the key pathway enzymes CysM, NrdH, and CysK in the L-cysteine ​​synthesis pathway using CRISPR / Cas9 gene editing technology. The modified channel proteins were combined with the pathway enzymes, and the optimal assembly method of the channel proteins and key pathway enzymes was screened based on the L-cysteine ​​production, thereby obtaining a strain that can produce high L-cysteine.

[0019] The present invention constructs an L-cysteine ​​production-export channel by building a multi-enzyme composite assembly system, effectively promoting the synthesis flux of L-cysteine ​​in a specific space, namely, near the cell membrane, accelerating the catalytic efficiency of the continuous reaction, and simultaneously accelerating the transport of L-cysteine ​​to the extracellular space, thereby accelerating the production and accumulation of L-cysteine.

[0020] As a preference, the genetically engineered bacteria that produces high L-cysteine ​​yield is strain E. coli W3110EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD / pTrc99a-cysE fUsing E. coli W3110 EYC as the starting strain, the short peptide RIAD was fused to the C-terminus of the channel protein EamA, and the short peptide RIDD was fused to the C-termini of the key pathway enzymes CysM and NrdH in the L-cysteine ​​synthesis pathway. The modified channel proteins and key pathway enzymes were combined via RIAD-RIDD binding to obtain an optimal multi-enzyme complex assembly system including the channel protein EamA, the key pathway enzyme CysM, and NrdH, and the above-mentioned optimal high-yield L-cysteine ​​genetically engineered bacteria were constructed.

[0021] In a third aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces high L-cysteine ​​yield, comprising the following steps:

[0022] (1) Using E. coli W3110 EYC as the starting strain, the short peptide RIAD encoding gene was inserted into one end of the eamA gene on its genome using CRISPR / Cas9 gene editing technology to construct the strain E. coli W3110 EYC::eamA RIAD .

[0023] (2) Using CRISPR / Cas9 gene editing technology, the short peptide RIDD encoding gene was inserted into the strain E. coli W3110 EYC::eamA RIAD The cysM gene and one end of the nrdH gene on the genome were constructed to obtain the strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD .

[0024] (3) Construction of vector pTrc99a-cysE f , and introduced into the strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD , construct the genetically engineered bacteria E.coli W3110 EYC::eamA that produces high L-cysteine RIAD ::cysM RIDD ::nrdH RIDD / pTrc99a-cysE f .

[0025] Preferably, the nucleotide sequence of the short peptide RIAD is shown as SEQ ID NO.1, and the nucleotide sequence of the short peptide RIDD is shown as SEQ ID NO.2.

[0026] Preferably, the nucleotide sequence of the eamA gene is shown as SEQ ID NO.3, and the nucleotide sequence of the eamB gene is shown as SEQ ID NO.4.

[0027] Preferably, the cysE f The nucleotide sequence of the gene is shown in SEQ ID NO.5, the nucleotide sequence of the gene cysK is shown in SEQ ID NO.6, the nucleotide sequence of the gene cysM is shown in SEQ ID NO.7, and the nucleotide sequence of the gene nrdH is shown in SEQ ID NO.8.

[0028] The fourth aspect of the present invention provides the use of the genetically engineered bacteria with high L-cysteine ​​production in the preparation of L-cysteine ​​by microbial fermentation.

[0029] Specifically, the application involves selecting a single colony of the genetically engineered strain and inoculating it into a 10ml LB medium test tube. The tube is then incubated in a shaker at 37°C and 150-200 rpm for 12-24 hours. The inoculum is then inoculated into 20-50ml of fermentation medium at a 1% volume concentration to initiate fermentation. After 4 hours of incubation, IPTG is added to a final concentration of 0.1mM. The fermentation temperature is 30°C, the rotation speed is 150-200 rpm, and the fermentation time is 2-4 days. After fermentation, the supernatant of the fermentation broth is separated and purified to obtain L-cysteine.

[0030] Preferably, the fermentation medium is composed of: 20-30 g / L glucose, 5-10 g / L (NH4)2SO4, 2-5 g / L KH2PO4, 35-10 g / L Na2S2O, 5-10 g / L yeast extract, 10-15 g / L Na2HPO4, 1-5 g / L peptone, 1 ml / L trace element solution, the solvent is deionized water, and the pH value is natural; the trace element solution is composed of: 300-500 g / L MgSO4·8H2O, 2-5 g / L MnSO4·8H2O, 2-5 g / L ZnSO4·7H2O, 2-8 g / L Fe2SO4, and the solvent is deionized water.

[0031] Beneficial Effects of the Invention: This invention proposes a strategy for constructing and applying a multi-enzyme complex assembly system to control the expression of the L-cysteine ​​biosynthesis pathway in Escherichia coli. First, by screening the key metabolic enzymes in the L-cysteine ​​biosynthesis pathway—CysE, CysM, NrdH, and CysK—and the L-cysteine ​​transport-related channel proteins EamA and EamB, rational metabolic modification targets were identified. Subsequently, two affinity peptides, RIDD and RIAD, were fused and expressed with the key pathway enzymes and transport proteins, respectively. Through combinatorial optimization, assembly of the pathway enzymes and channel proteins was achieved. The construction of this multi-enzyme complex assembly system will construct an L-cysteine ​​production-export pathway, effectively promoting the synthesis flux of L-cysteine ​​in a specific space, namely, near the cell membrane, and accelerating the catalytic efficiency of the continuous reaction. Furthermore, by assembling the L-cysteine ​​biosynthesis pathway with the cell membrane, this system can effectively enhance the ability of the channel proteins to recognize and capture L-cysteine, accelerating L-cysteine ​​transport to the extracellular space, and accelerating L-cysteine ​​production and accumulation. The present invention constructs a genetically engineered Escherichia coli strain with high L-cysteine ​​production through the construction and application strategy of a multi-enzyme complex assembly system, thereby achieving efficient synthesis of L-cysteine. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows:

[0033] E. coli W3110 EYC / pTrc99a-cysE f 、

[0034] E. coli strain W3110EYC / pTrc99a-cysE f -eamA,

[0035] E. coli strain W3110EYC / pTrc99a-cysE f -eamA RIAD 、

[0036] E. coli strain W3110EYC / pTrc99a-cysE f -eamB,

[0037] E. coli strain W3110EYC / pTrc99a-cysE f -eamB RIAD L-cysteine ​​production and OD 600 .

[0038] Figure 2 shows:

[0039] Strain E.coli W3110 EYC::cysE-RIDD / pTrc99a-cysE fDD 、

[0040] Strain E.coli W3110 EYC::cysK-RIDD / pTrc99a-cysE f 、

[0041] E. coli W3110 EYC::cysM-RIDD / pTrc99a-cysE f 、

[0042] Strain E.coli W3110 EYC::nrdH-RIDD / pTrc99a-cysE f L-cysteine ​​production and OD 600 .

[0043] Figure 3 shows:

[0044] strain E. coli W3110 EYC::eamA RIAD ::cysK RIDD / pTrc99a-cysE f 、

[0045] strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD / pTrc99a-cysE f 、

[0046] strain E. coli W3110 EYC::eamA RIAD ::nrdH RIDD / pTrc99a-cysE f 、

[0047] strain E. coli W3110 EYC::eamB RIAD ::cysK RIDD / pTrc99a-cysE f 、

[0048] strain E. coli W3110 EYC::eamB RIAD ::cysM RIDD / pTrc99a-cysE f 、

[0049] strain E. coli W3110 EYC::eamB RIAD ::nrdH RIDD / pTrc99a-cysE f L-cysteine ​​production and OD 600 .

[0050] Figure 4 shows the strain E. coli W3110 EYC::eamA AD ::cysM DD ::nrdH DD / pTrc99a-cysE f L-cysteine ​​production and OD 600 . DETAILED DESCRIPTION

[0051] The following describes the embodiments of the present invention by specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the case of no conflict, the features in the following examples and embodiments can be combined with each other. In the embodiments of the present invention, unless otherwise specified, the methods used are all conventional methods, and the reagents used can be obtained from commercial sources.

[0052] In the following examples, the final concentration of kanamycin in the culture medium is 50 mg / L, the final concentration of spectinomycin in the culture medium is 50 mg / L, and the final concentration of ampicillin in the culture medium is 100 mg / L. The parent strain E. coli W3110 EYC of the present invention is from the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20191026, which has been disclosed in CN 111019877A. The strain E. coli W3110 EYC was constructed using the strain E. coli W3110 as the chassis strain. The strain E. coli W3110 is from the Coli Genetic Stock Center of Yale University CGSC, with a deposit number of CGSC#4474, which has been disclosed in patents US2009 / 0298135A1 and US2010 / 0248311A1.

[0053] Example 1: Determination of L-cysteine ​​content

[0054] (1) Place 1 mL of bacterial culture in a 2 mL EP tube and centrifuge at 12,000 rpm for 1 minute to separate the supernatant and precipitate. The supernatant is used for the detection of L-cysteine, L-serine, and other metabolites.

[0055] (2) Weigh 0.27 g of CNBF and dissolve it in 10 mL of acetonitrile as Solution I. Mix 0.2 M boric acid solution and 0.05 M borax solution as mother solutions in a 4:1 ratio to prepare a pH 9.0 standard buffer solution, which is called Solution II. Dilute the sample to a concentration of 0-5 g / L and mix 100 μL of sample, 300 μL of Solution I, and 500 μL of Solution II. Incubate in a constant temperature oscillator at 40-60°C and 500-1000 rpm for 0.5-1 hour. Pass the sample through a membrane and place it in a liquid phase bottle for testing.

[0056] (3) The instrument was a Thermo Fisher UPLC ultrahigh pressure liquid chromatograph. The chromatographic column was a C18 column (4.6 × 250 mm, 5 μm); the UV detector was set at a wavelength of 260 nm; the injection volume was 10 μL; the column temperature was 30°C; the flow rate was 0.8 mL / min; and the mobile phase was a two-phase AB phase: pure acetonitrile in phase A and 50 mM HAc-NaAc buffer in phase B: acetonitrile: triethylamine = 82.8:17:0.2, pH = 4.9. The gradient elution program is shown in Table 1.

[0057] Table 1: Gradient elution program

[0058] Example 2: Effect of short peptide tag RIAD fused to the C-terminus of EamA and EamB on L-cysteine ​​production by E. coli W3110 EYC

[0059] In order to study the effect of short peptide tag RIAD fused to the C-terminus of EamA and EamB on the fermentation production of L-cysteine, different expression vectors containing strong promoter Trc and mutant CysE were constructed. f The expression plasmid pTrc99a-cysE encoding gene f 、pTrc99a-cysE f -eamA, pTrc99a-cysE f -eamA AD 、pTrc99a-cysE f -eamB, pTrc99a-cysE f -eamB AD The plasmid was introduced into the L-cysteine ​​engineered strain E. coli W3110 EYC (SS1), resulting in strains SS1-01, SS1-02, SS1-03, SS1-04, and SS1-05. Strains overexpressing EamA and EamB without the short peptide RIAD served as controls. After 48 hours of fermentation, the L-cysteine ​​titer in the fermentation supernatant was determined.

[0060] (1) Using the pTrc99a plasmid as a template and 99aline-F and 99aline-R as primers, PCR amplification was performed to obtain a linearized vector. The wild-type strain E. coli W3110 genome was used as a template and cysE-F and cysE-R as primers to amplify the gene cysE fragment. The PCR product was digested with DpnI. All PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified. According to the instructions of the One step clone kit (Vazyme Biotech, Nanjing, China), the linearized vector was connected to the gene fragment cysE, transformed into E. coli DH5α, and spread on an ampicillin-resistant plate. A single colony was picked and verified by colony PCR using primers 99a-VF and 99a-VR. The pTrc99a-cysE plasmid was obtained by sequencing verification. Using plasmid pTrc99a-cysE as a template, PCR was performed using primers T167A-F and T167A-R, G245S-F and G245S-R, and the clone was transformed into E. coli DH5α and plated on an ampicillin-resistant plate. Single colonies were picked and verified by colony PCR (primers 99a-VF and 99a-VR), and sequencing was performed to obtain pTrc99a-cysE containing the CysE mutant. f The primers are shown in Table 2.

[0061] (2) using pTrc99a-cysE f The plasmid was used as a template, and pE-line-F and pE-line-R were used as primers to amplify the linearized vector by PCR. The wild-type strain E. coli W3110 genome was used as a template, and eamA-F and eamA-R were used as primers to amplify the eamA fragment. The PCR product digestion and one-step cloning and connection of the fragment and the vector were the same as (1), and sequencing was verified to obtain pTrc99a-cysE f -eamA plasmid. Primers are shown in Table 2.

[0062] (3) using pTrc99a-cysE f -eamA plasmid as a template, pEA-line-F and pEA-line-R as primers, amplified by PCR to obtain a linearized vector. fThe eamA fragment was obtained using the -eamA plasmid as a template and primers pEA-eamA-F and pEA-eamA-R. The RIAD fragment was obtained using the pET28a-RIAD plasmid as a template and primers pEA-RIAD-F and pEA-RIAD-R. Fusion PCR was performed using the eamA and RIAD fragments as templates and primers pEA-eamA-F and pEA-RIAD-R to obtain the eamA-RIAD fragment. All PCR products were checked by 1.0% agarose gel electrophoresis and the PCR fragments were purified. The linearized vector was ligated with the gene fragment eamA-RIAD according to the instructions of the One Step Clone Kit (Vazyme Biotech, Nanjing, China), transformed into E. coli DH5α, and plated on ampicillin-resistant plates. Single colonies were picked and verified by colony PCR using primers 99a-VF and 99a-VR. Sequencing confirmed the resulting pTrc99a-cysE f -eamA AD Plasmids and primers are shown in Table 2.

[0063] (4) Construct plasmid pTrc99a-cysE using the same construction method as (2) f -eamB. Primers are shown in Table 2.

[0064] (5) Construct plasmid pTrc99a-cysE using the same construction method as (3) f -eamB AD The primers are shown in Table 2.

[0065] (6) Prepare the strain E. coli W3110 EYC (SS1) into competent cells, and insert the constructed pTrc99a-cysE f 、pTrc99a-cysE f -eamA, pTrc99a-cysE f -eamA AD 、pTrc99a-cysE f -eamB, pTrc99a-cysE f -eamB AD The plasmid was transformed into E. coli W3110EYC competent cells by chemical transformation to obtain the strain E. coli W3110EYC / pTrc99a-cysE f (SS1-01), strain E. coli W3110EYC / pTrc99a-cysE f -eamA(SS1-02), strain E.coli W3110EYC / pTrc99a-cysEf -eamA RIAD (SS1-03), strain E. coli W3110EYC / pTrc99a-cysE f -eamB(SS1-04), strain E.coli W3110EYC / pTrc99a-cysE f -eamB RIAD (SS1-05).

[0066] (7) Strains SS1-01, SS1-02, SS1-03, SS1-04, and SS1-05 were inoculated into 10 mL of LB medium and cultured overnight at 30-37°C and 150-200 rpm. 1 mL of the preculture was inoculated into a 500 mL shake flask containing 20-50 mL of fermentation medium. After 4-6 hours of culture, 0.1 mM IPTG was added and fermentation was carried out for 2-4 days. The fermentation medium was composed of 20-30 g / L glucose, 5-10 g / L (NH4)2SO4, 2-5 g / L KH2PO4, 5-10 g / L Na2S2O3, 5-10 g / L yeast extract, 10-15 g / L Na2HPO4, 1-5 g / L peptone, and 1 ml / L trace element solution. The solvent was deionized water and the pH value was natural. The trace element solution composition is: 300-500 g / L MgSO4·8H2O, 2-5 g / L MnSO4·8H2O, 2-5 g / L ZnSO4·7H2O, 2-8 g / L Fe2SO4, and the solvent is deionized water. The fermentation broth was tested according to the method in Example 1, and the OD 600 The L-cysteine ​​content in the fermentation supernatant is shown in FIG1 .

[0067] As shown in Figure 1, the yield of strain SS1-02 was 1.27 g / L, a 57% increase compared to the control, and the yield of strain SS1-04 increased by 32%. This indicates that enhancing the expression of L-cysteine ​​efflux proteins EamA and EamB can accelerate the efflux of L-cysteine, thereby increasing the titer of the product. There was no significant difference in L-cysteine ​​yield between strains SS1-03 and SS1-02, and between strains SS1-04 and SS1-05, proving that the addition of the short peptide tag RIAD does not seriously affect the normal function of the channel proteins EamA and EamB. These results lay the foundation for subsequent experiments.

[0068] Table 2: Primers for Example 2

[0069] Example 3: Effect of short peptide tag RIID fusion to the C-terminus of key pathway enzymes on L-cysteine ​​production by E. coli W3110 EYC

[0070] In order to study the effect of the addition of short peptide RIDD on the key pathway enzymes CysE, CysK, CysM, and NrdH, RIDD was fused to the CysE on the genome of strain SS1 by CRISPR / Cas9 gene editing technology. f , CysK, CysM and NrdH, seamlessly inserted before the stop codon.

[0071] (1) Using the pTarget plasmid (Addgene Plasmid #62226) as a template and cysE-RIDD-PTTB-F and cysE-RIDD-PTTB-R as primers, the gRNA was site-directedly mutagenized according to the pCas plasmid (Addgene Plasmid #62225) by PCR amplification. The PCR product was digested with DpnI. The digested product was transferred into E. coli DH5α and spread on a spectacle enzyme plate. Single colonies were picked for sequencing verification (primers pT-VF and pT-VR) to screen the successfully mutated △pTarget plasmid. The successfully mutated △pTarget plasmid was linearized using linearization primers PTL-F and PTL-R. The product was detected by 1.0% agarose gel electrophoresis and purified to obtain a linearized vector.

[0072] (2) Using the E. coli W3110 EYC genome as a template, PCR amplification was performed using primers cysE-RIDD-D1F and cysE-RIDD-D1R, cysE-RIDD-D3F and cysE-RIDD-D3R to obtain the cysE gene on the genome. f 500 bp upstream and downstream of the insertion site. Using plasmid pET28a-RIDD as a template, primers RIDD-D2F and RIDD-D2R were used to amplify the RIDD gene fragment. The three DNA fragments were fused by fusion PCR to obtain the Donor-RIDD fragment.

[0073] (3) According to the instructions of the One Step Clone Kit (Vazyme Biotech, Nanjing, China), the linearized vector in step (1) was ligated with the gene fragment Donor-RIDD and transformed into E. coli DH5α. The plasmid was then plated on a spectinomycin-resistant plate. A single colony was picked and verified by colony PCR using primers pT-VF and pT-VR. The pTD-RIDD plasmid was obtained by sequencing.

[0074] (4) Since the gene fragment is seamlessly inserted, the PAM site and 20bp also exist on the homologous sequences of 500bp upstream and downstream in the pTD-RIDD plasmid. Therefore, in order to prevent the Cas9 protein expressed by the pCas plasmid from recognizing and cutting the PTD, the 20bp on the homologous arm of the pTD-RIDD plasmid are replaced with synonymous codons. Using the plasmid pTD-RIDD as a template and cysE-RIDD-STTBF and cysE-RIDD-STTBR as primers, PCR amplification is performed to obtain pTD-RIDD-TB, and the PCR product is digested with DpnI. The digestion product is transformed into E. coli DH5α and spread on a spectacular enzyme plate. Single colonies are picked for sequencing verification (primers pT-VF and pT-VR) to screen the pTD-RIDD-TB plasmid with successful mutation.

[0075] (5) The E. coli W3110 EYC strain was prepared into a chemically competent state, and the pCas plasmid (Addgene Plasmid #62225) was transformed into the competent E. coli W3110 EYC by chemical transformation and plated on a kanamycin-resistant plate to obtain the strain E. coli W3110 EYC / pCas.

[0076] (6) Prepare the strain E.coli W3110 EYC / pCas into an electroporation competent state. After the plasmid PTD-RIDD-TB was electroporated into the E.coli W3110 EYC / pCas electroporation competent state, it was spread on a kanamycin and spectinomycin double-resistant plate, and a single colony was picked and verified by colony PCR using primers cysE-RIDD-VF and cysE-RIDD-VR. The successfully edited strain was screened to obtain the strain E.coli W3110 EYC::cysE f RIDD The primers are shown in Table 3.

[0077] (7) Pick a single positive colony and inoculate it into an LB tube containing 1mM IPTG and 0.05mg / L kanamycin, culture it at 30℃ overnight, streak it onto an LB plate containing 0.05mg / L kanamycin, culture it at 30℃ for 24h, pick a single colony and streak it onto an LB plate containing 0.05mg / L spectinomycin. If no single colony grows, the pTarget plasmid is successfully eliminated. Pick a single colony from which the pTarget plasmid has been successfully eliminated into an LB tube, culture it at 37℃ overnight, streak the bacterial solution onto an LB plate the next day, culture it at 37℃ for 12h, pick a single colony and streak it onto an LB plate containing 0.05mg / L kanamycin. If no single colony grows, the pCas plasmid is successfully eliminated, and finally the plasmid-free strain E.coli W3110 EYC::cysE is obtained. fRIDD .

[0078] (8) According to the above method, the short peptide RIDD was inserted into the C-terminus of the genes cysK, cysM and nrdH respectively. The strain E. coli W3110 EYC::cysK was obtained. RIDD , E. coli W3110 EYC::cysM RIDD , strain E.coli W3110EYC::nrdH RIDD . Introduce plasmid pTrc99a-cysE into the above strains respectively f -RIDD and plasmid pTrc99a-cysE f To verify the effect of RIDD on the pathway enzymes on the genome, the plasmid pTrc99a-cysE containing the RIDD fragment was also introduced. f -RIDD and plasmid pTrc99a-cysE without RIDD fragment f , in order to meet the requirements of the control variables. Strains SS1-06, SS1-07, SS1-08, and SS1-09 were inoculated into 10 mL of LB medium respectively, and cultured overnight at 30-37°C and 150-200 rpm. Inoculate 1 mL of pre-culture into a 500 mL shake flask containing 20-50 mL of fermentation medium, add 0.1 mM IPTG after culturing for 4-6 hours, and ferment for 2-4 days. The composition of the fermentation medium is the same as that in Example 2. The fermentation broth was tested according to the method in Example 1, and OD 600 The L-cysteine ​​content in the fermentation supernatant is shown in FIG2 .

[0079] As shown in Figure 2, the yields of strains SS1-07, SS1-08, and SS1-09 remained relatively stable compared to the control group, while the yield of strain SS1-06 dropped significantly by 57% to 0.3 g / L. This indicates that fusion of RIDD to the C-termini of cysK, cysM, and nrdH does not affect the normal activity of pathway enzymes, whereas fusion of RIDD to the C-terminus of CysE reduces the strain's ability to produce L-cysteine. This may be due to the fact that the short peptide RIDD affects the structure of the serine acetyltransferase CysE, resulting in reduced production of OAS, the direct precursor of L-cysteine, and thus the decreased yield.

[0080] Table 3: Primers of Example 3

[0081] Example 4: Studying the Effect of Combining Channel Proteins with Pathway Enzymes on L-Cysteine ​​Production by E. coli W3110 EYC and Screening the Optimal Combination

[0082] In order to screen the optimal combination, based on the results of Examples 2 to 3, pathway enzymes CysK, CysM, NrdH and channel proteins EamA and EamB were selected to construct an L-cysteine ​​multi-enzyme complex assembly system. Based on the strain E. coli W3110 EYC, RIAD was fused to the C-terminus of the channel proteins EamA and EamB to obtain the strain E. coli W3110 EYC::eamA RIAD (SS2) and strain E. coli W3110 EYC::eamB RIAD Then, based on strains SS2 and SS3, RIDD was fused to the C-termini of pathway enzymes CysK, CysM, and NrdH, respectively, to obtain strain SS2::cysK RIDD (SS2-1), SS2::cysM RIDD (SS2-2), SS2::nrdH RIDD (SS2-3) and SS3::cysK RIDD (SS3-1), SS3::cysM RIDD (SS2-2), SS3::nrdH RIDD (SS3-3).

[0083] (1) Using the pTarget plasmid (Addgene Plasmid #62226) as a template, PCR amplification was performed using primers eamA-RIAD-PTTBF and eamA-RIAD-PTTBR. Site-directed mutagenesis of the gRNA was performed according to PCR amplification of the pCas plasmid (Addgene Plasmid #62225). The PCR product was digested with DpnI. The digested product was transferred into E. coli DH5α and spread on a spectacle enzyme plate. Single colonies were picked for sequencing verification (primers pT-VF and pT-VR) to screen for successfully mutated pTarget plasmids. The successfully mutated pTarget plasmid was linearized using linearization primers PTL-F and PTL-R. The product was detected by 1.0% agarose gel electrophoresis and purified to obtain a linearized vector.

[0084] (2) Using the E. coli W3110 EYC genome as a template, primers eamA-RIAD-D1F and eamA-RIAD-D1R, and primers eamA-RIAD-D3F and eamA-RIAD-D3R were used to amplify 500 bp upstream and downstream of the insertion site of the gene eamA on the genome. Using the plasmid pET28a-RIAD as a template, primers RIAD-D2F and RIAD-D2R were used to amplify the fragment of the gene RIAD. The three DNA fragments were fused by fusion PCR to obtain the fragment Donor-RIAD.

[0085] (3) According to the instructions of the One Step Clone Kit (Vazyme Biotech, Nanjing, China), the linearized vector was ligated with the gene fragment Donor-RIAD and transformed into E. coli DH5α. The plasmid was then plated on a spectinomycin-resistant plate. A single colony was picked and verified by colony PCR using primers pT-VF and pT-VR. The pTD-RIAD plasmid was obtained by sequencing.

[0086] (4) Synonymous codon replacement was performed on the 20 bp homology arm of the pTD-RIAD plasmid. Using the plasmid pTD-RIAD as a template and primers eamA-RIAD-STTBF and eamA-RIAD-STTBR, PCR amplification was performed to obtain pTD-RIAD-TB. The PCR product was digested with DpnI. The digestion product was transformed into E. coli DH5α and spread on a spectacle enzyme plate. Single colonies were picked for sequencing verification (primers pT-VF and pT-VR) to screen for pTD-RIAD-TB plasmids with successful mutations.

[0087] (5) The E. coli W3110 EYC strain was prepared into a transfection competent state, and the pCas plasmid (Addgene Plasmid #62225) was transformed into the E. coli W3110 EYC transfection competent state by chemical transformation, and then plated on a kanamycin-resistant plate to obtain the strain E. coli W3110 EYC / pCas.

[0088] (6) Prepare the strain E. coli W3110 EYC / pCas into an electroporation competent state. After the plasmid pTD-RIAD-TB was electroporated into the E. coli W3110 EYC / pCas electroporation competent state, it was spread on a kanamycin and spectinomycin double-resistant plate, and a single colony was picked and verified by colony PCR using primers eamA-RIAD-VF and eamA-RIAD-VR. The successfully edited strain was screened to obtain the strain E. coli W3110 EYC::eamARIAD The primers are shown in Table 4.

[0089] (7) Pick a single positive colony and inoculate it into an LB tube containing 1mM IPTG and 0.05mg / L kanamycin, culture it at 30℃ overnight, streak it onto an LB plate containing 0.05mg / L kanamycin, culture it at 30℃ for 24h, pick a single colony and streak it onto an LB plate containing 0.05mg / L spectinomycin. If no single colony grows, the pTarget plasmid is successfully eliminated. Pick a single colony from which the pTarget plasmid has been successfully eliminated into an LB tube, culture it at 37℃ overnight, streak the bacterial solution onto an LB plate the next day, culture it at 37℃ for 12h, pick a single colony and streak it onto an LB plate containing 0.05mg / L kanamycin. If no single colony grows, the pCas plasmid is successfully eliminated, and finally the plasmid-free strain E.coli W3110 EYC::eamA is obtained. RIAD .

[0090] According to the above method, the short peptide RIAD was inserted into the C-terminus of the eamB gene of the strain E. coli W3110 EYC to obtain the strain E. coli W3110 EYC::eamB RIAD , primers are shown in Table 4. The short peptide RIDD was inserted into the strain E.coli W3110 EYC::eamA RIAD and E. coli W3110 EYC::eamB RIAD The C-termini of the genomic genes cysK, cysM and nrdH of the strain were cloned using primers as shown in Table 3 to obtain the following strains:

[0091] strain E. coli W3110 EYC::eamA RIAD ::cysK RIDD (SS2-1),

[0092] strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD (SS2-2),

[0093] strain E. coli W3110 EYC::eamA RIAD ::nrdH RIDD (SS2-3),

[0094] strain E. coli W3110 EYC::eamB RIAD ::cysK RIDD (SS3-1),

[0095] strain E. coli W3110 EYC::eamB RIAD ::cysMRIDD (SS3-2),

[0096] strain E. coli W3110 EYC::eamB RIAD ::nrdH RIDD (SS3-3).

[0097] Plasmid pTrc99a-cysE was introduced into the above strains respectively f The constructed strains SS2-1 / pE, SS2-2 / pE, SS2-3 / pE, SS3-1 / pE, SS3-2 / pE, and SS3-3 / pE were inoculated into 10 mL of LB medium and cultured overnight at 30-37°C and 150-200 rpm. 1 mL of the preculture was inoculated into a 500 mL shake flask containing 20-50 mL of fermentation medium. After 4-6 hours of culture, 0.1 mM IPTG was added and fermentation was carried out for 2-4 days. The fermentation medium was as described in Example 2. The fermentation broth was tested according to the method of Example 1, and the OD 600 The L-cysteine ​​content in the fermentation supernatant is shown in FIG3 .

[0098] As shown in Figure 3(a), compared with other pathway enzymes, the assembly of EamA and CysM has the best effect, and the production of L-cysteine ​​reaches 1.229 g / L, an increase of 46%. The production of EamA and NrdH is 1.212 g / L, an increase of 44%. The effect of assembling EamA and CysK is not obvious. Among them, CysM and NrdH are two important enzymes in the thiosulfate pathway. CysM catalyzes the reaction of OAS and thiosulfate to produce SSC, which is then reduced to L-cysteine ​​and sulfite (SO3 2- ). This may be due to the fact that L-cysteine ​​synthesis primarily relies on the thiosulfate pathway, which is more energy-efficient than the sulfate pathway. Therefore, selecting two key enzymes in the thiosulfate pathway, CysM and NrdH, to construct a substrate channel has a significant effect on improving L-cysteine ​​fermentation production. As shown in Figure 3(b), the assembly of any pathway enzyme with EamB has no significant effect on L-cysteine ​​production, likely due to the lack of complex formation between the pathway enzyme and EamB.

[0099] Table 4: Primers of Example 4

[0100] Example 5: Effect of assembling the channel protein EamA and the thiosulfate pathway enzymes cysM and nrdH on the production of L-cysteine ​​by the strain E. coli W3110 EYC.

[0101] According to the results of Example 4, in the optimal strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD Based on the above, RIDD was fused to the C-terminus of nrdH to obtain the strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD .

[0102] (1) The short peptide RIDD was inserted into the strain E. coli W3110EYC::eamA using the same method as in Example 3. RIAD ::cysM RIDD The C-terminus of the genomic gene nrdH was obtained to obtain the strain E. coli W3110EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD (SS4), primers are shown in Table 3. Transformed plasmid pTrc99a-cysE f , obtained strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD / pTrc99a-cysE f (SS4 / pE).

[0103] (2) E.coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD / pTrc99a-cysE f Inoculate into 10 mL of LB medium and culture overnight at 30-37°C and 150-200 rpm. Inoculate 1 mL of the preculture into a 500 mL shake flask containing 20-50 mL of fermentation medium. After 4-6 hours of culture, add 0.1 mM IPTG and ferment for 2-4 days. The fermentation medium is as described in Example 2. The fermentation broth is tested according to the method of Example 1, and the OD 600 The L-cysteine ​​content in the fermentation supernatant is shown in FIG4 .

[0104] As shown in Figure 4, L-cysteine ​​production increased to 1.52 g / L, a 79% increase compared to the starting strain. The results demonstrate that this strategy effectively increases L-cysteine ​​production by installing metabolic nodes in the L-cysteine ​​biosynthesis pathway, connecting previously physically separate metabolic pathways. The EamA-CysM-NrdH multienzyme complex acts as a link, synthesizing more L-cysteine ​​and directing it to the target pathway protein, streamlining L-cysteine ​​biosynthesis and increasing titer.

[0105] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection of the present invention.

Claims

1. A multi-enzyme complex assembly system for high-yield L-cysteine, characterized in that: The system is constructed by the following method: short peptide RIAD and short peptide RIDD are fused and expressed with at least one L-cysteine ​​channel protein and at least one L-cysteine ​​key pathway enzyme respectively, so that the L-cysteine ​​channel protein connected with the short peptide RIAD and the L-cysteine ​​key pathway enzyme connected with the short peptide RIDD are combined through RIAD-RIDD to obtain a multi-enzyme complex assembly system with high L-cysteine ​​production.

2. The multi-enzyme complex assembly system for high-yield L-cysteine ​​according to claim 1, characterized in that: The L-cysteine ​​channel proteins include EamA and EamB.

3. The multi-enzyme complex assembly system for high-yield L-cysteine ​​according to claim 1, characterized in that: The L-cysteine ​​key pathway enzymes include CysM, NrdH, and CysK.

4. A genetically engineered bacterium for high production of L-cysteine, characterized in that: It is constructed by the following method: Using the strain E. coli W3110 EYC as the starting strain, a multi-enzyme complex assembly system for high-yield L-cysteine ​​production as described in any one of claims 1 to 3 was constructed, and the overexpressed cysE f The plasmid of the gene is used to obtain the genetically engineered bacteria with high L-cysteine ​​production.

5. A genetically engineered bacterium for high production of L-cysteine ​​according to claim 4, characterized in that: The genetically engineered bacteria with high L-cysteine ​​production is strain E.coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD / pTrc99a-cysE f .

6. A method for constructing a genetically engineered bacterium with high L-cysteine ​​production as claimed in claim 5, characterized in that: The following steps are involved: (1) Using the strain E. coli W3110 EYC as the starting strain, the short peptide RIAD encoding gene was inserted into one end of the eamA gene on its genome using CRISPR / Cas9 gene editing technology to construct the strain E. coli W3110 EYC::eamA RIAD . (2) Using CRISPR / Cas9 gene editing technology, the short peptide RIDD encoding gene was inserted into the strain E. coli W3110 EYC::eamA RIAD The cysM gene and one end of the nrdH gene on the genome were constructed to obtain the strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD . (3) Construction of vector pTrc99a-cysE f , and introduced into the strain E. coli W3110 EYC::eamA RIAD ::cysM RIDD ::nrdH RIDD , construct the genetically engineered bacterium E.coli W3110 EYC::eamA that produces high L-cysteine RIAD ::cysM RIDD ::nrdH RIDD / pTrc99a-cysE f .

7. The method for constructing a genetically engineered bacterium with high L-cysteine ​​production according to claim 6, characterized in that: The nucleotide sequence of the short peptide RIAD is shown in SEQ ID NO.1, and the nucleotide sequence of the short peptide RIDD is shown in SEQ ID NO.

2.

8. The method for constructing a genetically engineered bacterium with high L-cysteine ​​production according to claim 6, characterized in that: The nucleotide sequence of the eamA gene is shown in SEQ ID NO.

3.

9. The method for constructing a genetically engineered bacterium with high L-cysteine ​​production according to claim 6, characterized in that: The cysE f The nucleotide sequence of the gene is shown in SEQ ID NO.5, the nucleotide sequence of the cysM gene is shown in SEQ ID NO.7, and the nucleotide sequence of the nrdH gene is shown in SEQ ID NO.

8.

10. Use of the genetically engineered bacteria with high L-cysteine ​​production according to claim 4 or 5 or the genetically engineered bacteria with high L-cysteine ​​production obtained by the method of any one of claims 6 to 9 in the preparation of L-cysteine ​​by microbial fermentation.

Images