A high-yield recombinant cysteine ​​strain, fermentation method and its application

Abstract

This invention relates to a high-yield recombinant cysteine ​​strain, a fermentation method, and their applications. The invention employs a growth-stage-dependent promoter to dynamically regulate the cysteine ​​efflux protein gene. ydeD The expression of the gene was controlled by promoters of varying strengths to regulate the expression of the cysteine ​​efflux protein mutant gene. yfiK G156S / N157S The expression of cysteine ​​was finally knocked out and transferred into the protein gene. yeaN Simultaneously integrate dynamic regulation ydeD Genes were used to construct a high-yield recombinant strain of cysteine, which can produce L-cysteine ​​using glycerol as a carbon source. The yield of L-cysteine ​​in shake flask fermentation reached 9.40 g / L, and the highest yield in batch fermentation reached 38.50 g / L, showing great potential for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and fermentation technology, specifically relating to a high-yield recombinant cysteine ​​strain, fermentation method and its application. Background Technology

[0002] L-cysteine ​​is a sulfur-containing amino acid that plays an important physiological role in metabolism, including maintaining cellular redox homeostasis, stabilizing protein conformation, and serving as a sulfur donor and precursor in the biosynthesis of various sulfur-containing compounds. It is widely used in medicine, food, cosmetics, and feed additives.

[0003] Currently, the production of L-cysteine ​​mainly uses chemical hydrolysis and keratin extraction methods, which not only cause environmental pollution but also have high production costs. Therefore, developing a green microbial fermentation method for L-cysteine ​​has significant research value and application prospects.

[0004] Microbial synthesis of L-cysteine ​​can solve the problem of its green production; however, two key issues remain unresolved. First, carbon flux is severely lost due to the low substrate selectivity of efflux proteins. In L-cysteine-producing strains, constitutive promoter overexpression of efflux proteins YdeD and YfiK is commonly used to increase yield. While this promotes product efflux, both proteins have poor substrate selectivity, resulting in the loss of precursor L-serine and other precursors along with cysteine. O O-acetylserine (OAS) is excreted from cells, leading to precursor loss and limiting further yield increases. Secondly, research on carbon source selection is insufficient. Most current studies on L-cysteine ​​biosynthesis use glucose as a substrate. Theoretical studies indicate that the metabolic pathway from glycerol to L-cysteine ​​is shorter than that from glucose, and glycerol, as a cheap byproduct of the biodiesel industry, has a cost advantage. Although preliminary studies show that glycerol may have potential, its highest reported yield (313.4 mg / L) is far below the optimal level for the glucose system, and there is a lack of process validation on a fermenter scale; its true industrialization advantages have not yet been verified.

[0005] Current technologies lack effective strategies for precisely regulating the expression of efflux proteins to reduce precursor carbon loss, and also lack sufficient validation of glycerol as a potentially efficient carbon source. Therefore, developing new metabolic engineering strategies to address carbon loss and optimizing fermentation processes using glycerol as a carbon source are crucial for constructing high-yield and more economically viable recombinant L-cysteine-producing strains. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects in the prior art and provide a high-yield recombinant strain of cysteine, a fermentation method and its application, which greatly improves the yield of cysteine ​​and can be fermented using glycerol as a carbon source, thereby reducing production costs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A high-cysteine-producing recombinant strain, using recombinant Escherichia coli W5E2A as the host strain, knocked out yeaN Genes and yeaN The gene locus integrates the L-cysteine ​​efflux protein YdeD, and simultaneously overexpresses the L-cysteine ​​efflux protein YfiK using the plasmid pACYCDuet-1 as a vector. G156SN157S The YfiK G156SN157S The nucleotide sequence is shown in SEQ ID NO.4.

[0008] As a further technical solution, it is also necessary to delete the plasmid pACYCDuet-1. lacI Gene expression cassettes, T7 promoter system and multiple cloning sites.

[0009] As a further technical solution, a growth-stage dependent promoter P is adopted. s143 P fic With P bolA Any of the genes that control ydeD Expression (preferred promoter P) s143 ).

[0010] As a further technical solution, the promoter P is adopted. GAPDH P trc1 P trc2 P tac Any of the regulatory genes YfiK G156SN157S Expression (preferred promoter P) trc1 ).

[0011] As a further technical solution, the method for constructing the recombinant *E. coli* W5E2A includes: using *E. coli* BW25113 as the basic chassis strain, knocking out the L-cysteine ​​degradation-related gene of the basic chassis strain, knocking out the carbon overflow node gene of the basic chassis strain, and integrating P into the knocked-out carbon overflow node-related gene site. J23119 - cysE T167A Expression cassette, gene knockout lafU and genes yjiP, And in genes lafU and genes yjiP site integration serA C83L / H344A / N346A / N364A Expression cassettes were used to obtain recombinant Escherichia coli W5E2A.

[0012] As a further technical solution, the L-cysteine ​​degradation-related genes include yhaM , tnaA and yciW .

[0013] As a further technical solution, L-cysteine ​​degradation-related genes in the basic chassis strains are sequentially knocked out. yhaM , tnaA and yciW。

[0014] As a further technical solution, the carbon overflow node-related genes include ldhA , poxB , dld , mgsA and gloA。

[0015] As a further technical solution, the carbon overflow node gene of the basic chassis strain is knocked out sequentially. ldhA , poxB , dld , mgsA and gloA。

[0016] As a further technical solution, gene knockout is performed sequentially. lafU and genes yjiP。

[0017] As a further technical solution, the aforementioned serA C83L / H344A / N346A / N364A The nucleotide sequence is shown in SEQ ID NO.9.

[0018] Application of the high-yield recombinant cysteine ​​strain in cysteine ​​production.

[0019] A method for producing cysteine, comprising fermenting the high-yield recombinant cysteine ​​strain to produce cysteine.

[0020] As a further technical solution, the fermentation medium used for fermentation includes: 30 g / L carbon source, 1~6 g / L yeast extract, Phenol red and 1 mL / L trace metal elements; The trace metal elements include .

[0021] As a further technical solution, the carbon source is glycerol and / or glucose (preferably glycerol).

[0022] As a further technical solution, the fermentation adopts a fed-batch fermentation method, which includes: using the fermentation medium as the initial medium, fermenting at 30°C for 48 hours; during fermentation, adjusting the aeration rate and stirring speed to maintain dissolved oxygen at around 30%; adding NH3·H2O to control the pH at 7.0; when the initial carbon source is exhausted, continuously adding 600 g / L of carbon source to maintain a carbon source concentration of 0-10 g / L; after 6 hours of fermentation, adding... As a sulfur source.

[0023] As a further technical solution, the carbon source in the initial culture medium is glycerol, and the carbon source for the supplementary feed is glycerol.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves precise control over product efflux (see...) Figure 1 The composition of the fermentation medium was optimized, solving the problems of precursor loss and carbon source utilization efficiency in L-cysteine ​​biosynthesis. The recombinant Escherichia coli obtained finally achieved an L-cysteine ​​yield of 9.40 g / L in shake-flask fermentation and 38.50 g / L in fed-batch fermentation in a 5 L fermenter, with a yield of 0.17 g / g glycerol, which is the highest level reported to date and has good prospects for industrial application. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the temporal separation and control of efflux protein activity in one embodiment of the present invention; Figure 2 This is a construction map of recombinant plasmid pYdeD01 in one embodiment of the present invention; Figure 3 This is a construction map of recombinant plasmid pYdeD02 in one embodiment of the present invention; Figure 4 This is a construction map of recombinant plasmid pYdeD03 in one embodiment of the present invention; Figure 5 This is a construction map of recombinant plasmid pYdeD04 in one embodiment of the present invention; Figure 6 This is a diagram showing the changes in system composition during fed-batch fermentation of recombinant Escherichia coli to produce L-cysteine ​​using glucose as a carbon source in one embodiment of the present invention. Figure 7 This is a diagram showing the changes in system composition during fed-batch fermentation of recombinant Escherichia coli to produce L-cysteine ​​using glycerol as a carbon source in one embodiment of the present invention. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0028] In this invention, 1. All commercially available products used in this invention, including PCR amplification enzymes, plasmids, DNA gel recovery kits, and column-based plasmid extraction cassettes, were operated according to the kit instructions. Preparation of competent *E. coli* cells: TAKARA kit; routine molecular biology experimental procedures such as nucleic acid agarose gel electrophoresis, water bath heat shock transformation, electroporation transformation, competent cell preparation, colony PCR, and bacterial genome extraction were performed according to *Molecular Cloning: A Laboratory Manual* (Fourth Edition). Plasmid construction and sequencing of PCR amplification products were performed by Suzhou Anshengda Co., Ltd.

[0029] 2. The culture medium involved in this invention LB solid medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar powder.

[0030] LB liquid medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L peptone.

[0031] Fermentation medium: 30 g / L carbon source, Phenol red and 1 mL / L trace metal element. Then adjust the pH to 7.0 with NaOH.

[0032] Trace metal elements: .

[0033] Antibiotic concentrations: 50 mg / L kanamycin, 33 mg / L spectinomycin.

[0034] Inducer concentration: In the CRISPR-associated transposase system, the final concentration of rhamnose added was 18 mM.

[0035] 3. The strain cultivation and fermentation methods involved in this invention are as follows: Single colonies with normal morphology were picked from newly transformed LB agar plates and transferred to 8 mL of LB medium, and incubated overnight at 37°C and 200 rpm. Subsequently, a 10% (v / v) inoculum was added to 250 mL shake flasks containing 30 mL of fermentation medium, and incubated at 30°C and 180–220 rpm. During fermentation, 14% (v / v) ammonia was added every 12 h to adjust the pH. Additionally, glucose or glycerol was added when the carbon source was depleted. After 72 h of fermentation, the fermentation broth was collected and samples were prepared for product analysis.

[0036] 4. Detection of cysteine ​​production involved in this invention: Take 1 mL of fermentation broth and centrifuge at 12000 rpm for 5 minutes to obtain supernatant 1 and precipitate. To detect L-cysteine, resuspend the precipitate in 1 mL of 0.5 M sulfuric acid and centrifuge again at 12000 rpm for 5 minutes to obtain supernatant 2. React supernatant 1 and supernatant 2 separately with an equal volume of 20 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) in 1 M NaOH solution for 10 minutes. Then, L-cysteine ​​is detected by pre-column derivatization HPLC using 4-chloro-3,5-dinitrotrifluorotoluene (CNBF).

[0037] The HPLC conditions were as follows: Agilent ZORBAX Eclipse XDB-C18 column (4.6×250mm, 5μm), detection wavelength 260 nm; AB biphase elution was used, with phase A being acetonitrile and phase B being 50 mM pH 4.9 triethylamine buffer:acetonitrile:triethylamine = 830:170:3 (volume ratio); column temperature was 30℃, and flow rate was 0.8 mL / min.

[0038] Measured using a spectrophotometer .

[0039] 5. The PCR reaction system involved in this invention is shown in Table 1: Table 1: PCR System

[0040] PCR reaction conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension at 4 kb / min; 32 cycles, stored at 4°C, and PCR products were recovered by gel extraction.

[0041] 6. The Gibson assembly reaction system involved in this invention is shown in Table 2: Table 2: Gibson assembly reaction system

[0042] Gibson assembly reaction conditions: 50°C, 30 min.

[0043] The amount of linearized carrier and the amount of inserted fragment can be calculated using the formula X / Y: X = (0.02 × number of base pairs in the cloning vector / concentration of the corresponding DNA fragment in gel recovery) ng; Y = (0.02 × number of base pairs in the inserted fragment / concentration of the corresponding DNA fragment in gel recovery) ng; 7. The overlap extension PCR system involved in this invention is shown in Table 3: Table 3: Overlap Extension PCR System

[0044] Overlap extension PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension at 4 kb / min; 32 cycles, stored at 4℃. A portion of the PCR product was taken for nucleic acid electrophoresis verification, and then the template plasmid was removed by enzyme digestion with demethylase Dpn I, followed by transformation verification.

[0045] 8. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0046] Example 1: Construction of recombinant strain W5E2A Using BW25113 as the base strain, the host strain was modified using the pEcCpf1 / pcrEG dual plasmid gene editing system.

[0047] Sequentially knock out genes related to L-cysteine ​​degradation yhaM , tnaA , yciW To reduce product degradation; based on the above-mentioned strains with knocked-out cysteine ​​degradation pathway genes, carbon overflow node genes were sequentially knocked out. ldhA , poxB , dld , mgsA , gloA and pflB Simultaneously, P is integrated at the corresponding site. J23119 - cysE T167A Expression boxes; finally knock them out one by one. lafU , yjiP Integration at two sites serA C83L / H344A / N346A / N364A Expression cassettes were used to enhance precursor supply to obtain Escherichia coli BW25113△ yhaM △ tnaA △yciW △ ldhA ::P J23119 - cysE T167A △ poxB ::P J23119 - cysE T167A △ dld ::P J23119 - cysE T167A △ mgsA ::P J23119 - cysE T167A △ gloA ::P J23119 - cysE T167A △ lafU ::P tac - serA C83L / H344A / N346A / N364A △ yjiP ::P tac - serA C83L / H344A / N346A / N364A (abbreviated as W5E2A); wherein, the CRISPR / Cpf1 gene editing system mentioned above can be found in the reference Zhu X, Wu Y, Lv X. Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli [J]. ACS Synthetic Biology, 2022(5):11.

[0048] The specific implementation process is as follows: (1) Knock out genes involved in the cysteine ​​degradation pathway.

[0049] In this embodiment, BW25113 was used as the basic chassis strain, and the Escherichia coli BW25113 genome was used as a template to construct... yhaM , tnaA and yciW The knockout cassette was used. Primers were used to amplify the original commercially available plasmid pEcgRNA by PCR to obtain the targeted... yhaM , tnaA and yciW gRNA expression plasmid N23- of gene sequence yhaM / tnaA / yciW ; The pEcCpf1 plasmid N23 plasmid and the above knockout cassette were transformed into Escherichia coli BW25113 by electroporation to obtain recombinant strains. The recombinant strains were plated on LB agar plates containing spectinomycin and kanamycin and incubated at 37°C for 12-20 hours.

[0050] Single colonies grown on the plate are picked and positive clones that have undergone correct homologous recombination are selected.

[0051] Colonies that were correctly verified by PCR were sent for sequencing (Suzhou Anshengda Company) to confirm the accuracy of the genes.

[0052] Positive clones with correct sequencing were inoculated into LB liquid medium containing 18 mM / L rhamnose and incubated overnight at 37°C to eliminate N23-. yeaN Plasmid. Verify plasmid removal by streaking onto a Cannabis-resistant plate and picking a single colony.

[0053] Colonies with successfully removed plasmids were selected, and single colonies were verified using antibiotic plates to confirm the elimination of the pEcCpf1 plasmid. Ultimately, a plasmid-free recombinant strain with complete promoter replacement was obtained.

[0054] (2) Multicopy integration at carbon overflow node gene loci cysE T167A Expression Box Using the pEcCpf1 / pcrEG dual plasmid gene editing system, and with the genome of E. coli BW25113 as a template, P was constructed. J23119 - cysE T167A Expression cassettes, based on the strains with knocked-out cysteine ​​degradation pathway genes, sequentially knocked out carbon overflow node genes. ldhA, poxB, dld, mgsA, gloA At the same time ldhA, poxB, dld, mgsA, gloA Site integration P J23119 - cysE T167A Expression box; PCR amplification of the original commercially available plasmid pEcgRNA was performed using primers to obtain targeted... ldhA, poxB, dld, mgsA, gloA gRNA expression plasmid N23- of gene sequence ldhA / poxB / dld / mgsA / gloA ; Subsequent transformation, screening, sequencing, and validation were the same as in Example 1. The constructed strain was named W5E.

[0055] (3) Integration of 2-HG to generate SerA mutants with reduced activity This embodiment uses the Escherichia coli BW25113 genome as a template to construct a system based on the strong promoter P. tac Driven serA C83L / H344A / N346A / N364A Expression box.

[0056] PCR amplification of the original commercially available plasmid pEcgRNA was performed using primers to obtain targeted... lafU , yjiP gRNA expression plasmid N23- of gene sequence lafU / yjiP; Using the pEcCpf1 / pcrEG dual plasmid gene editing system, the above expression cassettes were sequentially integrated into the W5 genome. lafU and yjiP The site, subsequent transformation, screening, sequencing, and validation were the same as in Example 1. The resulting recombinant strain was named W5E2A.

[0057] The primers used in the above construction are shown in Table 3. Table 3: Primer sequences

[0058] Example 2: Growth stages of different intensities depend on promoter regulation ydeD Gene expression 1. Constructing plasmid pYdeD01: Using pACYCDuet-1 as the backbone, the original plasmid was deleted. lacI Gene expression cassettes, the T7 promoter system, and multiple cloning sites for MCS1 and MCS2 were incorporated, and P was inserted. GAPDH Driven ydeD Gene expression cassettes (see Figure 2 ), and obtained plasmid pYdeD01.

[0059] Using laboratory-preserved *E. coli* BW25113 as a template, high-fidelity PCR amplification was performed using primers D01-F, yfiK-R and yfiK-F, D01-vR, respectively, to obtain two gene fragments. The purified linear gene fragment and the linearized vector fragment were mixed at a specific molar ratio. A commercially available homologous recombinase was used for the recombination reaction. See the Gibson assembly reaction system described below for details. P was constructed. GAPDH Driven ydeD Gene expression cassette.

[0060] 2. Promoter substitution: Utilizing homologous recombination, promoters P, which are dependent on the growth stage, are used respectively. s143 P fic and P bolA Replace the promoter P in the constitutive expression of pYdeD01 GAPDH The specific steps are as follows: Using pYdeD01 as a template, primers D02 / 03 / 04-F (sequences targeting P respectively) were used. s143 P fic P bolA (Design), D01-vR was used for high-fidelity PCR amplification to obtain linear gene fragments with homologous arms at both ends.

[0061] Using pYdeD01 as a template, primers D01-vF and D02 / 03 / 04-F (sequences targeting P respectively) were used. s143 P fic P bolA (Design) Perform reverse PCR to amplify the linearized vector, and construct the required primers as shown in Table 4.

[0062] Among them, promoter P s143、 P fic P bolA The nucleotide sequences are shown in SEQ ID NO. 1-3, respectively; Table 4: Primer Sequences

[0063] The purified linear gene fragment and the linearized vector fragment were mixed at a specific molar ratio. A commercially available homologous recombinase was used for the recombination reaction. See the Gibson assembly reaction system for details.

[0064] The recombinant reaction product was transformed into JM109 competent cells. The transformed bacterial culture was spread on LB agar plates containing chloramphenicol and incubated upside down at 37°C for 9–12 h.

[0065] Single colonies were picked, positive clones were screened, and the recombinant plasmid was sequenced to confirm that the promoter had been precisely replaced. ydeD The gene coding sequence is correct; the recombinant plasmid pYdeD02 was obtained. Figure 3 The promoter P used s143 ), pYdeD03 ( Figure 4 The promoter P used fic ), pYdeD04 ( Figure 5 The promoter P used bolA ).

[0066] pYdeD01, pYdeD02, pYdeD03, and pYdeD04 were transformed into W5E2A calcium-competent cells to obtain recombinant strains for L-cysteine ​​production, which were named CYS3-13, CYS4-1, CYS4-2, and CYS4-3, respectively. The obtained strains were then subjected to shake-flask fermentation according to the strain culture and fermentation methods described above.

[0067] Growth and yield were tested according to the cysteine ​​detection method, and the results are shown in Table 5.

[0068] Table 5: Promoter control based on different growth stages ydeD The effect of expression

[0069] Example 3: Regulation of YfiK by constitutive promoters of different strengths G156SN157S Gene expression This embodiment uses pYdeD01 as a template and utilizes homologous recombination, employing promoters P with different strengths. GAPDH P trc1 P trc2 P tac Regulation of YfiK G156SN157S Gene expression; among which, yfiK G156SN157S The nucleotide sequence of the gene is shown in SEQ ID NO.4. The specific operation is as follows: 1) Using Escherichia coli BW25113 as a template, high-fidelity PCR amplification was performed using primers yfiK-F / yfiK-R to obtain a linear yfiK gene fragment with homologous arms at both ends.

[0070] Using pYdeD01 as a template, reverse PCR was performed using primers K01-vF / R to amplify the vector backbone and P. GAPDH Linearized carrier.

[0071] The subsequent assembly, transformation and screening steps are the same as in Example 1; finally, plasmid pYfiK is obtained.

[0072] 2) Using pYfiK as a template, high-fidelity PCR amplification was performed using primers K01-pF / R to obtain a linear fragment, and the template was removed; subsequent transformation and screening were exactly the same as in Example 2; finally, plasmid pYfiK01 (corresponding to promoter P) was obtained. GAPDH ).

[0073] 3) Using pYfiK01 as a template, use primers K02 / 03 / 04-F (sequences targeting P respectively) trc1 P trc2 P tac The recombinant plasmids were designed and subjected to high-fidelity PCR amplification using K02 / 03 / 04-R to obtain linear fragments, and the template was removed. Subsequent transformation and screening were performed in complete accordance with the steps in Example 2. The resulting recombinant plasmids were named pYfiK02 (corresponding to promoter P). trc1 pYfiK03 (corresponding to the bootloader P) trc2 pYfiK04 (corresponding to the bootloader P) tac ).

[0074] The primers required for construction are shown in Table 6.

[0075] Among them, promoter P GAPDH P trc1 P trc2 P tac The nucleotide sequences are shown in SEQ ID NO.5-8, respectively.

[0076] Table 6: Primer Sequences

[0077] pYfiK01, pYfiK02, pYfiK03, and pYfiK04 were transformed into W5E2A calcium-competent cells to obtain L-cysteine-producing strains, which were named CYS4-4, CYS4-5, CYS4-6, and CYS4-7, respectively. The obtained strains were then subjected to shake-flask fermentation according to the strain culture and fermentation methods described above.

[0078] Growth and yield were tested according to the cysteine ​​detection method, and the results are shown in Table 7.

[0079] Table 7: Control using promoters with different strength compositions yfiK G156SN157S The effect of expression

[0080] Example 4: Combination overexpression of two custom-regulated L-cysteine ​​efflux protein genes The custom-regulated L-cysteine ​​efflux protein genes are as follows: promoter P trc1 Regulation yfiK G156SN157S Genes and P s143 - ydeD Expression box.

[0081] This embodiment, based on W5E2A, integrates P through the CRISPR / Cpf1 gene editing system. s143 - ydeD Expression box to yeaN Site; the specific operation is as follows: The original commercially available plasmid pEcgRNA was amplified by PCR using primer pair N23-4-8-F / R to obtain targeted... yeaN The gRNA expression plasmid N23-yeaN contains the gene sequence. Using primers 4-8-UP-F / R, 4-8-DH-F / R, and 4-8-F / R, the upstream and downstream homologous arms of the gene, P, were amplified by PCR. s143- ydeD Fragment; The above three PCR fragments (upstream homologous arm and downstream homologous arm, P) were used to... s143 - ydeD The fragment was bridged using a Gibson assembly kit and amplified using primers Y-4-8-F / R to obtain the P-type fragment for promoter substitution. s143 - ydeD The expression cassette and the primers required for its construction are shown in Table 8.

[0082] Table 8: Primer Sequences

[0083] pEcCpf1 plasmid, N23-yeaN plasmid, and P were converted by electroporation. s143 - ydeD The expression cassette fragment was transformed into the starting strain (recombinant strain W5E2A) to obtain the recombinant host, which was then plated on LB agar plates containing spectinomycin and kanamycin and incubated at 37°C for 12-20 hours.

[0084] Single colonies grown on the plate are picked and positive clones that have undergone correct homologous recombination are selected.

[0085] Colonies that were correctly verified by PCR were sent for sequencing (Suzhou Anshengda Company) to confirm the accuracy of the genes.

[0086] Positive clones with correct sequencing were inoculated into LB liquid medium containing 18 mM / L rhamnose and incubated overnight at 37°C to eliminate N23-. yeaN Plasmid. Verify plasmid removal by streaking onto a Cannabis-resistant plate and picking a single colony.

[0087] Colonies with successfully removed plasmids were selected, and single colonies were verified using antibiotic plates to confirm the elimination of the pEcCpf1 plasmid. Ultimately, a plasmid-free recombinant strain with complete promoter replacement was obtained.

[0088] The recombinant plasmid pYfiK02 was transformed into the plasmid-free recombinant strain with promoter replacement to obtain the engineered strain CYS4-8, namely: Escherichia coli BW25113△ yhaM △ tnaA △ yciW △ ldhA ::P J23119 - cysE T167A △ poxB ::P J23119 - cysE T167A △ dld ::P J23119 - cysE T167A△ mgsA ::P J23119 - cysE T167A △ gloA ::P J23119 - cysE T167A △ lafU ::P tac - serA C83L / H344A / N346A / N364A △ yjiP ::P tac - serA C83L / H344A / N346A / N364A △ yeaN ::P S143 - ydeD / pYfiK02.

[0089] The obtained strain was cultured and fermented in shake flasks according to the described strain culture and fermentation method. Growth and yield were detected using the cysteine ​​detection method, and the results are shown in Table 9.

[0090] Table 9: YdeD and YfiK G156SN157S Synergistic Enhancement of L-Cysteine ​​Production by Customized Expression Combinations

[0091] Example 5: Optimization of the amount of yeast extract added to the fermentation medium The engineered strain CYS4-8 was cultured on a plate for 12-20 h. Single colonies of appropriate size were picked and placed into a test tube containing 8 mL of LB liquid medium. The culture was carried out overnight at 37°C and 220 rpm to prepare the primary seed culture. The above-mentioned primary seed culture was inoculated at a rate of 10% (v / v) into 250 mL shake flasks containing 30 mL of fermentation medium. Different concentrations of yeast extract were added, and the flasks were incubated at 30°C and 180–220 rpm for 72 h. Samples were then taken to determine the cell density (OD). 600 The concentrations of L-cysteine ​​and L-cysteine ​​(the determination method is described in the cysteine ​​detection method) are shown in Table 10.

[0092] Table 10: Yeast extract concentrations at different levels and L-cysteine ​​production

[0093] Example 6: Synergistic optimization of carbon source compatibility and sulfur source addition amount Single colonies of recombinant strain CYS4-8 were used to prepare primary seed culture according to the method in Example 5; The above-mentioned primary seed culture was inoculated at an inoculation rate of 10% (v / v) into shake flasks containing 30 mL of eight different fermentation media (based on the optimization results of Example 4); the fermentation media were prepared according to the following eight combinations: Group A (glucose series): The carbon source is glucose (30 g / L), and the concentrations of ammonium thiosulfate are 30, 40, 50, and 60 mM, respectively; Group B (glycerol series): The carbon source is glycerol (30 g / L), and the concentrations of ammonium thiosulfate are 30, 40, 50, and 60 mM, respectively; Samples were taken after culturing at 30°C and in a shaker at 180–220 rpm for 72 h, and the cell density (OD) was measured. 600 The concentrations of L-cysteine ​​and L-cysteine ​​were measured, and the results are shown in Table 11.

[0094] Table 11: Results of different carbon sources and ammonium thiosulfate concentrations and L-cysteine ​​production

[0095] Example 7: Production of L-cysteine ​​by Feed-in Fermentation in a Fermenter Fresh single colonies of the engineered strain CYS4-8 constructed in Example 4 were inoculated into LB test tubes and cultured overnight at 37°C and 200 rpm to obtain primary seed.

[0096] Two mL of primary seed culture was inoculated into a shake flask containing 100 mL of the optimized fermentation medium from Example 5, and cultured at 37°C and 200 rpm until OD reached. 600 Reaching version 2.0 yields a level 2 seed.

[0097] Secondary seed was inoculated at 15% (v / v) into a bioreactor containing 2 L of optimized fermentation medium. Fermentation was carried out at 30°C, with dissolved oxygen maintained at approximately 30% by adjusting aeration rate and stirring speed, and pH controlled at 7.0 by adding 14% (v / v) NH3·H2O. When the initial carbon source was depleted, 600 g / L of carbon source was continuously added to maintain the carbon source concentration at 0–10 g / L. After 6 h of cultivation, automatic replenishment was performed. As a sulfur source.

[0098] The fermentation medium composition was optimized according to Example 6, containing 30 g / L carbon source, 5 g / L yeast extract, And 1 mL / L of trace metal elements.

[0099] Measurement Carbon source and L-cysteine ​​concentrations, and results of fed-batch fermentation with glucose as the carbon source are as follows: Figure 6As shown, the results of fed-batch fermentation using glycerol as the carbon source are as follows: Figure 7 As shown.

[0100] from Figure 6 - 7 The results showed that when the engineered bacterium CYS4-8 used glucose as a carbon source, the highest L-cysteine ​​yield was 22.16 g / L; when using glycerol as a carbon source, the highest L-cysteine ​​yield was 38.50 g / L, with a yield of 0.17 g / g glycerol (the weight of L-cysteine ​​obtained from consuming 1g of glycerol was 0.17g). These results indicate that the L-cysteine ​​yield was significantly higher when using glycerol as a carbon source than in the glucose system.

[0101] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A high-yield recombinant cysteine ​​strain, characterized in that, Using recombinant Escherichia coli W5E2A as the host strain, knockout yeaN Genes and yeaN The gene locus integrates the L-cysteine ​​efflux protein YdeD, and simultaneously overexpresses the L-cysteine ​​efflux protein YfiK using the plasmid pACYCDuet-1 as a vector. G156SN157S The YfiK G156SN157S The nucleotide sequence is shown in SEQ ID NO.

4.

2. The high-cysteine-producing recombinant strain according to claim 1, characterized in that, It is also necessary to delete the plasmid pACYCDuet-1 lacI Gene expression cassettes, T7 promoter system and multiple cloning sites.

3. The high-cysteine-producing recombinant strain according to claim 2, characterized in that, Using growth stage-dependent promoter P s143 P fic With P bolA Any of the genes that control ydeD The expression; Using promoter P GAPDH P trc1 P trc2 P tac Any of the regulatory genes YfiK G156SN157S The expression.

4. The high-cysteine-producing recombinant strain according to claim 1, characterized in that, The method for constructing the recombinant *E. coli* W5E2A includes: using *E. coli* BW25113 as the basic chassis strain, knocking out the L-cysteine ​​degradation-related gene of the basic chassis strain, knocking out the carbon overflow node gene of the basic chassis strain, and integrating P into the knocked-out carbon overflow node-related gene site. J23119 - cysE T167A Expression cassette, gene knockout lafU and genes yjiP, And in genes lafU and genes yjiP site integration serA C83L / H344A / N346A / N364A Expression cassettes were used to obtain recombinant Escherichia coli W5E2A.

5. The high-cysteine-producing recombinant strain according to claim 4, characterized in that, The L-cysteine ​​degradation-related genes include genes yhaM ,Gene tnaA and genes yciW ; The carbon overflow node-related genes include genes ldhA ,Gene poxB ,Gene dld ,Gene mgsA and genes gloA; The serA C83L / H344A / N346A / N364A The nucleotide sequence is shown in SEQ ID NO.

9.

6. The application of the high-yield recombinant cysteine ​​strain as described in any one of claims 1-5 in the production of cysteine.

7. A method for producing cysteine, characterized in that, Cysteine ​​is produced by fermentation of the high-yield recombinant cysteine ​​strain as described in any one of claims 1-5.

8. A method for producing cysteine ​​according to claim 6, characterized in that, The fermentation medium used for fermentation includes: 30 g / L carbon source, 1-6 g / L yeast extract, 8 mg phenol red and 1 mL / L trace metal elements; The trace metal elements include .

9. A method for producing cysteine ​​according to claim 8, characterized in that, The carbon source is glycerol and / or glucose.

10. A method for producing cysteine ​​according to claim 8, characterized in that, The fermentation employs a fed-batch fermentation method, which includes: using the fermentation medium as the initial medium, fermenting at 30°C for 48 hours; during fermentation, adjusting the aeration rate and stirring speed to maintain dissolved oxygen at approximately 30%; adding NH3·H2O to control the pH at 7.0; when the initial carbon source is depleted, continuously adding 600 g / L of carbon source to control the carbon source concentration at 0–10 g / L; after 6 hours of fermentation, adding... As a sulfur source.

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