High-yield l-cysteine engineering bacterium with accumulation of sulfur donor 3'-phosphoadenosine-5'-phosphosulfate, construction method therefor, and use thereof

Abstract

A high-yield L-cysteine engineering bacterium with accumulation of sulfur donor 3'-phosphoadenosine-5'-phosphosulfate, a construction method therefor, and the use thereof. By altering the copy number of key genes in the cysteine synthesis pathway in Escherichia coli, introducing heterologous genes, and enhancing the expression level of key genes in the cysteine synthesis pathway in Escherichia coli, the sulfur flux is further directed toward the synthesis of L-cysteine. On the basis of enhanced carbon flux in the chassis strain, to maintain carbon-sulfur balance in the strain, the sulfur flux is further directed toward the synthesis of L-cysteine, and the synthesis capacity of the sulfur donor PAPS in the sulfate pathway is enhanced to promote product synthesis, thereby finally obtaining a plasmid-free and antibiotic-free engineering strain for high-yield production of L-cysteine. Finally, the shake-flask titer of L-cysteine reaches 6.06 g / L, which is increased by 49.2% compared with the starting strain.

Description

A high-yield L-cysteine-producing engineered bacterium that accumulates sulfur donor 3'-adenosine-5'-phosphorylsulfate, its construction method and application Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a high-yield L-cysteine ​​engineered bacterium that accumulates sulfur donor 3'-adenosine-5'-phosphorylsulfate, its construction method, and its application. Background Technology

[0002] Cysteine ​​has two isomers: L-cysteine ​​and D-cysteine. The biologically active L-cysteine ​​is an important sulfur-containing amino acid that forms disulfide bonds to link protein chains into a three-dimensional network structure, thereby improving protein stability. Furthermore, studies have shown that L-cysteine ​​plays a crucial role in resisting oxidative stress in the periplasmic space of *E. coli*. Therefore, as an important sulfur-containing amino acid, L-cysteine ​​is widely used in food, cosmetics, pharmaceuticals, and animal feed. Currently, L-cysteine ​​is mainly produced through protein hydrolysis, chemical synthesis, enzymatic methods, and microbial fermentation. Given the continuously increasing market demand for L-cysteine ​​and the need for low-cost, high-efficiency, and green production, microbial fermentation is a superior method. However, due to the complex biosynthetic pathway of cysteine ​​within microorganisms, its large-scale industrial production still faces certain challenges. Summary of the Invention

[0003] To address the problem of low L-cysteine ​​yield in existing microbial fermentation synthesis technologies, this invention utilizes rational design and CRISPR-Cas9 gene editing technology to provide a genetically engineered bacterium that produces high levels of L-cysteine, its construction method, and its application in the microbial fermentation preparation of L-cysteine.

[0004] The technical solution adopted in this invention is: a method for constructing a high-yield L-cysteine ​​engineered bacterium, the method comprising: enhancing the synthesis capacity of 3'-adenosine-5'-phosphosulfate in a chassis strain;

[0005] The method for enhancing the synthesis ability of 3'-adenosine-5'-phosphosulfate includes at least one of the following:

[0006] (1) Introduce and overexpress the MET3 gene derived from Kluyveromyces lactis GG799 in the sclerotium;

[0007] (2) The N7489_009139 gene derived from Penicillium chrysogenum was introduced and overexpressed in the sclerotium;

[0008] (3) Overexpression of the cysD gene in the genome of the basal bacteria.

[0009] Please refer to Figure 1 for the L-cysteine ​​metabolic pathway diagram and modification sites. This invention relates to the sclerotium SSC8 (E. coli W3110, pgk / serA). f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f To maintain carbon-sulfur balance in the strain after enhancing carbon flux, a comprehensive systemic metabolic engineering strategy was employed. Using CRISPR / Cas9 gene editing technology, heterologous genes MET3 (cysD / N, encoding ATP sulfate) and N7489_009139 (CYSC, encoding APS kinase) were introduced via gene knock-in to enhance the expression levels of key genes in the cysteine ​​synthesis pathway in *E. coli* (engineered strain SSC8-2YJ1). Simultaneously, the expression of the cysD gene in the *E. coli* genome was further strengthened to continue enhancing the sulfur flux to L-cysteine ​​synthesis. This, in turn, promoted product synthesis by enhancing the synthesis of the sulfur donor 3'-phosphoadenosine-5'-phosphorylsulfate (PAPS). Ultimately, a plasmid-free, antibiotic-free engineered strain, SSC8-2YJ3, was obtained for high L-cysteine ​​production.

[0010] Table 1: Genes involved in gene editing and corresponding pathways

[0011] Preferably, the method for enhancing the synthesis capacity of 3'-adenosine-5'-phosphate sulfate includes: constructing the MET3 gene and the N7489_009139 gene into the pTrc99a plasmid and then transforming it into a substrate bacteria for overexpression. Specifically, using engineered bacteria SSC8 as the substrate bacteria, the pTrc99a plasmid is used to overexpress the heterologous genes MET3 and N7489_009139, enhancing the expression intensity of MET3 and N7489_009139, to obtain engineered bacteria SSC8 derivative, pTrc99a-MET3-N7489_009139, denoted as engineered bacteria TKA1. Further preferably, both the MET3 gene and the N7489_009139 gene are regulated by the Trc promoter. Specifically, using engineered strain SSC8 as the substrate strain, the heterologous genes MET3 and N7489_009139 were overexpressed using the pTrc99a plasmid. Both genes were regulated by the Trc promoter to enhance gene expression intensity, resulting in engineered strain SSC8 derivative, pTrc(MET3,N7489_009139), denoted as engineered strain TKA3.

[0012] Preferably, the method for enhancing the synthesis capacity of 3'-adenosine-5'-phosphoryl sulfate includes: using CRISPR-Cas9-mediated gene editing technology to replace the original pseudogene yjhw on the genome of the substrate bacteria with the MET3 gene and the N7489_009139 gene, followed by overexpression. Specifically, using engineered strain SSC8 as the substrate bacteria, CRISPR-Cas9-mediated gene editing technology is used to replace the original pseudogene yjhw on the genome of the starting strain with the MET3 and N7489_009139 genes derived from pRSFDuet to enhance the expression intensity of MET3 and N7489_009139, resulting in engineered strain SSC8 derivative, yjhw::MET3-N7489_009139, denoted as engineered strain SSC8-2YJ1.

[0013] Preferably, the method for enhancing the synthesis of 3'-adenosine-5'-phosphosulfate includes: using CRISPR-Cas9-mediated gene editing technology to replace the in situ promoter of the cysD gene in the genome of the substrate bacteria with a Trc promoter and then overexpressing it. Specifically, using engineered bacteria SSC8 as the substrate bacteria, CRISPR-Cas9-mediated gene editing technology is used to replace the in situ promoter of the cysD gene in its genome with Ptrc, resulting in engineered bacteria SSC8 derivative, cysD::Ptrc, denoted as engineered bacteria SSC8-2YJ2.

[0014] Preferably, the method for enhancing the synthesis of 3'-adenosine-5'-phosphosulfate includes: using CRISPR-Cas9-mediated gene editing technology to replace the in situ promoter of the cysD gene in the genome of *Saccharomyces cerevisiae* with a Trc promoter and then overexpressing it; and replacing the original pseudogene yjhw in the genome of *Saccharomyces cerevisiae* with the MET3 gene and the N7489_009139 gene and then overexpressing them. Specifically, using engineered strain SSC8 as *Saccharomyces cerevisiae*, CRISPR-Cas9-mediated gene editing technology is used to replace the in situ promoter of the cysD gene in its genome with a Ptrc promoter to obtain engineered strain SSC8. The derivative, cysD::Ptrc, is denoted as engineered strain SSC8-2YJ2. Using engineered strain SSC8-2YJ2 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene yjhw on the genome of the starting strain with the MET3 and N7489_009139 genes regulated by the Ptrc promoter derived from pTrc99A, thereby enhancing the expression intensity of MET3 and N7489_009139, resulting in engineered strain SSC8derivative, cysD::Ptrc / yjhw::Ptrc:MET3(KI),N7489_009139(KI), denoted as engineered strain SSC8-2YJ3, which is the high-yielding L-cysteine ​​engineered strain.

[0015] Preferably, the fungus in the chassis is: E. coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE fThe aforementioned basal bacteria is the strain CYS(E. coli W3110 pgk / serA) published in Bo Zhang, & Yuguo Zheng, (2023). Spatiotemporal Gene Expression by a Genetic Circuit for Chemical Production in Escherichia coli. ACS Synthetic Biology, 12 3, 768–779. f / serB / serC / cysE f This was obtained by modifying the strain CYS ( / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR). Specifically, it utilizes CRISPR-Cas9-mediated gene editing technology, using strain CYS as the starting strain. The eamA, eamB, and tolC genes in its genome were expressed, and the ydjN gene was knocked out (this process is described in CN 117431257 A, which includes a detailed construction procedure). Subsequently, the yjiP, yeeJ, ycdN, ydeU, ylbE, and yjhE genes were knocked out, and cysE was introduced at the knockout site. f Gene (cysE) f The gene has been documented in the aforementioned literature.

[0016] Preferably, the nucleotide sequence of the MET3 gene is shown in SEQ ID NO.1; and the nucleotide sequence of the N7489_009139 gene is shown in SEQ ID NO.2.

[0017] The present invention also provides engineered bacteria with high L-cysteine ​​production obtained by the method described.

[0018] Preferably, the high-yield L-cysteine ​​engineered bacteria is any one of the following:

[0019] (1) E. coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysEf / ΔycdN - cysE f / ΔydeU - cysE f / ΔylbE - cysE f / ΔyjhE - cysE f / yjhw::MET3 - N7489_009139, denoted as engineered strain SSC8 - 2YJ1;

[0020] (2)E.coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP - cysE f / ΔyjiR - cysE f / ΔyeeJ - cysE f / ΔycdN - cysE f / ΔydeU - cysE f / ΔylbE - cysE f / ΔyjhE - cysE f / cysD::Ptrc, denoted as engineered strain SSC8 - 2YJ2;

[0021] (3)E.coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP - cysE f / ΔyjiR - cysE f / ΔyeeJ - cysE f / ΔycdN - cysE f / ΔydeU - cysE f / ΔylbE - cysE f / ΔyjhE - cysE f / cysD::Ptrc / yjhw::Ptrc:MET3(KI),N7489_009139(KI), denoted as engineered strain SSC8 - 2YJ3;

[0022] (4) E. coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f / pTrc(MET3,N7489_009139).

[0023] The present invention also provides the application of the aforementioned high-L-cysteine-producing engineered bacteria in the microbial fermentation preparation of L-cysteine.

[0024] Preferably, the application includes: inoculating the high-yield L-cysteine ​​engineered bacteria into a fermentation medium, fermenting at 30°C and 180 rpm for 48 h, and then separating and purifying the fermentation broth to obtain the L-cysteine ​​after fermentation.

[0025] Preferably, the shake-flask fermentation medium consists of the following components: glucose 42 g / L, peptone 1 g / L, ammonium sulfate 9 g / L, sodium thiosulfate pentahydrate 7 g / L, disodium hydrogen phosphate decahydrate 2.52 g / L, yeast extract 8 g / L, potassium dihydrogen phosphate 1 g / L, 1 ml / L trace element solution, and 1 ml / L vitamin B1 solution, with deionized water as the solvent and a natural pH. The trace element solution consists of: manganese sulfate octahydrate 5 g / L, magnesium sulfate heptahydrate 500 g / L, ferrous sulfate heptahydrate 5 g / L, and zinc sulfate 5 g / L, with deionized water as the solvent.

[0026] Preferably, the application includes: filling a 500mL shake flask with 20-50mL of fermentation medium, sterilizing at 104℃ for 45min, inoculating the high-L-cysteine-producing engineered bacterial strain into a 10mL test tube, culturing at 37℃ and 180rpm for 16-18h, inoculating at a 2% inoculum into a shake flask, fermenting at 30℃ and 180rpm for 48h, adjusting the pH with 1g / L CaCO3, and simultaneously adding IPTG to a final concentration of 1mM and VB1 to a final concentration of 5mg / L to obtain the fermentation broth, and separating and purifying the fermentation broth to obtain the L-cysteine.

[0027] The beneficial effects of this invention are:

[0028] This invention utilizes CRISPR / Cas9 gene editing technology to replace the in situ promoter of the cysD gene in the genome of an existing engineered strain, further enhancing the expression level of the cysD gene, which encodes a key enzyme in the L-cysteine ​​biogeneration pathway. Furthermore, it introduces a dual gene heterologously encoding ATP sulfatase and APS kinase to enhance the supply of the sulfonyl donor PAPS. By knocking the gene into the pseudogene site of the chassis strain, a high-yielding strain, SSC8-2YJ3, without plasmids and requiring no antibiotics during fermentation, was obtained, representing a 49.2% increase in yield compared to the starting strain. Attached Figure Description

[0029] Figure 1 shows the L-cysteine ​​metabolic pathway and modified sites of this invention.

[0030] Figure 2 shows the changes in OD600 and L-cysteine ​​titer of TKA1 and TKA2 in Example 1.

[0031] Figure 3 shows the changes in OD600 and L-cysteine ​​titer of SSC8-2YJ1 in Example 2.

[0032] Figure 4 shows the changes in OD600 and L-cysteine ​​titer of TKA3 in Example 3.

[0033] Figure 5 shows the changes in OD600 and L-cysteine ​​titer of SSC8-2YJ2 in Example 4.

[0034] Figure 6 shows the changes in OD600 and L-cysteine ​​titer of SSC8-2YJ3 in Example 5. Detailed Implementation

[0035] The following specific embodiments illustrate the implementation of the present invention. 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, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0036] In the following examples, the final concentration of spectinomycin and kanamycin in the culture medium was 0.05 mg / L.

[0037] The parent strain E. coli W3110 described in this invention was deposited at the Coli Genetic Stock Center (CGSC) of Yale University on August 5, 1975, with accession number CGSC#4474, and has been disclosed in patents US 2009 / 0298135 A1 and US 2010 / 0248311 A1.

[0038] HPLC determination of L-cysteine ​​content:

[0039] Chromatographic conditions: C18 column, detection wavelength 260 nm; column temperature 30 °C;

[0040] Sample preparation: Centrifuge 1 mL of fermentation broth to separate the supernatant and precipitate. Resuspend the precipitate in 0.5 M sulfuric acid, mix well, let stand for 3-5 minutes, centrifuge, and then neutralize with 1 M sodium hydroxide. Separate the supernatant and precipitate. Take 100 μL of the supernatant and add 500 μL of borate-borax buffer and 300 μL of derivatization reagent, respectively. React at 60℃ and 600 rpm in the dark for 1 hour. Take 200-400 μL of the sample solution for HPLC analysis.

[0041] Mobile phase: A. pure acetonitrile, B. acetonitrile / 50mM sodium acetate buffer (17 / 83), adjusted to pH 4.9;

[0042] Data acquisition time: 23 minutes.

[0043] The primer sequence information used in the examples is shown in Table 2.

[0044] Table 2: Primer sequences

[0045] Example 1: Construction and shake-flask fermentation of TKA1 and TKA2

[0046] SSC8(E.coli W3110,pgk / serA) f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f Using the pTrc99a plasmid as the starting strain, the heterologous genes MET3 and N7489_009139 (nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2) were overexpressed to enhance the strength of PAPS synthesis in the strain.

[0047] (1) Obtaining heterologous genes MET3 and N7489_009139: The nucleotide and protein sequences of MET3 (encoding ATP sulfurylase) from Kluyveromyces lactis GG799 and N7489_009139 (encoding APS kinase) from Penicillium chrysogenum were obtained from NCBI. The sequences were obtained by PCR amplification using pET28-KAST / APSK and pET-KF / R and pETA-F / R primers, respectively. After verification by nucleic acid gel electrophoresis, the PCR products were digested with Dpn I digestive enzyme at 37℃ for 3 h. After purification with Clean up kit and verification by sequencing, the correct KAST / APSK fragment was obtained and used for subsequent ligation of overexpression plasmid.

[0048] (2) Construction of pTrc99a-KAST-APSK plasmid: First, using the initial pTrc99a plasmid as a template, linearization was achieved by PCR using 99a-PLine-F / R primers. After verification by nucleic acid gel electrophoresis, the product was digested with Dpn I digestive enzyme at 37℃ for 3 h. Second, using the KAST / APSK fragment as a template, and with 99a-KF / R and 99a-AF / R primers, the KAST fragment (K1) with homologous arms on the upper part of the pTrc99a plasmid and homologous arms on the lower part of the APSK fragment, and the APSK fragment (A1) with homologous arms on the upper part of the KAST fragment and homologous arms on the lower part of the pTrc99a plasmid were amplified by PCR. After verification by nucleic acid gel electrophoresis, the three fragments were digested with Dpn I digestive enzyme at 37℃ for 3 h, purified by a Clean up kit, and then... (One-step clone kit, Vazyme Biotech, Nanjing, China) The instructions describe how to ligate the pTrc99a vector, K1, and A1 fragments via fusion PCR. The PCR product is then transformed into E. coli DH5α. After screening with kanamycin sulfate plates and sequencing verification, the correct pTrc99a-KAST-APSK plasmid is obtained. The plasmid is then extracted and overexpressed in SSC8 spores to obtain TKA1.

[0049] (3) Construction of pTrc99a-KAST-Linker-APSK plasmid: First, using the KAST / APSK fragment as a template and 99a-KF and 99a-KL-R, 99a-LA-F and 99a-AR as primers, the KAST fragment (K2) with the homologous arm on the upper part of the pTrc99a plasmid and the homologous arm on the lower part of the Linker-APSK fragment, and the APSK fragment (A2) with the homologous arm on the upper part of the KAST-Linker fragment and the homologous arm on the lower part of the pTrc99a plasmid were amplified by PCR. After verification by nucleic acid gel electrophoresis, the fragments were digested with Dpn I digestive enzyme at 37℃ for 3 h, purified with Clean Up kit, and then used for later use. Second, using the linearized pTrc99a fragment from (2), the KAST fragment (K2) was amplified by PCR. (One-step clone kit, Vazyme Biotech, Nanjing, China) The instructions describe how to ligate the pTrc99a vector, K2, and A2 fragments via fusion PCR. The PCR product is then transformed into E. coli DH5α. After screening with kanamycin sulfate plates and sequencing verification, the correct pTrc99a-KAST-Linker-APSK plasmid is obtained. The plasmid is then extracted and overexpressed in SSC8 basal bacteria to obtain TKA2.

[0050] (4) Shake-flask fermentation: TKA1 and TKA2, with the starting strain SSC8 as the control group, were inoculated into 10 mL of LB medium and cultured at 37℃ and 200 rpm as pre-cultures. After 8-12 h, 1 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium at a 2% inoculation rate. The flask was then cultured in a constant temperature shaker at 30℃ and 180 rpm for 48 h for bacterial fermentation. After fermentation, 1 mL of the fermentation broth was taken to determine the OD. 600 Simultaneously, for sample processing and HPLC detection, OD... 600 The L-cysteine ​​content in the fermentation broth is shown in Figure 2.

[0051] As shown in the figure, overexpression of the heterologous gene has a certain impact on cysteine ​​production. A comparison between TKA1 and TKA2 revealed that the addition of the linker actually decreased production, while a comparison between TKA1 and SSC8 showed a slight increase in production. However, OD... 600 The yield was reduced (the results of repeatability experiments were consistent), which may be due to the resistance of the overexpression plasmid, which has a certain inhibitory effect on the growth of the strain. Based on this result, it can be seen that the expression of heterologous genes to enhance the synthesis of ATP sulfatase and APS kinase is effective in increasing the yield. Therefore, the strain was subsequently modified in terms of genome.

[0052] LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent: deionized water, pH: natural.

[0053] Shake-flask fermentation medium: glucose 42 g / L, peptone 1 g / L, ammonium sulfate 9 g / L, sodium thiosulfate pentahydrate 7 g / L, disodium hydrogen phosphate decahydrate 2.52 g / L, yeast extract 8 g / L, potassium dihydrogen phosphate 1 g / L, 1 ml / L trace element solution, 1 ml / L vitamin B1 solution, solvent is deionized water, pH is natural; the trace element solution composition is: manganese sulfate octahydrate 5 g / L, magnesium sulfate heptahydrate 500 g / L, ferrous sulfate heptahydrate 5 g / L, zinc sulfate 5 g / L, solvent is deionized water.

[0054] Example 2: Construction and shake-flask fermentation of SSC8-2YJ1

[0055] Using SSC8 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to replace the original pseudogene yjhw on the starting strain genome with the MET3 and N7489_009139 genes (nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2) derived from pRSFDuet via gene knock-in, thereby enhancing the ability to synthesize PAPS and the intensity of sulfate metabolism.

[0056] (1) Construction of pTarget-yjhw plasmid: Using pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed using pT-yjhw-F / R primers. After verification by nucleic acid gel electrophoresis, the PCR product was digested with Dpn I digestive enzyme at 37℃ for 3 h, and then transformed into E. coli DH5α. After screening by spectrozin plate and sequencing verification, the correct pTarget-yjhw plasmid was obtained and used for subsequent ligation of Donor DNA.

[0057] (2) Construction of pTD-KAST-APSK plasmid: First, using the E. coli W3110 genome as a template, the upstream portion of the donor DNA was amplified using yjhw-S5F / R primers (F1), and the downstream portion of the donor DNA was amplified using yjhw-X5F / R primers (F2). Then, using pTrc99a-KAST-APSK as a template, the MET3 and N7489_009139 gene fragments were amplified using PTD-TKA-F / R primers (F3); plasmid pTarget-yjhw was incubated at 37℃ for 8 hours with Xba I and Pst I, and the DNA fragments were recovered using a Clean Up kit; according to... The instructions for the (One-step clone kit, Vazyme Biotech, Nanjing, China) link the pTarget-yjhw vector, fragments F1, F2, and F3 together, and verify the pTD-KAST-APSK plasmid through sequencing.

[0058] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into SSC8 (E. coli W3110, pgk / serA). f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f In this method, single colonies were transferred to LB tubes containing 0.05 mg / L kanamycin and cultured overnight at 30°C. Then, 1% (v / v) of the culture was inoculated into 250 mL shake flasks containing 50 mL of LB medium, and 500 μl of 1 mol / L L-arabinose was added. The cells were cultured at 150 rpm and 30°C until the OD600 reached 0.4–0.6. The cells were then collected by centrifugation at 4000 rpm and 4°C for 10 min to prepare electrocompetent cells. For detailed procedures, please refer to the description in (Molecular Cloning: A Laboratory Manual, 3rd ed Edition, 99-102).

[0059] (4) Using a pipette, a suitable amount of pTD-KAST-APSK plasmid (about 200 ng) was mixed with 100 μl of electroporation competent cells and transferred together into a pre-cooled 2 mm electroporation cuvette. After incubating on ice for about 1-2 min, electroporation transformation was performed using an electroporator (MicroPluser™, BIO-RAD). Immediately after electroporation, 800 μl of LB medium was added and gently aspirated, then transferred to a 2 mL Eppendorf tube. After recovery at 30°C for 3-4 h, the medium was plated onto LB solid plates containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin. The plates were incubated upside down at 30°C for 12-16 h. Colony PCR was performed using yjhw-KA-F / R as primers. If a fragment of about 2530 bp could be successfully cloned, it would prove that the colony was positive for SSC8-2YJ1 (SSC8 derivative, yjhw::MET3-N7489_009139).

[0060] (5) Plasmid elimination: Positive single colonies were picked using an inoculation loop and inoculated into LB liquid tubes containing 1 mM IPTG and 0.05 mg / L kanamycin. The tubes were incubated overnight at 30°C. The next day, the bacterial suspension was streaked onto LB agar plates containing 0.05 mg / L kanamycin and incubated at 30°C for 24 hours. When the bacteria reached a certain size, a portion of single colonies were picked and streaked onto LB agar plates containing 0.05 mg / L spectinomycin. Single colonies that could not be streaked onto LB agar plates containing 0.05 mg / L spectinomycin indicated successful elimination of the pTarget-yjhw plasmid. Single colonies successfully eliminated from the pTarget-yjhw plasmid were picked and incubated overnight at 37°C in LB tubes to eliminate the pCas plasmid. The next day, the bacterial culture was streaked onto LB plates and incubated at 37°C for 12 hours. Some single colonies were then streaked onto LB plates containing 0.05 mg / L kanamycin. Single colonies that could not be streaked onto LB plates containing 0.05 mg / L kanamycin were considered to have successfully eliminated the pCas plasmid, resulting in the plasmid-free strain SSC8-2YJ1 (SSC8 derivative, yjhw::MET3-N7489_009139).

[0061] (6) The constructed SSC8-2YJ1 production strain was subjected to shake-flask testing and detection according to the method in Example 1(4), with SSC8 as the control group. OD 600 The L-cysteine ​​content in the fermentation broth is shown in Figure 3.

[0062] As shown in the figure, increasing the copy number of heterologous MET3 and N7489_009139 genes through gene knock-in did not affect bacterial growth. Compared with SSC8, the L-cysteine ​​titer of SSC8-2YJ1 increased to 4.84 g / L in shake flasks. This may be because the introduction of the heterologous dual genes enhanced the sulfate pathway, promoting the synthesis of sulfur donors and thus promoting the biosynthesis of L-cysteine ​​in the strain.

[0063] Example 3: Construction and shake-flask fermentation of TKA3

[0064] Starting with SSC8, the heterologous genes MET3 and N7489_009139 (nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2) were overexpressed using the pTrc99a plasmid. The expression of both genes was regulated by the Trc promoter to enhance the strength of PAPS synthesis in the strain.

[0065] (1) Construction of pTrc99a-Trc(KAST / APSK) plasmid: First, using pTrc99a-KAST-APSK plasmid as a template, and 99a-KF and 99a-TK-R, 99a-TA-F and 99a-AR as primers, the KAST fragment (K3) containing the upper homologous arm of the pTrc99a plasmid with the Trc promoter and the lower homologous arm of the APSK fragment with the Trc promoter, and the APSK fragment (A3) containing the upper homologous arm of the KAST fragment and the Trc promoter and the lower homologous arm of the pTrc99a plasmid were amplified by PCR. After verification by nucleic acid gel electrophoresis, the three fragments were digested with Dpn I digestive enzyme at 37℃ for 3 h, purified by Clean up kit, and then... (One-step clone kit, Vazyme Biotech, Nanjing, China) The instructions describe ligating the pTrc99a vector, K3, and A3 fragments via fusion PCR. The PCR product was transformed into E. coli DH5α, screened using kanamycin sulfate plates, and sequenced to verify the correct pTrc99a-Trc(KAST / APSK) plasmid. The plasmid was then extracted and overexpressed in SSC8 spores to obtain TKA3.

[0066] (2) The constructed TKA3 production strain was used as a control group, with the TKA1 constructed in Example 1 as the control group, and shake-flask tests and detections were performed according to the method in Example 1(4). OD 600 The L-cysteine ​​content in the fermentation broth is shown in Figure 4.

[0067] As shown in the figure, the addition of dual Trc promoters to heterologous dual genes in TKA1 using overexpression plasmids resulted in higher cell growth concentrations. Compared to TKA1, the L-cysteine ​​titer in TKA3 increased to 4.93 g / L in shake flasks. This may be because the Trc promoter has an enhancing effect on the gene encoding APS kinase, thereby promoting the biosynthesis of L-cysteine ​​in the strain.

[0068] Example 4: Construction and shake-flask fermentation of SSC8-2YJ2

[0069] Using SSC8 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the in situ promoter of the cysD gene in its genome with Ptrc (nucleotide sequence shown in SEQ.ID NO.3) to enhance the ability of SSC8 chassis bacteria to synthesize PAPS and the intensity of sulfate metabolism.

[0070] (1) Construction of pTarget-cysD::Ptrc plasmid: pTarget F plasmid (Addgene Plasmid#62226) was used as a template and pT-cysD-F / R was used as primers for PCR amplification. The PCR product was digested with Dpn I at 37℃ for 3 h. The PCR product of pTarget-cysD::Ptrc was amplified with linearized primers pTarget-PLine-F / R. The pTarget-cysD::Ptrc linearized vector was purified with a Clean up kit and used for subsequent ligation of Donor DNA.

[0071] (2) Construction of pTD-cysD::Ptrc plasmid: Using the E. coli W3110 genome as a template, the upstream portion of the donor DNA was amplified using cysD-S5F / R primers (F1), and the downstream portion of the donor DNA was amplified using cysD-X5F / R primers (F2). Using pTrc99a plasmid as a template, fragments containing homologous arms of the upstream and downstream portions of the donor DNA were amplified using Trc-F / R primers (F3), according to... (One-step clone kit, Vazyme Biotech, Nanjing, China) The instructions link the pTarget-cysD::Ptrc linearized vector, fragments F1, F2, and F3 together, following the same construction steps as in Example 2.

[0072] (2) The pTD-cysD::Ptrc plasmid was obtained.

[0073] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into SSC8 competent cells. The preparation method of SSC8 competent cells is the same as in Example 2 (3).

[0074] (4) SSC8-2YJ2 positive colonies were constructed using the same method as in Example 2(4).

[0075] (5) Plasmid elimination: The implementation method is the same as in Example 2(5), and plasmid-free SSC8-2YJ2 is obtained.

[0076] (6) The constructed SSC8-2YJ2 production strain was subjected to shake-flask testing and detection according to the method in Example 2(6), with SSC8 as the control group. OD 600 The content of L-cysteine ​​in the fermentation broth is shown in Figure 5.

[0077] As shown in the figure, after replacing the in situ promoter of the cysD gene with Ptrc through gene editing, L-cysteine ​​production increased to 5.74 g / L. The original cysD gene expression level was insufficient; by using a strong promoter to increase expression level, the sulfate pathway was enhanced, thus promoting the increase in L-cysteine ​​production.

[0078] Example 5: Construction and shake-flask fermentation of SSC8-2YJ3

[0079] Using the engineered bacterium SSC8-2YJ2 prepared in Example 4 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene yjhw on the genome of the starting strain by gene knock-in, with the MET3 and N7489_009139 genes respectively regulated by the Ptrc promoter derived from pTrc99A replacing the original pseudogene yjhw, in order to enhance the ability to synthesize PAPS and the intensity of sulfate metabolism.

[0080] (1) Constructing pTarget-yjhw plasmid: The construction method is the same as in Example 2(1).

[0081] (2) Construction of pTD-yjhw-Trc(KAST / APSK) plasmid: First, the upstream portion (F1) and downstream portion (F2) of the donor DNA were obtained using the method in Example 2(2). Then, using the plasmid constructed in Example 3(1) as a template, the KAST fragment with the pTrc promoter and the APSK fragment with the Trc promoter (F3) were amplified using 99a-TKTA-F / R primers. The PCR fragments were purified using a Clean Up kit to obtain F1, F2, and F3; according to The instructions for the (One-step clone kit, Vazyme Biotech, Nanjing, China) link the pTarget-yjhw vector, fragments F1, F2, and F3 together, and verify the pTD-yjhw-Trc(KAST / APSK) plasmid through sequencing.

[0082] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SSC8-2YJ2 competent cells obtained in Example 4. The preparation method of SSC8-2YJ2 electrotransfer competent cells was the same as in Example 2 (3).

[0083] (4) SSC8-2YJ3 positive colonies were constructed using the same method as in Example 2(4).

[0084] (5) Plasmid elimination: The implementation method is the same as in Example 2 (5), and plasmid-free SSC8-2YJ3 is obtained.

[0085] (6) The constructed SSC8-2YJ3 production strain was used as a control group, with the SSC8-2YJ2 constructed in Example 4 as the control group, and shake-flask tests and detections were performed according to the method in Example 2(6). OD 600 The L-cysteine ​​content in the fermentation broth is shown in Figure 6.

[0086] As shown in the figure, even after enhancing the key genes in the sulfate pathway, further increasing the copy number of heterologous MET3 and N7489_009139 genes at pseudogene sites in the genome via gene knock-in still resulted in a significant increase in L-cysteine ​​production, approximately 6.06 g / L. This demonstrates the importance of sulfur flux in the pathway for L-cysteine ​​synthesis, and that enhancing the sulfate pathway can promote increased L-cysteine ​​production in shake flasks.

[0087] The OD values ​​of the engineered strains constructed in Examples 1-5 after shake-flask testing were obtained. 600The results of L-cysteine ​​content in the fermentation broth are shown in Table 3. Compared with the method of introducing heterologous dual genes by plasmid and using dual Trc promoter regulation (engineered strain TKA3), this invention attempts to introduce heterologous dual genes into the chassis strain (engineered strain SSC8-2YJ1) by knocking the genes into the pseudogene site and overexpressing them. The results show that the heterologous dual genes can be well expressed at the pseudogene site yjhw, thereby enhancing the sulfate pathway and promoting the synthesis of sulfur donors. Its effect on increasing L-cysteine ​​production (18.9%) is similar to that of the plasmid introduction method (21.3%). Furthermore, based on the engineered strain SSC8-2YJ1, this invention utilizes CRISPR-Cas9-mediated gene editing technology to further enhance the expression level of the cysD gene, which encodes a key enzyme in the L-cysteine ​​bioproduction pathway. This resulted in a plasmid-free, high-yielding strain SSC8-2YJ3 that requires no antibiotics during fermentation. Compared to the engineered strain SSC8-2YJ1, its L-cysteine ​​yield increased by 25.3%, and compared to the starting strain, it increased by 49.2%. This improvement was unexpected, achieving a remarkable technical effect.

[0088] Table 3: OD of different strains 600 and L-cysteine ​​production

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for constructing a high-yield L-cysteine-producing engineered bacterium, characterized in that, The method includes: enhancing the ability to synthesize 3'-adenosine-5'-phosphosulfate in chassis strains; The method for enhancing the synthesis ability of 3'-adenosine-5'-phosphosulfate includes at least one of the following: (1) Introduce and overexpress the MET3 gene derived from Kluyveromyces lactis GG799 in the sclerotium; (2) The N7489_009139 gene derived from Penicillium chrysogenum was introduced and overexpressed in the sclerotium; (3) Overexpression of the cysD gene in the genome of the basal spores.

2. The method of claim 1, wherein, The method for enhancing the synthesis of 3'-adenosine-5'-phosphosulfate includes: using CRISPR-Cas9-mediated gene editing technology to replace the in situ promoter of the cysD gene in the genome of *Bacillus subtilis* with the Trc promoter and then overexpressing it.

3. The method of claim 1, wherein, The method for enhancing the synthesis of 3'-adenosine-5'-phosphoryl sulfate includes: using CRISPR-Cas9-mediated gene editing technology, replacing the original pseudogene yjhw on the genome of the fungus with the MET3 gene and the N7489_009139 gene, and then overexpressing them.

4. The method of claim 1, wherein, The method for enhancing the synthesis of 3'-adenosine-5'-phosphosulfate includes: constructing the MET3 gene and the N7489_009139 gene into the pTrc99a plasmid and then transfecting it into the sclerotium for overexpression.

5. The method of claim 1, wherein, The method for enhancing the synthesis of 3'-adenosine-5'-phosphosulfate includes: using CRISPR-Cas9-mediated gene editing technology, replacing the in situ promoter of the cysD gene in the genome of *Bacillus subtilis* with the Trc promoter and then overexpressing it; and replacing the original pseudogene yjhw on the genome of *Bacillus subtilis* with the MET3 gene and the N7489_009139 gene and then overexpressing them.

6. The method of claim 1, wherein, The chassis bacterium is: E.coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f .

7. The method of claim 1, wherein, The nucleotide sequence of the MET3 gene is shown in SEQ ID NO.1; the nucleotide sequence of the N7489_009139 gene is shown in SEQ ID NO.

2.

8. The engineered bacteria that produce high levels of L-cysteine ​​as described in any one of claims 1 to 7.

9. The engineered bacterium for high L-cysteine ​​production as described in claim 8, characterized in that, It can be any of the following: (1)E.coli W3110,pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f / yjhw::MET3-N7489_009139; (2)E.coli W3110,pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f / cysD::Ptrc: (3) E. coli W3110, pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f / cysD::Ptrc / yjhw::Ptrc:MET3(KI),N7489_009139(KI); (4)E.coli W3110,pgk / serA f / serB / serC / cysE f / cysB / ΔsdaA / ΔsdaB / ΔtdcG / Δyham / ΔtnaA / ΔyciW / ΔgpmA / ΔpykF / ΔpoxB / ΔmetR / eamA / eamB f / tolC / ΔydjN / ΔyjiP-cysE f / ΔyjiR-cysE f / ΔyeeJ-cysE f / ΔycdN-cysE f / ΔydeU-cysE f / ΔylbE-cysE f / ΔyjhE-cysE f / pTrc(MET3,N7489_009139)。 10. The application of the high-L-cysteine-producing engineered bacteria as described in claim 8 in the microbial fermentation preparation of L-cysteine.

Images