CN113493745B - Genetically engineered bacteria that produce cephalosporin C and their construction methods - Google Patents

Abstract

The invention discloses genetically engineered bacteria that produce cephalosporin C and their construction methods. By knocking out the Acrpn4 gene in the Cephalosporium acremonium C10 strain (a high-yield strain of cephalosporin C), the present invention greatly improves the production of cephalosporin C and enhances the survival ability of the bacteria during the fermentation process, thereby reducing the risk of cephalosporin C. The production cost of element C is of great significance. The invention has important application value.

Description

Genetically engineered bacteria that produce cephalosporin C and their construction methods

Technical field

The invention belongs to the field of microorganisms, and specifically relates to genetically engineered bacteria that produce cephalosporin C and their construction methods.

Background technique

Cephalosporium acremonium is an industrially produced strain of cephalosporin C (CPC). Cephalosporin C and penicillin both belong to the β-lactam antibiotics, but cephalosporin C is structurally different from penicillin. It is composed of a dihydrothiazine ring connected to a β-lactam ring, replacing the thiazole ring in penicillin. , it is precisely because of this difference that cephalosporin C has stronger stability, can kill many penicillin-resistant bacteria, and is not easily destroyed by penicillinase. Due to these advantages, cephalosporin antibiotics have become widely used anti-infective drugs in clinical practice. In the domestic pharmaceutical market, cephalosporin antibiotics account for more than 40% of anti-infective drug sales.

Contents of the invention

The object of the present invention is to provide genetically engineered bacteria that produce cephalosporin C.

The present invention first protects the method for preparing engineering bacteria for producing cephalosporin C, which may include the following steps: reducing the expression amount and/or activity of AcRpn4 protein in Cephalosporium acremonium, thereby obtaining the engineering bacteria for producing cephalosporin C. Engineering bacteria;

The AcRpn4 protein may be as follows a1) or a2) or a3):

a1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

a2) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 2;

a3) A protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the protein represented by a1) or a2).

The present invention also protects a method for prolonging the survival time of Cephalosporium acremonium, which may include the following steps: reducing the expression amount 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 AcRpn4 protein in Cephalosporium acremonium" can be achieved by homologous recombination, RNA interference or gene site-directed editing and other common methods in this field. The expression level and/or activity of AcRpn4 protein in mold.

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

The encoding gene of the AcRpn4 protein (i.e. AcRpn4 gene) can be the following J1) or J2) or J3) or J4):

J1) The coding region is the DNA molecule shown in SEQ ID NO: 1;

J2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1;

J3) A DNA molecule that has 75% or more identity with the nucleotide sequence defined by J1) or J2), originates from Cephalosporium acremonium and encodes the AcRpn4 protein;

J4) A DNA molecule derived from Cephalosporium acremonium and encoding the AcRpn4 protein that hybridizes to the nucleotide sequence defined by J1) or J2) under stringent conditions.

In any of the above methods, the gene encoding the AcRpn4 protein is deleted by homologous recombination; the homologous recombination fragment contains the upstream homology arm and the downstream homology arm of the gene encoding the AcRpn4 protein. ; The length of the upstream homology arm and the downstream homology arm of the gene encoding the AcRpn4 protein are both 2-4kb (such as 2-3kb, 3-4kb, 2kb, 3kb or 4kb).

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

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

More specifically, in the present invention, the gene encoding the AcRpn4 protein is deleted by introducing a recombinant vector into Cephalosporium acremonium. The recombinant vector can be a small DNA fragment between the restriction endonuclease KpnI and ApaI recognition sites of the vector pAgHB, which is replaced with the DNA molecule shown in SEQ ID NO: 3, and the restriction endonuclease AscI and PacI recognition sites. The small DNA fragment between the points is replaced with the DNA molecule shown in SEQ ID NO: 4 to obtain the recombinant plasmid.

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

Engineering bacteria for producing cephalosporin C prepared by any of the above methods also belong to the protection scope of the present invention.

The application of any of the above-mentioned engineering bacteria in the production of cephalosporin C or cephalosporin C upstream and downstream products also falls within the protection scope of the present invention.

The present invention also protects a method for producing cephalosporin C, which may include the following steps: fermenting and cultivating any of the above-mentioned engineering bacteria, collecting the fermentation products, and obtaining cephalosporin C therefrom.

In the above method, the fermentation culture conditions may be: 26-30°C (such as 26-28°C, 28-30°C, 26°C, 28°C or 30°C) shaking culture for 5-10d (such as 5-7d, 7 -10d, 5d, 7d or 10d). The rotation speed of shaking culture can be 200-250rpm (such as 200-220rpm, 220-250rpm, 200rpm, 220rpm or 250rpm).

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

The composition of the MDFA medium can be as follows: Dissolve 36.0g Sucrose, 3.2g DL-methionine, 7.5g L-Asparagine and 144.0mL Solution III in an appropriate amount of distilled water, and adjust the pH value to 7.4 with NaOH (need to add per liter About 3.3gNaOH), then add distilled water to make the total volume 932mL, sterilize at 121℃ for 20min; then add 20.0mLSolution I (sterilized at 115℃ for 30min), 40.0mL Solution II (sterilized at 121℃ for 20min) and 8.0mLSolution IV (filter sterilized).

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

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

Solution III: Combine 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 were dissolved in an appropriate amount of distilled water, and then diluted to 1L with distilled water.

By knocking out the Acrpn4 gene in the Cephalosporium acremonium C10 strain (a high-yield strain of cephalosporin C), the present invention greatly improves the production of cephalosporin C, enhances the survival ability of bacteria during the fermentation process, and contributes to the reduction of cephalosporin C. The production cost of C is of great significance. The invention has important application value.

Description of the drawings

Figure 1 shows the schematic diagram of homologous recombination and the identification results of Acrpn4 knockout mutant strains.

Figure 2 shows the comparison of cephalosporin C production in Cephalosporium acremonium C10 strain and ΔAcrpn4.

Figure 3 is a comparison of the dry weight of Cephalosporium acremonium C10 strain and ΔAcrpn4.

Figure 4 is a comparison of the growth and viability of Cephalosporium acremonium C10 strain and ΔAcrpn4.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

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

The test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified.

The quantitative experiments in the following examples were repeated three times, and the results were averaged.

Cephalosporium acremonium C10 strain is a strain that produces cephalosporin C and is recorded in the following documents: Liu G, CasqueiroJ, O, Cardoza RE, Gutiérrez S, Martín JF. Targeted inactivation of themecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremoniumchrysogenum C10 but not formethionine induction of thecephalosporin genes. J Bacteriol. 2001, 183( 5):1765-1772.

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

LB liquid culture medium: Dissolve 10.0g tryptone, 5.0g yeast extract and 10.0g NaCl in an appropriate amount of distilled water, adjust the pH value to 7.0 with NaOH, then adjust the volume to 1L with distilled water, and sterilize at 121°C for 20 minutes.

LB solid plate: Dissolve 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl and 12g agar in an appropriate amount of distilled water, adjust the pH value to 7.0 with NaOH, then adjust the volume to 1L with distilled water, sterilize at 121°C for 20 minutes; wait Cool to 55°C, pour into a sterile petri dish (diameter: 9cm), and cool naturally.

MM culture medium: Combine 10.0mL K solution, 20.0mL LM-N solution, 1.0mL1% (w/v) CaCl ₂ ·2H ₂ O solution, 2.5mL20% (w/v) (NH ₄ ) ₂ SO ₄ solution and appropriate amount Mix with distilled water, then add distilled water to bring the total volume to 986mL, and sterilize at 121°C for 20 minutes; when using, add 4mL of 50% (w/v) glucose solution (sterile) and 10mL of 0.01% (w/v) FeSO ₄ solution (without bacteria). The solute and concentration of K solution are K ₂ HPO ₄ 205g/L and KH ₂ PO ₄ 145g/L. The solvent is distilled water and the pH value is 7.0. The solute and concentration of the MN solution are MgSO ₄ ·7H ₂ O 30g/L and NaCl 15g/L, the solvent is distilled water, and the pH value is natural.

IM culture medium: mix 10.0mL K solution, 20.0mLM-N solution, 1.0mL1% (w/v) CaCl ₂ ·2H ₂ O solution, 2.5mL20% (w/v) (NH ₄ ) ₂ SO ₄ solution, 40.0 Mix mL MES solution with a concentration of 1 mol/L, 5.0 mL glycerin and an appropriate amount of distilled water, then add distilled water to make the total volume 986 mL, and sterilize at 121°C for 20 min; when using, add 4 mL 50% (w/v) glucose solution (sterile) and 10 mL of 0.01% (w/v) FeSO ₄ solution (sterile) and AS. The amount of AS added is 2 μL of AS solution with a concentration of 0.1 mol/L per mL of IM culture medium.

CM plate: mix 10.0mL K solution, 20.0mL LM-N solution, 1.0mL1% (w/v) CaCl ₂ ·2H ₂ O solution, 2.5mL20% (w/v) (NH ₄ ) ₂ SO ₄ solution, 40.0mL Mix MES solution with a concentration of 1 mol/L, 5.0 mL glycerin, 15 g agar and an appropriate amount of distilled water, then add distilled water to make the total volume 986 mL, and sterilize at 121°C for 20 minutes; after cooling to 55°C, add 2 mL 50% (w/v) Glucose solution (sterile), 10mL 0.01% (w/v) FeSO ₄ solution (sterile) and 4mL AS solution with a concentration of 0.1mol/L, mix well; pour into a sterile petri dish (diameter 9cm), Cool naturally.

LPE plate: Dissolve 10.0gCaCl ₂ , 1.0g glucose, 1.5gNaCl, 2.0gYeast Extract and 25.0gAgar in an appropriate amount of distilled water, adjust the pH value to 6.8 with NaOH, then dilute to 1L with distilled water, sterilize at 121°C for 20min; wait for cooling to 55°C, pour into a sterile petri dish (diameter: 9cm), and cool naturally.

TSA screening plate: Dissolve 17.0g Tryptone, 2.5gGlucose, 5.0gNaCl, 2.5gK ₂ HPO ₃ ·3H ₂ O, 3.0g Tryptone soya broth and 15g agar in an appropriate amount of distilled water, then adjust the volume to 1L with distilled water, and sterilize at 121°C for 20 minutes. ; After cooling to 55°C, add thisporin and hygromycin so that the concentration of thisporin in the system is 500 μg/mL and the concentration of hygromycin in the system is 50 μg/mL. Pour into sterile culture Dish (9cm in diameter) and allowed to cool naturally.

Bleomycin plate: Dissolve 17.0g Tryptone, 2.5gGlucose, 5.0gNaCl, 2.5gK ₂ HPO ₃ ·3H ₂ O, 3.0g Tryptone soya broth and 15g agar in an appropriate amount of distilled water, then adjust the volume to 1L with distilled water, and sterilize at 121°C Bacteria for 20 minutes; after cooling to 55°C, add bleomycin so that the concentration of bleomycin in the system is 20 μg/mL, pour into a sterile petri dish (diameter: 9cm), and cool naturally.

MDFA culture medium: Dissolve 36.0g Sucrose, 3.2g DL-Methionine, 7.5g L-Asparagine and 144.0mL Solution III in an appropriate amount of distilled water, adjust the pH value to 7.4 with NaOH (approximately 3.3gNaOH is added per liter), and then add Distill water to bring the total volume to 932mL and sterilize at 121°C for 20 minutes; then add 20.0mLSolution I (sterilized at 115°C for 30min), 40.0mLSolution II (sterilized at 121°C for 20min) and 8.0mLSolution IV (filtered and sterilized).

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

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

Solution III: Combine 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 were dissolved in an appropriate amount of distilled water, and then diluted to 1L with distilled water.

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

The names of the primers involved in the following examples and their nucleotide sequences 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. Extract the total RNA of Cephalosporium acremonium C10 strain, and then reverse-transcribe to obtain the cDNA of Cephalosporium acremonium C10 strain.

2. Use the cDNA of Cephalosporium acremonium strain C10 as a template and use the primer pair consisting of Acrpn4-F: 5′-ATGATATCCGTCTCCCATCCA-3′ and Acrpn4-R: 5′-TCAGCCCCCACCACGTCGGCGGA-3′ to obtain PCR amplification product.

3. Sequence the PCR amplification products. The sequencing results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ IDNO: 1.

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

2. Amino acid sequence analysis of AcRpn4 protein

The C-terminus of AcRpn4 protein contains three zinc finger domains. The amino acid sequence of AcRpn4 protein was blast aligned. The results show that the amino acid sequence of AcRpn4 protein is very conserved in fungi; especially the three zinc finger domains are highly conserved in fungi, with a homology of 61% with the homologous protein derived from Tolypocladiumophioglossoides CBS 100239 and the same as that derived from Saccharomyces cerevisiae. The homology of the source protein is 49%, the homology with the homologous protein derived from MetarhiziumanisopliaeARSEF 549 is 62%, and the homology with the homologous protein derived from Purpureocilliumlilacinum is 56%.

Example 2. Obtainment of Acrpn4 knockout mutant strain and its application in the production of cephalosporin C

1. Construction of recombinant plasmid pAgHB::Acrpn4LR

1. Use the genomic DNA of Cephalosporium acremonium strain C10 as a template and use the primer pair consisting of Acrpn4-LB-F and Acrpn4-LB-R to perform PCR amplification to obtain a 2831 bp DNA fragment A upstream of the Acrpn4 gene.

2. Connect DNA fragment A to the pEASY-Blunt vector (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain recombinant plasmid A.

The specific steps are as follows: connect DNA fragment A to the pEASY-Blunt vector, transform, spread on LB solid plate, and culture at 37°C overnight; pick the transformants and inoculate them into 3 mL LB liquid culture medium, and culture overnight at 37°C and 220 rpm with shaking. Extract the plasmid to obtain recombinant plasmid A.

3. Sequence the recombinant plasmid A. The sequencing results showed that recombinant plasmid A contained the DNA molecule shown in SEQ ID NO: 3.

4. Digest recombinant plasmid A with restriction endonucleases KpnI and ApaI, and recover the 2831bp upstream fragment.

5. Use restriction endonucleases KpnI and ApaI to digest the vector pAgHB (Li J, Pan Y, Liu G. Disruption of the nitrogen regulatory geneAcareAinAcremoniumchrysogenumleads toreduction of cephalosporin production and repressionof nitrogenmetabolism. Fungal Genet. Biol. 2013, 61, 69–79 .), and recovered the 7.9kb vector backbone 1.

6. Connect the upstream fragment and vector backbone 1 to obtain the recombinant plasmid pAgHB::Acrpn4L.

7. Use the genomic DNA of Cephalosporium acremonium strain C10 as a template and use the primer pair consisting of Acrpn4-RB-F and Acrpn4-RB-R to perform PCR amplification to obtain a 2951 bp DNA fragment B downstream of the Acrpn4 gene.

8. Connect DNA fragment B and pEASY-Blunt vector to obtain recombinant plasmid B.

9. Sequence the recombinant plasmid B. The sequencing results showed that recombinant plasmid B contained the DNA molecule shown in SEQ ID NO: 4.

10. Digest recombinant plasmid B with restriction endonucleases AscI and PacI, and recover the 2951bp downstream fragment.

11. Use restriction endonucleases AscI and PacI to digest the recombinant plasmid pAgHB::Acrpn4L, and recover about 10.75kb vector backbone 2.

12. Connect the downstream fragment to vector backbone 2 to obtain the recombinant plasmid pAgHB::Acrpn4LR.

The recombinant plasmid pAgHB::Acrpn4LR was sequenced. According to the sequencing results, the structure of the recombinant plasmid pAgHB::Acrpn4LR is described as follows: the small DNA fragment between the restriction endonuclease KpnI and ApaI recognition sites of the vector pAgHB is replaced with the DNA molecule shown in SEQ ID NO: 3, The small DNA fragment between the recognition sites of restriction endonuclease AscI and PacI is replaced with the DNA molecule shown in SEQ ID NO: 4 to obtain a recombinant plasmid.

The recombinant plasmid pAgHB::Acrpn4LR contains about 3 kb of homology arms upstream and downstream of the Acrpn4 gene, which can be used to knock out the Acrpn4 gene through homologous recombination.

2. Obtaining and identifying Acrpn4 knockout mutant strains

1. Use the Agrobacterium-mediated transformation (ATMT) method (documented in the following literature: Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S. Agrobacterium-mediated transformation of Fusariumoxysporum: an efficient tool for insertional mutagenesis and genetransfer.Phytopathology2001, 91(2):173-80.) The recombinant plasmid pAgHB::Acrpn4LR was introduced into the C10 strain of Cephalosporium acremonium. After homologous recombination, several recombinant Cephalosporium acremonium strains were obtained. Specific steps are as follows:

(1) Transform the recombinant plasmid pAgHB::Acrpn4LR into Agrobacterium competent cells to obtain recombinant Agrobacterium.

(2) After completing step (1), inoculate a single clone of the recombinant Agrobacterium into 3 mLMM medium, and culture it with shaking at 28°C and 220 rpm for 2 days to obtain culture liquid 1.

(3) After completing step (2), dilute culture liquid 1 with IM medium to obtain a diluted liquid with an OD ₆₀₀ value of 0.2-0.3; then incubate the diluted liquid with shaking at 28°C and 220rpm for 6-8 hours to obtain an OD ₆₀₀ value The culture bacterial liquid 2 is about 0.6.

(4) Inoculate the C10 strain of Cephalosporium acremonium on the LPE plate and culture it at 28°C for 7 days; then add 3 mL of sterile ddH ₂ O, scrape off the spores and hyphae with a cotton swab, and remove the spores and fungi with a sterile cut tip. Collect the mixture of silk into a centrifuge tube (1.5 mL specification); finally centrifuge, discard excess ddH ₂ O, and obtain a spore suspension.

(5) After completing step (4), mix the spore suspension and 500 μL of bacterial culture solution 2 to obtain a mixed solution; then spread the mixed solution on a CM plate and incubate it upside down at 24°C for 3 days to obtain a co-culture.

(6) After completing step (5), transfer the co-culture to a TSA screening plate and culture it at 28°C for 3-5 days until transformants appear on the TSA screening plate.

(7) After completing step (6), transfer the transformant single clones to TSA screening plates respectively, culture at 28°C, select the positive transformants (Hyg ^R ) growing on hygromycin, and copy and transfer to bleomycin. plate; the bleomycin-sensitive transformant is the recombinant Cephalosporium acremonium.

The schematic diagram of homologous recombination is shown in A in Figure 1.

2. Identification of recombinant Cephalosporium acremonium

(1) Using the genomic DNA of the recombinant Cephalosporium acremonium as a template, PCR amplification was performed using the primer pair consisting of Acrpn4-out-F and Acrpn4-out-R, and the corresponding PCR amplification product 1 was obtained.

(2) Using the genomic DNA of the recombinant Cephalosporium acremonium as a template, PCR amplification was performed using a primer pair consisting of Acrpn4-in-F and Acrpn4-in-R to obtain the corresponding PCR amplification product 2.

If the PCR amplification product 1 of a strain contains a DNA fragment of 2.1kb and does not contain a DNA fragment of 2.6kb, and the PCR amplification product 2 contains a DNA fragment of 1.1kb, then the strain is the top Cephalosporium strain C10;

If the PCR amplification product 1 of a strain contains a DNA fragment of 2.6 kb and does not contain a DNA fragment of 2.1 kb, and the PCR amplification product 2 does not contain any DNA fragment, then the strain is an Acrpn4 knockout mutation. strains.

An Acrpn4 knockout mutant strain (referred to as ΔAcrpn4 or Acrpn4DM) was selected for subsequent experiments.

The identification results of Acrpn4DM are shown in B in Figure 1 (PCR1 is PCR amplification product 1, PCR2 is PCR amplification product 2, C10 is Cephalosporium acremonium C10 strain, NC is blank control).

3. Comparison of cephalosporin C production and bacterial cell dry weight in Cephalosporium acremonium C10 strain and ΔAcrpn4

The strain to be tested is Cephalosporium acremonium C10 strain or ΔAcrpn4.

The experiment was repeated three times independently, with two parallels for each strain each time, and the results were averaged. Specific steps are as follows:

1. Inoculate the strain to be tested on the LPE plate and culture it at 28°C for 7-10 days.

2. After completing step 1, scrape the spores of the strain to be tested on 3 LPE plates and inoculate them into an Erlenmeyer flask (specification: 250 mL) containing 40 mL of MDFA medium. Shake and culture at 28°C and 220 rpm for 2 days to obtain a seed liquid.

3. After completing step 2, inoculate 4 mL of seed solution into an Erlenmeyer flask (specification: 250 mL) containing 40 mL of MDFA medium, and culture with shaking at 28°C and 220 rpm for 7 days. Take 1 mL of fermentation broth every day to detect cephalosporin C content and bacterial dry weight.

(1) HPLC detection of cephalosporin C content in fermentation broth

(1-1) Take a sterile centrifuge tube (1.5 mL specification), add 10 mg of cephalosporin C standard, dissolve it with ddH ₂ O (sterile), and then adjust the volume to 1 mL with ddH ₂ O (sterile) , shake well, and prepare a cephalosporin C stock solution with a concentration of 10 mg/mL. Take the cephalosporin C stock solution and dilute it with water to obtain cephalosporin C standard solutions with concentrations of 0.75, 1.00, 2.50, 5.00, 7.50, and 10.00 mg/mL. The peak areas of cephalosporin C standard solutions of different concentrations were detected by HPLC and repeated three times. Using the cephalosporin C concentration as the abscissa and the peak area as the ordinate, draw a cephalosporin C standard curve.

The instrument used for HPLC detection was Waters ACQUITY UPLC H-Classsystem with UV detector. The detection conditions are as follows: ACQUITY UPLC BEH AmideColumn, 1.7μm, 2.1mm×100mm; 98-10% CAN (acetonitrile) in H ₂ O over 12min; 0.2mL/min; 25°C.

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

(1-2) Take the fermentation broth, centrifuge, and collect the supernatant.

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

The test results are shown in Figure 2 (C10 is the C10 strain of Cephalosporium acremonium). The results showed that compared with the C10 strain of Cephalosporium acremonium, the content of cephalosporin C in the fermentation broth of ΔAcrpn4 increased significantly; the content of cephalosporin C in ΔAcrpn4 reached the highest on the 6th day of fermentation, which was 2.4g/L; Cephalosporium acremonium The content of cephalosporin C in strain C10 reached the highest level on the 5th day of fermentation, which was 0.789g/L. It can be seen that compared with the C10 strain of C. acremonium, the cephalosporin C production in ΔAcrpn4 is increased by 2 times.

(2) HPLC detection of dry weight of bacteria in fermentation broth

(2-1) Take the fermentation liquid, centrifuge, and collect the bacteria.

(2-2) Take the bacterial cells collected in step (2-1), rinse them with ddH ₂ O first, then place them in a blast drying oven and dry them at 80°C to constant weight.

(2-3) Take the bacterial cells obtained in step (2-2) and calculate the dry weight of the bacterial cells in the fermentation broth.

The test results are shown in Figure 3 (C10 is the C10 strain of Cephalosporium acremonium). The results showed that compared with the C10 strain of C. acremonium, the dry weight of bacteria in the fermentation broth of ΔAcrpn4 increased slightly.

4. Comparison of the growth and survival status of Cephalosporium acremonium C10 strain and ΔAcrpn4

The strain to be tested is Cephalosporium acremonium C10 strain or ΔAcrpn4.

1. Inoculate the strain to be tested on the LPE plate and culture it at 28°C for 7-10 days.

2. After completing step 1, scrape the spores of the strain to be tested on 3 LPE plates and inoculate them into an Erlenmeyer flask (specification: 250 mL) containing 40 mL of MDFA medium. Shake and culture at 28°C and 220 rpm for 2 days to obtain a seed liquid.

3. After completing step 2, inoculate 4 mL of seed liquid into an Erlenmeyer flask (specification: 250 mL) containing 40 mL of MDFA culture medium, and culture with shaking at 28°C and 220 rpm for 6 days to obtain a fermentation broth.

4. After completing step 3, take the fermentation liquid, centrifuge, and collect the bacteria.

5. After completing step 4, take the bacterial cells and wash them three times with PBS buffer (the purpose is to wash away impurities on the bacterial cells) to obtain a bacterial sample.

6. After completing step 5, place a small amount of bacterial sample on a glass slide, drop 5-10 μLPI (propidium iodide) staining solution onto the bacterial cells, and stain at room temperature for 5-10 minutes.

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

PI staining solution: Take an appropriate amount of PI mother solution and dilute it with ddH ₂ O to obtain a PI staining solution with a concentration of 20 mg/mL.

7. After completing step 6, rinse the bacterial cells twice with PBS buffer, and then observe the coloring of the cells under a 40× fluorescence microscope.

The observation results are shown in Figure 4 (C10 is the C10 strain of Cephalosporium acremonium, Acrpn4DM is ΔAcrpn4; BF is the image of the visual field observed in the normal light mode, that is, bright field and no fluorescence; PI is the visual field of fluorescence observation after propidium iodide staining. image; Merge is a superposition merging effect, that is, merging the image of the bright field field of view and the image of the fluorescence field of view into one image). The results showed that compared with the C10 strain of C. acremonium, the PI staining fluorescence of ΔAcrpn4 was weakened, that is, the growth time of ΔAcrpn4 was prolonged. It can be seen that Acrpn4 protein has an impact on bacterial growth, that is, Acrpn4 gene knockout can slow down the death rate of bacterial cells.

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> Genetically engineered bacteria that produce cephalosporin C and their construction methods

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1911

<212> DNA

<213> 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 gtggggggctg a 1911

<210> 2

<211> 636

<212> PRT

<213> 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

ctggggggcga 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.