CN113493745B - Genetically engineered bacterium for producing cephalosporin C and construction method thereof - Google Patents

Abstract

The invention discloses a genetically engineered bacterium for producing cephalosporin C and a construction method thereof. According to the invention, the Acrpn4 gene is knocked out from the Cephalosporium acremonium C10 strain (cephalosporin C high-yield strain), so that the yield of the cephalosporin C is greatly improved, the survival ability of thalli in the fermentation process is enhanced, and the method has important significance in reducing the production cost of the cephalosporin C. The invention has important application value.

Description

Genetically engineered bacterium for producing cephalosporin C and construction method thereof

Technical Field

The invention belongs to the field of microorganisms, and in particular relates to a genetically engineered bacterium for producing cephalosporin C and a construction method thereof.

Background

Cephalosporium acremonium is an industrially produced strain of cephalosporin C (CPC for short). Cephalosporin C and penicillin belong to beta-lactam antibiotics, but cephalosporin C is structurally different from penicillin in that a dihydrothiazine ring is connected with a beta-lactam ring to replace a thiazole ring in penicillin, and the cephalosporin C has stronger stability, can kill a plurality of penicillin-resistant bacteria and is not easily damaged by penicillin enzymes. Due to these advantages, cephalosporin antibiotics are widely used as anti-infective drugs in clinic. In the domestic medical market, cephalosporin antibiotics account for more than 40% of sales of anti-infective drugs.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium for producing cephalosporin C.

The invention first protects a method for preparing engineering bacteria for producing cephalosporin C, which can comprise the following steps: reducing the expression quantity and/or activity of AcRpn4 protein in the cephalosporans, thereby obtaining the engineering bacteria for producing cephalosporin C;

the AcRpn4 protein may be a 1) or a 2) or a 3) as follows:

a1 Amino acid sequence is SEQ ID NO: 2;

a2 In SEQ ID NO:2 or/and C terminal of the protein shown in the specification;

a3 A) a protein having the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in a 1) or a 2).

The invention also provides a method for prolonging the survival time of the cephalosporium acremonium, which can comprise the following steps: reducing the expression level and/or activity of the AcRpn4 protein in Cephalosporium acremonium.

In any of the above methods, the "reducing the expression level and/or activity of the AcRpn4 protein in cephalosporans" can be achieved by methods commonly used in the art, such as homologous recombination, RNA interference, or site-directed gene editing.

In any of the above methods, the "reducing the expression level and/or activity of the AcRpn4 protein in cephalosporanges" can be achieved by knocking out the gene encoding the AcRpn4 protein in the acremonium (i.e., the AcRpn4 gene). The knockout includes knocking out the entire AcRpn4 gene, as well as knocking out a partial segment of the AcRpn4 gene.

The coding gene of the AcRpn4 protein (i.e., the AcRpn4 gene) can be J1) or J2) or J3) or J4) as follows:

j1 A) the coding region is SEQ ID NO:1, a DNA molecule shown in fig. 1;

j2 Nucleotide sequence is SEQ ID NO:1, a DNA molecule shown in fig. 1;

j3 A DNA molecule derived from Cephalosporium acremonium and encoding said AcRpn4 protein having 75% or more identity with the nucleotide sequence defined in J1) or J2);

j4 Under stringent conditions with the nucleotide sequence defined in J1) or J2), a DNA molecule derived from cephalosporium acremonium and encoding said AcRpn4 protein.

In any of the above methods, the coding gene of the AcRpn4 protein is knocked out by homologous recombination; the homologous recombination fragment contains an upstream homology arm and a downstream homology arm of the coding gene of the AcRpn4 protein; the upstream homology arm and the downstream homology arm of the coding gene of the AcRpn4 protein are each 2-4kb (e.g., 2-3kb, 3-4kb, 2kb, 3kb or 4 kb) in length.

The nucleotide sequence of the upstream homology arm of the coding gene of the AcRpn4 protein can be shown as SEQ ID NO: 3.

The nucleotide sequence of the downstream homology arm of the coding gene of the AcRpn4 protein can be shown as SEQ ID NO: 4.

More specifically, in the invention, the coding gene of the AcRpn4 protein is knocked out by introducing a recombinant vector into Cephalosporium acremonium. The recombinant vector may be one in which a small DNA fragment between the recognition sites of restriction enzymes kpnl and ApaI of vector pAgHB is replaced by SEQ ID NO:3, the DNA small fragment between the recognition sites of restriction enzymes AscI and PacI is replaced by the DNA fragment shown in SEQ ID NO:4, and the recombinant plasmid obtained.

Any of the above-mentioned Cephalosporium acremonium may be a Cephalosporium acremonium C10 strain.

Engineering bacteria for producing cephalosporin C prepared by any of the above methods also fall within the scope of the present invention.

The application of any of the engineering bacteria in the production of cephalosporin C or cephalosporin C upstream and downstream products also belongs to the protection scope of the invention.

The invention also provides a method for producing cephalosporin C, which can comprise the following steps: fermenting and culturing any engineering bacteria, and collecting fermentation products to obtain cephalosporin C.

In the above method, the conditions of the fermentation culture may be: the culture is carried out by shaking at 26-30deg.C (such as 26-28deg.C, 28-30deg.C, 26 deg.C, 28 deg.C or 30deg.C) for 5-10d (such as 5-7d, 7-10d, 5d, 7d or 10 d). The rotational speed of the shaking culture may be 200-250rpm (e.g., 200-220rpm, 220-250rpm, 200rpm, 220rpm, or 250 rpm).

In the above method, the medium for fermentation culture may be an MDFA medium.

The composition of the MDFA medium may be as follows: 36.0g of Sucrose, 3.2g of DL-methionine, 7.5g of g L-Asparagine and 144.0mL of Solution III are dissolved in a proper amount of distilled water, the pH value is adjusted to 7.4 by NaOH (about 3.3g of NaOH is required to be added per liter), then distilled water is added to ensure that the total volume is 932mL, and the sterilization is carried out for 20min at 121 ℃; after that, 20.0mL of Solution I (sterilized at 115℃for 30 min), 40.0mL of Solution II (sterilized at 121℃for 20 min) and 8.0mL of Solution IV (sterilized by filtration) were added.

Solution I:50% (w/v) glucose solution.

Solution II:50% (w/v) glycerol solution.

Solution III: will 136.22g K ₂ HPO ₄ ·3H ₂ O、102.00g KH ₂ PO ₄ 、11.50g Na ₂ SO ₄ ·10H ₂ O、2.40g MgSO ₄ ·7H ₂ O、0.20g ZnSO ₄ ·7H ₂ O、0.20g MnSO ₄ ·H ₂ O、0.05g CuSO ₄ ·5H ₂ O and 0.50g CaCl ₂ ·2H ₂ O was dissolved in a proper amount of distilled water, and then distilled water was used to fix the volume to 1L.

According to the invention, the Acrpn4 gene is knocked out from the Cephalosporium acremonium C10 strain (cephalosporin C high-yield strain), so that the yield of the cephalosporin C is greatly improved, the survival ability of thalli in the fermentation process is enhanced, and the method has important significance in reducing the production cost of the cephalosporin C. The invention has important application value.

Drawings

FIG. 1 is a schematic diagram of homologous recombination and the results of identification of Acrpn4 knockout mutant strains.

FIG. 2 shows a comparison of cephalosporin C production in Cephalosporium acrtn C10 strain and DeltaAcrpn 4.

FIG. 3 shows a comparison of cell dry weights of Cephalosporium acrtn C10 strain and ΔAcrpn4.

FIG. 4 shows the comparison of the growth and viability of Cephalosporium acreage C10 strain and DeltaAcrpn 4.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

The Cephalosporium acremonium C10 strain is a cephalosporin C-producing strain and is described in the following literature: liu G, casqueiro J,O，Cardoza RE，Gutiérrez S，Martín JF.Targeted inactivation of the mecB gene，encodingcystathionine-gamma-lyase，shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesisinAcremoniumchrysogenum C10but not formethionineinduction of the cephalosporin genes.J Bacteriol.2001，183(5):1765-1772.。

the media and plates involved in the following examples are as follows:

LB liquid medium: 10.0g of tryptone, 5.0g of yeast extract and 10.0g of NaCl are dissolved in a proper amount of distilled water, the pH value is adjusted to 7.0 by NaOH, and then distilled water is used for constant volume to 1L, and sterilization is carried out for 20min at 121 ℃.

LB solid plate: dissolving 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl and 12g agar in a proper amount of distilled water, adjusting pH to 7.0 with NaOH, then fixing volume to 1L with distilled water, and sterilizing at 121deg.C for 20min; after cooling to 55 ℃, pouring into a sterile petri dish (diameter of 9 cm), and naturally cooling.

MM medium: 10.0mL of K solution, 20.0mL of mLM-N solution, 1.0mL of 1% (w/v) CaCl ₂ ·2H ₂ O solution, 2.5mL20% (w/v) (NH) ₄ ) ₂ SO ₄ Mixing the solution with a proper amount of distilled water, adding distilled water to make the total volume be 986mL, and sterilizing at 121 ℃ for 20min; at the time of use, 4mL of 50% (w/v) glucose solution (sterile) and 10mL of 0.01% (w/v) FeSO were added ₄ Solutions (sterile). The solute of the K solution and the concentration thereof are K ₂ HPO ₄ 205g/L and KH ₂ PO ₄ 145g/L, distilled water as solvent, and pH value of 7.0. The solute of the M-N solution and the concentration thereof are MgSO ₄ ·7H ₂ 30g/L of O and 15g/L of NaCl, distilled water is used as the solvent, and the pH value is natural.

IM medium: 10.0mL of K solution, 20.0mL of mLM-N solution, 1.0mL of 1% (w/v) CaCl ₂ ·2H ₂ O solution, 2.5mL20% (w/v) (NH) ₄ ) ₂ SO ₄ Mixing the solution, 40.0mL of MES solution with the concentration of 1mol/L, 5.0mL of glycerol and a proper amount of distilled water, adding distilled water to ensure that the total volume is 986mL, and sterilizing for 20min at 121 ℃;at the time of use, 4mL of 50% (w/v) glucose solution (sterile) and 10mL of 0.01% (w/v) FeSO were added ₄ Solution (sterile) and AS, the amount of AS added was 2. Mu.L of AS solution at a concentration of 0.1mol/L per mL of IM medium.

CM plate: 10.0mL of K solution, 20.0mL of mLM-N solution, 1.0mL of 1% (w/v) CaCl ₂ ·2H ₂ O solution, 2.5mL20% (w/v) (NH) ₄ ) ₂ SO ₄ Mixing the solution, 40.0mL of MES solution with the concentration of 1mol/L, 5.0mL of glycerol, 15g of agar and a proper amount of distilled water, adding distilled water to make the total volume be 986mL, and sterilizing at 121 ℃ for 20min; after cooling to 55℃2mL of a 50% (w/v) glucose solution (sterile) and 10mL of 0.01% (w/v) FeSO were added ₄ Mixing the solution (sterile) and 4mL of AS solution with the concentration of 0.1 mol/L; pouring into sterile petri dish (diameter 9 cm), and naturally cooling.

LPE plate: 10.0g CaCl ₂ Dissolving 1.0g glucose, 1.5g NaCl, 2.0g Yeast Extract and 25.0g Agar in a proper amount of distilled water, adjusting pH to 6.8 with NaOH, then fixing volume to 1L with distilled water, and sterilizing at 121deg.C for 20min; after cooling to 55 ℃, pouring into a sterile petri dish (diameter of 9 cm), and naturally cooling.

TSA screening plates: 17.0g of Tryptone, 2.5g of Glucose, 5.0g of NaCl and 2.5g of K ₂ HPO ₃ ·3H ₂ Dissolving O, 3.0. 3.0gTryptone soya broth and 15g of agar in a proper amount of distilled water, and sterilizing with distilled water to 1L at 121 ℃ for 20min; after cooling to 55 ℃, the thiamycin and the hygromycin are added, the concentration of the thiamycin in the system is 500 mug/mL, the concentration of the hygromycin in the system is 50 mug/mL, and the mixture is poured into a sterile culture dish (with the diameter of 9 cm) and naturally cooled.

Bleomycin plates: 17.0g of Tryptone, 2.5g of Glucose, 5.0g of NaCl and 2.5g of K ₂ HPO ₃ ·3H ₂ Dissolving O, 3.0. 3.0gTryptone soya broth and 15g of agar in a proper amount of distilled water, and sterilizing with distilled water to 1L at 121 ℃ for 20min; after cooling to 55 ℃, bleomycin is added and the concentration of the bleomycin in the system is 20 mug/mL, poured into a sterile petri dish (diameter of 9 cm) and naturally cooled.

MDFA medium: 36.0g of Sucrose, 3.2g of DL-methionine, 7.5g of g L-Asparagine and 144.0ml of LSolution III are dissolved in a proper amount of distilled water, the pH value is adjusted to 7.4 by NaOH (about 3.3g of NaOH is required to be added per liter), distilled water is then added to ensure that the total volume is 932mL, and sterilization is carried out for 20min at 121 ℃; then 20.0mLSolution I (sterilized at 115℃for 30 min), 40.0mLSolution II (sterilized at 121℃for 20 min) and 8.0mLSolution IV (sterilized by filtration) were added.

Solution I:50% (w/v) glucose solution.

Solution II:50% (w/v) glycerol solution.

Solution III: will 136.22g K ₂ HPO ₄ ·3H ₂ O、102.00g KH ₂ PO ₄ 、11.50g Na ₂ SO ₄ ·10H ₂ O、2.40g MgSO ₄ ·7H ₂ O、0.20g ZnSO ₄ ·7H ₂ O、0.20g MnSO ₄ ·H ₂ O、0.05g CuSO ₄ ·5H ₂ O and 0.50g CaCl ₂ ·2H ₂ O was dissolved in a proper amount of distilled water, and then distilled water was used to fix the volume to 1L.

Solution IV：2％(w/v)Fe(NH ₄ ) ₂ (SO ₄ ) ₂ ·6H ₂ O solution.

The names and nucleotide sequences of the primers referred to in the following examples are shown in Table 1.

TABLE 1

EXAMPLE 1 cloning of Acrpn4 Gene and amino acid sequence analysis of Acrpn4 protein

1. Cloning of Acrpn4 Gene

1. Extracting total RNA of the Cephalosporium acremonium C10 strain, and then carrying out reverse transcription to obtain cDNA of the Cephalosporium acremonium C10 strain.

2. Using cDNA of Cephalosporium acremonium C10 strain as template, adopting Acrpn4-F:5'-ATGATATCCGTCTCCCATCCA-3' and Acrpn4-R: and 5'-TCAGCCCCCACCACGTCGGCGGA-3', performing PCR amplification to obtain a PCR amplification product.

3. The PCR amplification product was sequenced. Sequencing results show that the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO: 1.

SEQ ID NO:1 is the nucleotide sequence of Acrpn4 gene. The Acrpn4 gene codes Acrpn4 protein, and the amino acid sequence of the Acrpn4 protein is shown in SEQ ID NO: 2. The theoretical relative molecular mass of the AcRpn4 protein is 70KDa.

2. Amino acid sequence analysis of AcRpn4 protein

The C-terminus of the AcRpn4 protein contains three zinc finger domains. Blast alignment was performed on the amino acid sequence of the AcRpn4 protein. The results show that the amino acid sequence of the AcRpn4 protein is very conserved in fungi; in particular, the three zinc finger domains are highly conserved in fungi, with 61% homology to the Tolypocladiumophioglossoides CBS 100239 source, 49% homology to the Saccharomyces cerevisiae source, 62% homology to the metarziumamiosaliaearsef 549 source, and 56% homology to the purpeociliumililacinum source.

EXAMPLE 2 acquisition of Acrpn4 knockout mutant Strain and its use in production of cephalosporin C

1. Construction of recombinant plasmid pAgHB: acrpn4LR

1. The genome DNA of the Cephalosporium acremonium C10 strain is used as a template, and a primer pair consisting of Acrpn4-LB-F and Acrpn4-LB-R is adopted for PCR amplification, so that a 2831bp DNA fragment A at the upstream of the Acrpn4 gene is obtained.

2. The DNA fragment A was ligated to pEASY-Blunt vector (Beijing full gold Biotechnology Co., ltd.) to obtain recombinant plasmid A.

The method comprises the following specific steps: connecting the DNA fragment A with a pEASY-Blunt vector, transforming, coating on an LB solid plate, and culturing overnight at 37 ℃; picking up the transformant and inoculating the transformant in 3mL of LB liquid medium, culturing overnight at 37 ℃ under shaking at 220rpm, and extracting plasmids to obtain recombinant plasmids A.

3. The recombinant plasmid A was sequenced. Sequencing results show that the recombinant plasmid A contains SEQ ID NO:3, and a DNA molecule shown in 3.

4. The recombinant plasmid A was digested with restriction enzymes KpnI and ApaI, and 2831bp upstream fragment was recovered.

5. Vector pAgHB (Li J, pan Y, liu G.Disruption of the nitrogen regulatory geneAcareAinAcremoniumchrysogenumleads to reduction of cephalosporin production and repressionof nitrogen metapolism.Fungal Genet.biol.2013, 61, 69-79.) was digested with restriction enzymes KpnI and ApaI, and a 7.9kb vector backbone 1 was recovered.

6. The upstream fragment and the vector backbone 1 were ligated to give the recombinant plasmid pAgHB:: acrpn4L.

7. The genome DNA of the Cephalosporium acremonium C10 strain is used as a template, and a primer pair consisting of Acrpn4-RB-F and Acrpn4-RB-R is adopted for PCR amplification, so that a 2951bp DNA fragment B downstream of the Acrpn4 gene is obtained.

8. And (3) connecting the DNA fragment B with the pEASY-Blunt vector to obtain a recombinant plasmid B.

9. Sequencing the recombinant plasmid B. Sequencing results show that the recombinant plasmid B contains SEQ ID NO:4, and a DNA molecule shown in the following formula.

10. The recombinant plasmid B was digested with the restriction enzymes AscI and PacI, and a 2951bp downstream fragment was recovered.

11. The recombinant plasmid pAgHB::: acrpn4L was digested with restriction enzymes AscI and PacI, and the vector backbone 2 of about 10.75kb was recovered.

12. The downstream fragment and the vector backbone 2 were ligated to give the recombinant plasmid pAgHB:: acrpn4LR.

The recombinant plasmid pAgHB:Acrpn 4LR was sequenced. According to the sequencing result, the structure of the recombinant plasmid pAgHB:Acrpn4LR is described as follows: the DNA fragment between the restriction enzyme kpnl and ApaI recognition sites of vector pAgHB was replaced by the DNA fragment of SEQ ID NO:3, the DNA small fragment between the recognition sites of restriction enzymes AscI and PacI is replaced by the DNA fragment shown in SEQ ID NO:4, and the recombinant plasmid obtained.

The recombinant plasmid pAgHB contains about 3kb homology arms on the upstream and downstream of the Acrpn4 gene in the Acrpn4LR, and is used for knocking out the Acrpn4 gene through homologous recombination.

2. Acquisition and identification of Acrpn4 knockout mutant strains

1. The recombinant plasmid pAgHB:: acrpn4LR was introduced into the Cephalosporium acremonium C10 strain by the Agrobacterium-mediated transformation (ATMT) method (described in the following literature: mullins ED, chen X, romain P, raina R, geiser DM, kang S. Agrobacterium-mediated transformation of Fusariumoxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology2001, 91 (2): 173-80) and subjected to homologous recombination to obtain several recombinant Cephalosporium acremonium strains. The method comprises the following specific steps:

(1) The recombinant plasmid pAgHB: acrpn4LR was transformed into Agrobacterium competent cells to obtain recombinant Agrobacterium.

(2) After the step (1) is completed, the recombinant agrobacterium monoclonal is inoculated in a 3mLMM culture medium, and is subjected to shaking culture at 28 ℃ and 220rpm for 2d, so as to obtain a culture solution 1.

(3) After the step (2) is completed, the culture solution 1 is diluted by an IM culture medium to obtain OD ₆₀₀ A dilution with a value of 0.2-0.3; then the diluted solution is subjected to shaking culture at 28 ℃ and 220rpm for 6 to 8 hours to obtain OD ₆₀₀ A value of about 0.6.

(4) Inoculating the Cephalosporium acremonium C10 strain on an LPE plate, and culturing for 7d at 28 ℃; then 3mL of sterile ddH was added ₂ O, scraping spores and hyphae by using a cotton swab, and collecting a mixed solution of the spores and the hyphae into a centrifuge tube (with the specification of 1.5 mL) by using a sheared sterile gun head; finally, centrifuging, discarding the excess ddH ₂ O, to obtain spore suspension.

(5) After the step (4) is completed, uniformly mixing the spore suspension and 500 mu L of culture bacteria liquid 2 to obtain a mixed liquid; then, the mixture was spread on a CM plate and cultured upside down at 24℃for 3d to obtain a co-culture.

(6) After completion of step (5), the co-cultures were transferred to TSA screening plates and incubated at 28℃for 3-5d until transformants were present on the TSA screening plates.

(7) After the completion of step (6), the transformants were individually transferred to TSA screening plates, cultured at 28℃and positive transformants (Hyg) grown on hygromycin were selected ^R ) Photocopying is transferred to a bleomycin plate; the bleomycin-sensitive transformant is recombinant Cephalosporium acremonium.

The homologous recombination is schematically shown in FIG. 1A.

2. Identification of recombinant Cephalosporium acremonium

(1) And respectively taking the genome DNA of the recombinant cephalosporium as a template, and adopting a primer pair consisting of Acrpn4-out-F and Acrpn4-out-R to carry out PCR amplification to obtain a corresponding PCR amplification product 1.

(2) And respectively taking the genome DNA of the recombinant cephalosporium as a template, and adopting a primer pair consisting of Acrpn4-in-F and Acrpn4-in-R to carry out PCR amplification to obtain a corresponding PCR amplification product 2.

If the PCR amplified product 1 of a certain strain contains a DNA fragment with the size of 2.1kb and does not contain a DNA fragment with the size of 2.6kb, and the PCR amplified product 2 contains a DNA fragment with the size of 1.1kb, the strain is a Cephalosporium acremonium C10 strain;

if the PCR amplification product 1 of a strain contains a DNA fragment of 2.6kb in size and does not contain a DNA fragment of 2.1kb in size, and the PCR amplification product 2 does not contain any DNA fragment, the strain is an Acrpn4 knockout mutant strain.

One Acrpn4 knockout mutant strain (abbreviated as ΔAcrpn4 or Acrpn4 DM) was selected for subsequent experiments.

The results of the identification of Acrpn4DM are shown in FIG. 1B (PCR 1 is PCR amplification product 1, PCR2 is PCR amplification product 2, C10 is Cephalosporium acremonium C10 strain, NC is blank).

3. Comparison of Cephalosporium acremonium C10 Strain and Cephalosporium C yield and cell dry weight in ΔAcrpn4

The strain to be tested is Cephalosporium acrtn C10 strain or DeltaAcrpn 4.

Experiments were independently repeated 3 times, two replicates per strain, and the results averaged. The method comprises the following specific steps:

1. the strain to be tested is inoculated on an LPE plate and is subjected to stationary culture at 28 ℃ for 7-10 days.

2. After the step 1 was completed, spores of the strain to be tested on 3 LPE plates were scraped and inoculated into a triangular flask (250 mL in specification) containing 40mL of MDFA medium, and shake-cultured at 28℃and 220rpm for 2 days to obtain a seed solution.

3. After completion of step 2, 4mL of the seed solution was inoculated into a triangular flask (250 mL in size) containing 40mL of MDFA medium, and cultured at 28℃for 7 days with shaking at 220 rpm. 1mL of fermentation broth was taken daily for detection of cephalosporin C content and dry weight of the cells.

(1) HPLC (high Performance liquid chromatography) detection of cephalosporin C content in fermentation liquor

(1-1) A sterile centrifuge tube (1.5 mL) was taken, 10mg of cephalosporin C standard was added, followed by ddH ₂ O (sterile) dissolving, and then ddH ₂ O (sterile) was fixed to 1mL and shaken well to prepare cephalosporin C mother liquor at a concentration of 10 mg/mL. The cephalosporin C mother solution is diluted with water to obtain cephalosporin C standard solutions with the concentration of 0.75 mg/mL, 1.00 mg/mL, 2.50 mg/mL, 5.00 mg/mL, 7.50 mg/mL and 10.00mg/mL, respectively. The peak areas of different concentrations of cephalosporin C standard solution were measured by HPLC and repeated 3 times. And drawing a cephalosporin C standard curve by taking the concentration of cephalosporin C as an abscissa and the peak area as an ordinate.

The instrument used for HPLC detection was Waters ACQUITY UPLC H-Classsystem with an ultraviolet detector. The detection conditions were as follows: ACQUITY UPLC BEH AmideColumn the number of the individual pieces of the plastic,1.7 μm,2.1 mm. Times.100 mm;98-10% CAN (acetonitrile) in H ₂ O over 12min；0.2mL/min；25℃。

The cephalosporin C standard curve is: y=5658.4x+61483 (R ² = 0.9937); where y is the peak area and x is the cephalosporin C concentration.

(1-2) collecting fermentation broth, centrifuging, and collecting supernatant.

(1-3) detecting the supernatant by HPLC, obtaining the content of cephalosporin C in the supernatant according to the corresponding peak area and cephalosporin C standard curve, and further obtaining the content of cephalosporin C in the fermentation broth.

The results are shown in FIG. 2 (C10 is the strain Cephalosporium acremonium C10). The results show that compared with the strain Cephalosporium acrn C10, the content of cephalosporin C in the fermentation liquor of the DeltaAcrpn 4 is obviously increased; the content of the DeltaAcrpn 4 reaches the highest value at the 6 th day of fermentation, and is 2.4g/L; the content of cephalosporin C in the strain Cephalosporium acremonium C10 reaches the highest value at the 5 th day of fermentation, which is 0.789g/L. As a result, the yield of cephalosporin C in DeltaAcrpn 4 was increased by 2 times as compared with the Cephalosporium acremonium C10 strain.

(2) HPLC detection of dry weight of thallus in fermentation liquor

(2-1) collecting fermentation broth, centrifuging, and collecting thalli.

(2-2) taking the cells collected in the step (2-1), first using ddH ₂ O flushing, then placing in a blast drying box, and drying at 80 ℃ to constant weight.

(2-3) taking the thalli obtained in the step (2-2), and calculating the dry weight of the thalli in the fermentation liquid.

The results are shown in FIG. 3 (C10 is the strain Cephalosporium acremonium C10). The results showed a slight increase in dry weight of cells in the fermentation broth of Δacrpn4 compared to the strain of cefprozil C10.

4. Comparison of growth and survival states of Cephalosporium acremonium C10 Strain and DeltaAcrpn 4

The strain to be tested is Cephalosporium acrtn C10 strain or DeltaAcrpn 4.

1. The strain to be tested is inoculated on an LPE plate and is subjected to stationary culture at 28 ℃ for 7-10 days.

2. After the step 1 was completed, spores of the strain to be tested on 3 LPE plates were scraped and inoculated into a triangular flask (250 mL in specification) containing 40mL of MDFA medium, and shake-cultured at 28℃and 220rpm for 2 days to obtain a seed solution.

3. After the completion of step 2, 4mL of the seed solution was inoculated into a flask (250 mL in size) containing 40mL of MDFA medium, and the culture was performed at 28℃and 220rpm for 6 days with shaking, to obtain a fermentation broth.

4. And 3, after the step is finished, taking fermentation liquor, centrifuging and collecting thalli.

5. After the step 4 is completed, the thalli is taken and washed 3 times by PBS buffer solution (the aim is to wash out impurities on the thalli), and a thalli sample is obtained.

6. After the step 5 is completed, a small amount of thallus sample is placed on a glass slide, 5-10 mu LPI (propidium iodide) staining solution is dripped on the thallus, and the thallus is stained for 5-10min at room temperature.

PI mother liquor: with 1mL ddH ₂ O dissolves PI to give a PI mother liquor with a concentration of 5 mg/mL.

PI staining solution: taking a proper amount of PI mother liquor, using ddH ₂ O was diluted to give a PI staining solution with a concentration of 20 mg/mL.

7. After completion of step 6, the cells were rinsed 2 times with PBS buffer, and then the staining of the cells was observed under a 40X fluorescence microscope.

The observation results are shown in FIG. 4 (C10 is the strain Cephalosporium acrn C10, acrpn4DM is DeltaAcrpn 4, BF is the normal light mode, that is, the image of the field of view observed in the bright field and in the absence of fluorescence, PI is the image of the field of view observed in fluorescence after propidium iodide staining, and Merge is the superposition and merging effect, that is, the image of the bright field of view and the image of the fluorescent field of view are merged into one image). The results show that PI staining fluorescence of Δacrpn4 is reduced, i.e. the Δacrpn4 growth time is prolonged, compared to the strain of cefprozil C10. Thus, acrpn4 protein has an effect on the growth of the bacterial cells, namely, the knockout of Acrpn4 gene can slow down the death rate of the bacterial cells.

<110> institute of microorganisms at national academy of sciences

<120> genetically engineered bacterium for producing cephalosporin C and method for constructing the same

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1911

<212> DNA

<213> Cephalosporium acremonium Cephalosporium acremonium

<400> 1

atgatatccg tctcccatcc accataccta cgccaggatc cacacaacaa ccaggaccag 60

cagcaaaacg ccaacggccg gcccgcatac ccttctcaac acagcctctc gtcgagccag 120

tcttcacatt acccgacgac agctccgtcg ttcgacctcg atcacatcaa ctctctagcc 180

actggtgctc ttcccaacgc cccgatgcag gcggacgcct ggggtaacct tgcgcaatac 240

cagcctgacc agttgggacg tctcccgcac cagcgcgagt cttcgctctc atccatgggc 300

tcgactgggc cagcctcgcc ctacaaccag aacatctcga accctcagat cgccatcacc 360

gactccacag gggaggagct ttccgacatg cacgcgcagg acgttggcag ccagaacagc 420

tcgttctacc agctggccaa gtcggctggc aacccgtact cgggttacca gaacttcgac 480

cagacagtcc ctgagatggc gtatcccgtc accatccaag ggccccgaag caagccccgc 540

accgaccgcg gtcttttgcc cgcggccgaa cttgccggca gttccaacag gtcccatccc 600

gcgtcggtgg cgagctcttt gacgggcgac tcgcctgcga ctccatcggc cggtggcgag 660

aacgacgcag ccgagaagcg cagaaaaggt aggcaccctt ttgctcgaac ggaagcggcc 720

agcgagagct cactattccc ccgtctagaa ggctatggga atgtccctaa gctcgaccgc 780

accctgaccg acgcgtacgg tgatgaactg tacagtccca acttcaccat tacttccacg 840

cccccaagtc ggcctcagca accatccact gcttctccgc caaacgatat cttcagccag 900

cgccttaacg ctgcaaacag ccaacatctg agcatcggga actcgccagc ctcccgcgac 960

aggtcatcgc cgttccgcgc gctgtcgccc tttgcgaact ctgccaacca cgacttcgca 1020

acctctcaag gcgccacgct aaatagcgcc cagcggatgc gggagcagtc caaggcccag 1080

caagacgcac agatgttcag gcaacgggtg gcgccgggaa ctgagcccga gaccccgaag 1140

accatctccc ccaaggacgc cattctagag ttcaatgagc ctgaggagac cgccgacttc 1200

cctctgttcc ctccgcactc ggccaacttc aatatggatc agctggccaa gatgatgccc 1260

ccccaggggc accatcagcc atttatggcc gacgggggaa acgggggaca aggtgaccgg 1320

ttcaactatc taccatcaca tatgcccact ggtattcagg tccctcagca atacccgttc 1380

gtcgcccgga tgcagcccaa ctccgatcct tcgccgccgc gcctcagtac gtctggctct 1440

agcccggcct cgggcggtag cactccggcg tatttggcgc gcccccctgg aactgctgcc 1500

gatggaggga catacacctg cacgtaccac ggctgcaccc tccgattcga aacaccagca 1560

cttctgcaga agcacaagcg ggagggtcac aggcagagcc atgggttggc ggctccccgg 1620

ccgcatgaaa cgggtatgac gtcgagcctg gtgaacagcc aggctggccc gcatcgttgc 1680

gatcgcatca acccgagcac cggcaagccg tgtcagacag tcttctcgcg accctacgat 1740

ctcacgaggc atgaggacac catccacaac gcccgcaagc agaaggtccg atgcgacctc 1800

tgcacagaag agaagacatt cagccgtgcg gacgcattga cacggcacta cagggtgtgc 1860

caccccgaca tggagctgcc aggaaagctc cgccgacgtg gtgggggctg a 1911

<210> 2

<211> 636

<212> PRT

<213> Cephalosporium acremonium Cephalosporium acremonium

<400> 2

Met Ile Ser Val Ser His Pro Pro Tyr Leu Arg Gln Asp Pro His Asn

1 5 10 15

Asn Gln Asp Gln Gln Gln Asn Ala Asn Gly Arg Pro Ala Tyr Pro Ser

20 25 30

Gln His Ser Leu Ser Ser Ser Gln Ser Ser His Tyr Pro Thr Thr Ala

35 40 45

Pro Ser Phe Asp Leu Asp His Ile Asn Ser Leu Ala Thr Gly Ala Leu

50 55 60

Pro Asn Ala Pro Met Gln Ala Asp Ala Trp Gly Asn Leu Ala Gln Tyr

65 70 75 80

Gln Pro Asp Gln Leu Gly Arg Leu Pro His Gln Arg Glu Ser Ser Leu

85 90 95

Ser Ser Met Gly Ser Thr Gly Pro Ala Ser Pro Tyr Asn Gln Asn Ile

100 105 110

Ser Asn Pro Gln Ile Ala Ile Thr Asp Ser Thr Gly Glu Glu Leu Ser

115 120 125

Asp Met His Ala Gln Asp Val Gly Ser Gln Asn Ser Ser Phe Tyr Gln

130 135 140

Leu Ala Lys Ser Ala Gly Asn Pro Tyr Ser Gly Tyr Gln Asn Phe Asp

145 150 155 160

Gln Thr Val Pro Glu Met Ala Tyr Pro Val Thr Ile Gln Gly Pro Arg

165 170 175

Ser Lys Pro Arg Thr Asp Arg Gly Leu Leu Pro Ala Ala Glu Leu Ala

180 185 190

Gly Ser Ser Asn Arg Ser His Pro Ala Ser Val Ala Ser Ser Leu Thr

195 200 205

Gly Asp Ser Pro Ala Thr Pro Ser Ala Gly Gly Glu Asn Asp Ala Ala

210 215 220

Glu Lys Arg Arg Lys Gly Arg His Pro Phe Ala Arg Thr Glu Ala Ala

225 230 235 240

Ser Glu Ser Ser Leu Phe Pro Arg Leu Glu Gly Tyr Gly Asn Val Pro

245 250 255

Lys Leu Asp Arg Thr Leu Thr Asp Ala Tyr Gly Asp Glu Leu Tyr Ser

260 265 270

Pro Asn Phe Thr Ile Thr Ser Thr Pro Pro Ser Arg Pro Gln Gln Pro

275 280 285

Ser Thr Ala Ser Pro Pro Asn Asp Ile Phe Ser Gln Arg Leu Asn Ala

290 295 300

Ala Asn Ser Gln His Leu Ser Ile Gly Asn Ser Pro Ala Ser Arg Asp

305 310 315 320

Arg Ser Ser Pro Phe Arg Ala Leu Ser Pro Phe Ala Asn Ser Ala Asn

325 330 335

His Asp Phe Ala Thr Ser Gln Gly Ala Thr Leu Asn Ser Ala Gln Arg

340 345 350

Met Arg Glu Gln Ser Lys Ala Gln Gln Asp Ala Gln Met Phe Arg Gln

355 360 365

Arg Val Ala Pro Gly Thr Glu Pro Glu Thr Pro Lys Thr Ile Ser Pro

370 375 380

Lys Asp Ala Ile Leu Glu Phe Asn Glu Pro Glu Glu Thr Ala Asp Phe

385 390 395 400

Pro Leu Phe Pro Pro His Ser Ala Asn Phe Asn Met Asp Gln Leu Ala

405 410 415

Lys Met Met Pro Pro Gln Gly His His Gln Pro Phe Met Ala Asp Gly

420 425 430

Gly Asn Gly Gly Gln Gly Asp Arg Phe Asn Tyr Leu Pro Ser His Met

435 440 445

Pro Thr Gly Ile Gln Val Pro Gln Gln Tyr Pro Phe Val Ala Arg Met

450 455 460

Gln Pro Asn Ser Asp Pro Ser Pro Pro Arg Leu Ser Thr Ser Gly Ser

465 470 475 480

Ser Pro Ala Ser Gly Gly Ser Thr Pro Ala Tyr Leu Ala Arg Pro Pro

485 490 495

Gly Thr Ala Ala Asp Gly Gly Thr Tyr Thr Cys Thr Tyr His Gly Cys

500 505 510

Thr Leu Arg Phe Glu Thr Pro Ala Leu Leu Gln Lys His Lys Arg Glu

515 520 525

Gly His Arg Gln Ser His Gly Leu Ala Ala Pro Arg Pro His Glu Thr

530 535 540

Gly Met Thr Ser Ser Leu Val Asn Ser Gln Ala Gly Pro His Arg Cys

545 550 555 560

Asp Arg Ile Asn Pro Ser Thr Gly Lys Pro Cys Gln Thr Val Phe Ser

565 570 575

Arg Pro Tyr Asp Leu Thr Arg His Glu Asp Thr Ile His Asn Ala Arg

580 585 590

Lys Gln Lys Val Arg Cys Asp Leu Cys Thr Glu Glu Lys Thr Phe Ser

595 600 605

Arg Ala Asp Ala Leu Thr Arg His Tyr Arg Val Cys His Pro Asp Met

610 615 620

Glu Leu Pro Gly Lys Leu Arg Arg Arg Gly Gly Gly

625 630 635

<210> 3

<211> 2831

<212> DNA

<213> Artificial sequence

<400> 3

gtcgaatgcc gagaacagtc cgactgccag acacggtcat gcctgcggtg tctattaggt 60

agagaatact ccgaatccca gagggaaaca gccggtcact ggtatattga tggttgggtg 120

ctccggaagc tcagatgtgc gcatgaagaa cggtattgta cgagatagat gcacgataag 180

tccgccgaag cgtcggccag aactcgtgac cccaagtgag tacggcagta ttagctactg 240

ggtgtcccaa caccgaaata ttatacaata ctacacaagt acaggataca tgtactgcag 300

accacggcca gaagaggcca atactccatc cgtactctgg actaaggcgt gtgcactatc 360

tggcgagctg gggaaagggt cgggacccct cggatcatcg gatgtcttcc cgaacctacc 420

aataaccgta aaacccgacc cgttgcgcat tgcactcaca gccgcgcagt tagcgaccta 480

gctgccaagt cgctagcagg cggcttcggc ggcatgcccg gtgcggccca ggcgtaaatt 540

cgatagactt gcgactctgc gaggaaccct atacgagaat ctcactggcc acctactagt 600

acatgctaca tgtacctaga accagagggc cggatctgcg gtcaggtgcc gggctgcacg 660

aattgtggcg cagccctcca acctcgagtc atgattggat gaatctccga tgcgtgcgcc 720

aaacaacttt gagcaccctt cttccagacc ttggaaaatc tggaaagcga atcaactaga 780

gccgaccgga atgcccgatc aatttttttt ttcaccaaaa attctgcctg gacctgcatc 840

tgtcaacacc taccgcggat atgcactccg accctatcaa tcattgcggt gcatctaggc 900

aggtcaaaaa ttttcacggt cgccgcactc gtgtattgac cgactgtgtt gaggcttgct 960

tgactcacgg caggagcagc ctgtcaatgc caagctgctc cacttgagtc gtgaggttaa 1020

tgtgcagtgg tggccattga gagtagcagt gactgttaag tcatgcggtc aggccactgc 1080

ggtccagaat catcccacgc aaccgcccag gctacgagat accacgacac aacgatacat 1140

acgagcggac ggtactgtat ctctgtaggc cgcggtcacg cgcactgcgg cacgcggtcg 1200

tggttcgtgg ctcgtggtcg gaggaatcat tcgagtctcg agttgccgat gcccaacact 1260

cgtatgtgaa tgccaagtac tgtacctagg tagggactcg gagtgacttc taggatgctt 1320

cgtttgcttc acttggccga gattgattgc acgcaagcgg acggggagaa ttattcgggg 1380

gtcgctcggc gagctggaat ccgccggcac acacacacac acacacacac acacacacct 1440

tggtgactcc gttgtttttt cggacagagt tagggccgga ataatcataa caaatcaccc 1500

ccaatgtaca aggtagtgcg gtaaggtagt tgctgataca ttcccaacac ctttgccatc 1560

ggcataacga ggttgtcgct accttagtaa ctaacgcgaa cgccgagtta tggcctttga 1620

tattgcttac gtgtgcaagc cggatataag gcggaggccg gccatgccgt agaatgtggc 1680

agaagtgtgg ctgcccaaac attagtaacc caacgagagc tctttttcag ccggtttcgg 1740

gtggctgagc accagaaggg cttcggttcc gggacgggaa ccaataatcg tcgcggtgat 1800

acgtcacgcc cgtcactcgt tcgcaacttg tcagtaccca acggacaatc aatcgggtct 1860

atgcatcatt gacccgcaga cgactctgct aaatccatga tgtgtacgga cgtacttacg 1920

gccgttaaat ccatctggag accagctcac agctcacggg gaaaggaaca ctagacggtg 1980

ctgagcgatg catctccgga tcatatcgca tcgccagctg agttgttagg tagagagcat 2040

tgtcgtacct aagtatgttg atgcacacac agccttggac gccggtagtg gattcgcgtc 2100

ttgcccggcg acgtcgtcga tgcggccatc ggcttgcgag gggacagtgt aatatatatc 2160

atccgtcgct aacagccaaa tgtctggcgc gggaagttga tggaatgctc tggtgtcttc 2220

cactacctca cttcgtggct ttcatgggat acggatacgc agccttccac ctttacggcc 2280

aaggagtcgc cgacgtaggt aaaatgtaca ccgggcgtct gaattgccag ccgatgacat 2340

tcggatcagc ttgccaaagt gatatgtaac tgtagtctaa gtcgtttaca gccgggtgct 2400

tacctcgtac cagcgggaca gtatttaatt cgggggccgg attgcggatg gaggccatat 2460

gatgcgggta agtgcttcgg cttccaaagg actcatctgt gtgggtgtgg gtggggaacc 2520

tgaaaagcct ctgacggagc gtaggatgca ccccacccca cacccccctt aagacacggt 2580

gatcggcgca tgcttttggg tgctctaagt actgtagttc gtacggtacg gggtggggtg 2640

cgtggatgac agtgccgtca ctaagaggtg cgtgcctgtc cggtcccccg agagggctgt 2700

ccggggccgc cggcgtgttg tcatcatggc cctataatac aagcactgtc ctcccaaatc 2760

tttttcgcca cacgatccta actctaaact cgatcttcca acttatttca tctcctgctt 2820

tttgaggcat c 2831

<210> 4

<211> 2951

<212> DNA

<213> Artificial sequence

<400> 4

tttacctccc ttggcgcacg gttcctttcc cccgccttcc catcatcctt ggctgctgag 60

gaacgacatg cacacaaaca tcatttacga ccggccgacg gccgtgaagg gagcgctgat 120

cccccagggc cctgagacag ggccactaac acgttggtta ttggggtttc cctcgaagga 180

acaaggcaac gttagtgggg tggcaatgat gccccatgac gtggacgggc tatttgtggt 240

tcggcatcat ctgtccaatc acaaccggag attggtctga gtggggcaca gggtcctgca 300

gggtccggaa tggacacaat caccgcctgc tgggtgacgt cactgccatc ctcctgtctc 360

cccgctgaca tcacgccgcc tggccgttcg cgacaaggag ccggaaatct gtttgacccg 420

ctgaacattc ctgctacctg acgtcagtcg atctcacatt tcaaccgcga cccattcgct 480

gttattacct actcctgagc gcatatccat attggcgcta gtgtggccgg ttcgggaatg 540

agccgtctct ctactattac gacactcgca gcctcccggc gtccccattt tcttacggcg 600

ttatgatccc ttcacgattt tcatggcggt atagaaacaa gtgtgcctta ttatttgctg 660

ttctttttat agttttcttt gcacctccta ttgcttccag agcggcacac agcttgtttt 720

ctggtctagg ttgcgatgtg atgatagtca acaatttcga gcgagcgggt gttcgggttc 780

cacacaaaat cgggttagaa aacgaagcga atttgtttta atctaaatgg agagaggtta 840

cggtctagct ttgcaaaaag aaaataaaga gagacgagcg ggtaacgcgg atgggtagtg 900

tattatagtc ctaatttgta taataccggc caggcacaca ggccctagag tcgacagcat 960

cgaagaactg aatttcaagc attcgacgga atcatcatcg tcctgcatgt gaaccagtgt 1020

agagcagcag agaaggagct gtgcatggat tcaacgctgg tgatgtcaag gatctcagtg 1080

cgaaccgtac cttgtagcat ttggctgccc aggatgatat catcatccca cctttgcttg 1140

taccagccgc agtatcgcga atgtatatgt aatatatccc agtggaaatc ctggactgat 1200

gagatcacac ttctgcccac ttccgttccg atgcaatact agtttgtcag gagagatctc 1260

atccaccttg gaggatgctc gctccatttt gagtaggatt gttgttgcta ccttcttgcg 1320

tggagtgccg gaggacggtg ggatgtgttg acgtattgtc ggtaagccag ccacaggccg 1380

acgttcatct gtgtgtccaa ctacatgcat tgccggtcaa agcggagact cgagccaggg 1440

ctgggggcga cttgaaatca gttttgaggg agaccaaaat gataaacaga ggcgagtcgg 1500

cagcgtctga atatacacct ccttcctttg ctgtgcagtt gtctcgtaag aggcacttgg 1560

gtcgctttgg tgggatcaag taggcgatcg tcgtggaagt atagtcgaga actcaactga 1620

ctttttattg caacttttac ttttcctttc taagccaaat cataccatct ttccatatcg 1680

cgcccccgta aacgtgcgtt ctgatgtcgt gcgtgactta tcgtatcgtt gccggagacg 1740

ggtagcatga gccgaaatcc ctgtcggctt ccattggcag actaaggtgt tcagtcatga 1800

gaagcttctc tgcccagaac agcgctgcga gcatcttcgt acacgggtgt gattcctgtc 1860

catgcattga actggatcga tgagcgttag cgagccgcgg agagcatgag gatcggtgcg 1920

accaacctgg taccacccct gagaagacaa aacttcgagc ccaggaatcg ttgtccactt 1980

gccagcctcc tcagccattt gcatggcagg agtctggcgc ggtttatatg ccatttccag 2040

aaggacacga gaatcatctt gtgcgggttg tttgttcagc gcaacagcta gaacctcgcg 2100

cagcttggga tcgatcggcc tgtctgctgg aatggtgctg ataattacac caggacctgc 2160

agccaaacct tcacagtcgg atgtgctcga gagaacctga agcccgtagc cggcggggaa 2220

gtccgatatg agcgcctttg ccttgaccgg gtcccgcgca gcaacgtgga ttgggttgaa 2280

acccatcgag tgcagcgcaa agatggcggc tcgggtcgtg cctccagagc cgaccaccat 2340

ggccggcccg ccgtcatctc gctgtacaac tcctgccctc cgtagaacat agaccatgcc 2400

cttccagtca gtattgtctc cgagcaagcg ccgcccattt ctgctgccat cctctccgac 2460

cggtattacg gtattgacgg cgccaatggt gcgcgccgcc tccgtgagat catcaacacg 2520

gttcatgatg tctattttga gtggaatagt caccgaagcg ccgccgaaat cggggctctg 2580

gagaaccgcg tccacgtcag caatatcgtc tgtttccagg cggtggtact tgtgtggaag 2640

gccggcctgc tcaaagagcg tgttgtgaag agccggcgag cgagaggcag aaatgggttt 2700

cccgaacaga tagaaattct tcggctcggt ttctcccagc agggctaggc cctggttgat 2760

gtctgccgca gacagctgcc cgggagctgc cttgaagggg aggtccggat gcgacacagg 2820

ggtgagaaat ccgttcaaga tccgactgag cctgccggct gttcccatgt tgagagcaat 2880

catcggcgtc ttttgggcag ccagcatctt ggccttgaac ctcgccaggt caaagttgtc 2940

ctccaccgac t 2951

Claims (9)

1. A method for preparing an engineered bacterium for the production of cephalosporin C comprising the steps of: reducing the expression quantity and/or activity of AcRpn4 protein in the cephalosporans, thereby obtaining the engineering bacteria for producing cephalosporin C;

the amino acid sequence of the AcRpn4 protein is shown as SEQ ID NO:2 is shown in the figure;

the Cephalosporium acremonium is Cephalosporium acremonium C10 strain.

2. A method for prolonging the survival time of cephalosporium acremonium, comprising the following steps: reducing the expression level and/or activity of the AcRpn4 protein of claim 1 in the cephalosporanic fungus of claim 1.

3. A method according to claim 1 or 2, characterized in that: the 'reducing the expression quantity and/or activity of the AcRpn4 protein in the cephalosporanges' is realized by knocking out the coding gene of the AcRpn4 protein in the cephalosporanges.

4. A method as claimed in claim 3, wherein: the nucleotide sequence of the coding gene of the AcRpn4 protein is shown as SEQ ID NO: 1.

5. A method as claimed in claim 3, wherein: the coding gene of the AcRpn4 protein is knocked out in a homologous recombination mode;

the homologous recombination fragment contains an upstream homology arm and a downstream homology arm of the coding gene of the AcRpn4 protein;

the length of the upstream homology arm and the downstream homology arm of the coding gene of the AcRpn4 protein is 2-4kb.

6. The method of claim 5, wherein:

the nucleotide sequence of the upstream homology arm of the coding gene of the AcRpn4 protein is shown as SEQ ID NO:3 is shown in the figure;

the nucleotide sequence of the downstream homology arm of the coding gene of the AcRpn4 protein is shown as SEQ ID NO: 4.

7. An engineered bacterium for the production of cephalosporin C produced by the process of claim 1.

8. The use of the engineering bacteria of claim 7 for producing cephalosporin C or cephalosporin C upstream and downstream products.

9. A method for producing cephalosporin C comprising the steps of: fermenting and culturing the engineering bacteria of claim 7, and collecting fermentation products to obtain cephalosporin C.