CN107201318B

CN107201318B - Recombinant bacterium for producing cephalosporin C and application thereof

Info

Publication number: CN107201318B
Application number: CN201710623482.XA
Authority: CN
Inventors: 刘钢; 刘佳佳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2017-07-27
Filing date: 2017-07-27
Publication date: 2020-04-21
Anticipated expiration: 2037-07-27
Also published as: CN107201318A

Abstract

The invention discloses a recombinant bacterium for producing cephalosporin C and application thereof. The preparation method of the recombinant bacterium comprises the following steps: introducing a substance for improving the expression and/or activity of adenosylmethionine synthetase into the fermentation strain to obtain the recombinant strain. Experiments prove that by adopting the recombinant bacterium to produce CPC, the addition of methionine in a culture medium is omitted, and the yield of CPC is improved. Compared with the original strain cephalosporium acremonium CGMCC3.3795, the recombinant strain has stronger CPC synthesis capacity. The recombinant bacterium provided by the invention has important application value in the aspects of improving the yield of CPC, reducing the production cost and the like.

Description

Recombinant bacterium for producing cephalosporin C and application thereof

Technical Field

The invention relates to the field of applied industrial microorganisms, in particular to a recombinant bacterium for producing cephalosporin C and application thereof.

Background

Cephalosporin C (CPC) is a main raw material for producing 7-aminocephalosporanic acid (7-ACA), an important intermediate of Cephalosporin antibiotics. Currently, CPC is obtained by fermentation mainly using Acremonium chrysogenum in industry. The biosynthesis pathway of CPC in Cephalosporium acremonium is basically and clearly researched, and the genetic operation system of Cephalosporium acremonium is relatively mature, so that a good foundation is laid for directionally transforming the important industrial fungus by utilizing a genetic engineering means.

Methionine can significantly stimulate the production of CPC in cephalosporium acremonium, has long been used as an accelerant for the production of CPC in cephalosporium acremonium, and has been used in industrial production. With the continuous increase of the fermentation level of CPC of Acremonium acremonium, methionine becomes an important factor influencing the production cost of CPC. Therefore, it would be of great value to obtain a strain that could still increase the fermentation level of CPC without adding methionine to the medium.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the fermentation level of CPC when methionine is not added into the culture medium.

In order to solve the technical problems, the invention firstly provides a recombinant bacterium.

The preparation method of the recombinant bacterium provided by the invention can be as follows: and improving the content and/or activity of adenosylmethionine synthetase in the fermentation strain to obtain the recombinant strain.

In the recombinant bacterium, the amino acid sequence of the adenosylmethionine synthetase is shown as a sequence 2 in a sequence table.

In the above recombinant bacterium, the method for increasing the content and/or activity of adenosylmethionine synthetase in the producing bacterium may be to introduce a nucleic acid molecule encoding adenosylmethionine synthetase into the producing bacterium.

In the recombinant bacterium, the step of introducing the nucleic acid molecule encoding the adenosylmethionine synthetase into the initial bacterium can be realized by introducing a recombinant vector into the initial bacterium; the recombinant vector may be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding an adenosylmethionine synthetase into an expression vector.

In any of the recombinant bacteria, the outgrowth bacteria can be cephalosporium acremonium. The Acremonium acremonium can be specifically Acremonium CGMCC 3.3795.

The nucleic acid molecule encoding adenosylmethionine synthetase as described above can be specifically represented by sequence 1 in the sequence table.

Any of the above-described nucleic acid molecules encoding an adenosylmethionine synthetase also falls within the scope of the present invention.

Also within the scope of the present invention is an expression cassette, a recombinant vector or a recombinant microorganism comprising any of the above-described nucleic acid molecules encoding an adenosylmethionine synthetase.

The expression cassette can be expression cassette a; the expression cassette a comprises a promoter, a nucleic acid molecule encoding an adenosylmethionine synthetase and a terminator. The promoter may be a CaMV35S promoter, a NOS promoter or an OCS promoter. The terminator may be a NOS terminator or an OCS polyA terminator.

The nucleotide sequence of the expression cassette A can be shown as a sequence 4 in a sequence table. In the expression cassette a: the 1 st to 2093 th positions of the sequence 4 in the sequence table from the 5' end are promoters, the 2109 th to 3293 th positions are nucleic acid molecules for coding adenosylmethionine synthetase, and the 3319 th to 3838 th positions are terminators.

The recombinant vector may be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding an adenosylmethionine synthetase into an expression vector. The expression vector may be plasmid pAg 1-G418-P-T. The recombinant vector can be specifically a recombinant plasmid pAg 1-AcsamsoE. The recombinant plasmid pAg1-AcsamsoE can be specifically a recombinant plasmid obtained by replacing a small fragment between restriction enzymes PacI and AscI of a plasmid pAg1-G418-P-T with a DNA molecule shown in a sequence 1 of a sequence table. The recombinant plasmid pAg1-Acsamso expresses adenosylmethionine synthetase shown in sequence 2 of the sequence table.

The recombinant microorganism can be obtained by introducing the recombinant vector into the starting microorganism.

The starting microorganism may be a yeast, bacterium, algae or fungus. The bacteria may be gram positive or gram negative bacteria. The gram-negative bacterium may be Agrobacterium tumefaciens (Agrobacterium tumefaciens). The Agrobacterium tumefaciens (Agrobacterium tumefaciens) may specifically be Agrobacterium tumefaciens AGL-1.

The recombinant microorganism can be AGL-1/pAg1-Acsamso OE specifically; AGL-1/pAg1-AcsamsoE is a recombinant Agrobacterium obtained by introducing the recombinant plasmid pAg1-AcsamsoE into Agrobacterium tumefaciens AGL-1.

In order to solve the technical problems, the invention also provides a microbial inoculum which contains any one of the recombinant bacteria.

The recombinant bacterium described above, or any nucleic acid molecule encoding an adenosylmethionine synthetase described above, or an expression cassette, a recombinant vector or a recombinant microorganism comprising a nucleic acid molecule encoding an adenosylmethionine synthetase described above, or the use of the microbial inoculum for producing cephalosporin C is also within the scope of the present invention.

In order to solve the technical problems, the invention also provides a method for producing cephalosporin C, which can comprise the step of carrying out fermentation culture on any one of the recombinant bacteria to obtain cephalosporin C.

In the above method, the fermentation medium used in the fermentation culture may be a methionine-free medium or a methionine-containing medium. The fermentation medium used in the fermentation culture can be specifically an original MDFA fermentation medium or a modified MDFA fermentation medium. The fermentation culture inoculum size is 5-15% (such as 5-10%, 10-15%, 5%, 15% or 10%). The culture condition of the fermentation culture is that the fermentation culture is carried out for 100 h-160 h (such as 100 h-120 h, 120 h-144 h, 144 h-160 h, 100h, 120h, 160h or 144h) at 25 ℃ -30 ℃ (such as 25 ℃ -28 ℃, 28 ℃ -30 ℃, 25 ℃, 30 ℃ or 28 ℃).

In the above method, the "fermenting and culturing the recombinant bacterium" may further include preparing a seed solution. The steps for preparing the seed liquid are as follows: and (5) culturing the recombinant strain in a shake flask to obtain a seed solution. The shake flask culture medium used for the shake flask culture may be a methionine-containing medium. The shake flask culture medium used for shake flask culture can be specifically the original MDFA fermentation medium. The shake flask culture medium used for shake flask culture can be specifically the modified MDFA fermentation medium. The initial biomass in the shake flask culture is 1X 10⁷cfu/40mL～5×10⁷cfu/40mL (e.g., 1X 10)⁷cfu/40mL～3×10⁷cfu/40mL、3×10⁷cfu/40mL～5×10⁷cfu/40mL、1×10⁷cfu/40mL、5×10⁷cfu/40mL or 3X 10⁷cfu/40 mL). The shake flask culture inoculum size is 5-15% (e.g. 5-10%, 10-15%, 5%, 15% or 10%). The culture conditions of the shake flask culture are 25-30 ℃ (such as 25-28 ℃, 28-30 ℃, 25 ℃, 30 ℃ or 28 ℃), 180 rpm-260 rpm (such as 180 rpm-220 rpm, 220 rpm-260 rpm, 180rpm, 260rpm or 220rpm), and 40 h-60 h (such as 40 h-48 h, 48 h-60 h, 40h, 60h or 48 h).

Any of the above methionine may be particularly DL-type methionine.

The preparation method of any one of the original MDFA fermentation culture media comprises the following steps: mixing sucrose 36g, DL-methionine 3.2g, L-asparagine 7.5g, and Fe (NH)₄)₂(SO₄)₂·6H₂0.16g of O, 20mL of the solution I, 40mL of the solution II, 144mL of the solution III and 8mL of the solution IV are dissolved in distilled water, the volume is adjusted to 1L by using the distilled water, and the pH value is adjusted to 7.4.

The preparation method of any one of the improved MDFA fermentation culture media comprises the following steps: mixing sucrose 36g, L-asparagine 7.5g, and Fe (NH)₄)₂(SO₄)₂·6H₂0.16g of O, 20mL of the solution I, 40mL of the solution II, 144mL of the solution III and 8mL of the solution IV are dissolved in distilled water, the volume is adjusted to 1L by using the distilled water, and the pH value is adjusted to 7.4 to obtain the culture medium.

Experiments prove that the recombinant bacterium provided by the invention is used for producing CPC, so that the addition of methionine in a culture medium is omitted, and the yield of CPC is improved. Compared with the original strain (i.e. the cephalosporium acremonium CGMCC 3.3795), the recombinant strain has stronger CPC synthesis capacity. The recombinant bacterium provided by the invention has important application value in the aspects of improving the yield of CPC, reducing the production cost and the like.

Drawings

FIG. 1 is a schematic diagram of the construction of recombinant plasmid pAg 1-AcsamsoE.

FIG. 2 is the identification of transformants which are pseudotransgenic for the AcsamsOE gene.

FIG. 3 is a graph showing the effect of methionine on the growth of recombinant bacterium AcsamseOE-1.

FIG. 4 shows the fermentation of recombinant AcsamsoE-1 and the determination of CPC production.

FIG. 5 is a transcriptional analysis of CPC biosynthesis-related genes of recombinant bacterium AcsamseOE-1 during fermentation.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Cephalosporium acremonium CGMCC3.3795 is described in the following documents: the functional research of hexokinase coding gene Achka in Neem, Chengyun, Yuxuan, Liu, Cephalosporium acremonium, Jun Xun, 2017, 36 (3): 323-331.

Bacillus subtilis CGMCC 1.1630 is described in the following references: long L, Yang J, An Y, Liu G.Disction of a glutamatere reducing gene in Acremonium chrysogenum leads to reduction of bits growth, centrosporisorium and antibiotic activity of yield. 114-22. hereinafter, Bacillus subtilis CGMCC 1.1630 is simply called Bacillus subtilis.

Agrobacterium tumefaciens AGL-1 is described in the following documents: CH Khang, SY Park, HS Rho, YH Lee, SKang. Filamethonous fungi (Magnaporthe grisea and Fusarium oxysporum). Methods in molecular Biology, 2006, 344: 403-.

The Pfaffl's process is described in the following documents: pan Y, Wang L, He X, Tian Y, Liu G, TanH. SabR enhancement process via adjustment of the transcriptional level of sang, a path-specific regulatory gene in Streptomyces and microorganisms. BMC Microbiol.2011Jul 20; 11:164.

The vector pEASY-Blunt is a product of Beijing Quanyu gold Biotechnology Co., Ltd, and the product catalog number is CB 101. PrimeScript^TMThe RT kit and 2 XSSYBR Premix Ex Taq are both products of TaKaRa. The thermocycler Mastercycler is a product of the company Eppendorf. The penicillinase is a product of Taixi love (Shanghai) chemical industry development limited company, and the product catalog number is P0310-25 ML. CPC-Zn is a product of Sigma company and has a product catalog number of C3270.

The nucleotide sequence of the plasmid pAg1-G418-P-T (circular) is shown as the sequence 3 in the sequence table.

In the following examples, the plates and media involved are as follows:

TSA resistant plates: mixing tryptone 17g, soytone 3g, glucose 2.5g, sodium chloride 5g, and K₂HPO₃·3H₂Dissolving O2.5g and 15g of agar powder in 1L of distilled water, and adjusting the pH value to 7.0; sterilizing at 115 deg.C under high pressure for 20 min; cooling to 60 deg.C, adding G418 and ThieladinSodium, so that the concentration of G418 and the concentration of the thienamycin sodium in the system are both 200 mug/mL; finally pouring the mixture into a culture dish, and naturally cooling.

LPE plate: dissolving 10g of calcium chloride, 1g of glucose, 1.5g of sodium chloride, 2g of yeast extract and 25g of agar powder in 1L of distilled water, and adjusting the pH value to 6.8; autoclaving at 115 deg.C for 20min, pouring into culture dish, and naturally cooling.

TSA plates without methionine: mixing tryptone 17g, soytone 3g, glucose 2.5g, sodium chloride 5g, and K₂HPO₃·3H₂Dissolving O2.5g and agar powder 15g in 1L distilled water, and adjusting pH to 7.0; sterilizing at 115 deg.C under high pressure for 20 min; then pouring into a culture dish, and naturally cooling.

Methionine-containing TSA plates: mixing tryptone 17g, soytone 3g, glucose 2.5g, sodium chloride 5g, and K₂HPO₃·3H₂Dissolving O2.5g, DL-methionine 3.2g and agar powder 15g in 1L distilled water, and adjusting pH to 7.0; sterilizing at 115 deg.C under high pressure for 20 min; then pouring into a culture dish, and naturally cooling.

LB resistant plates: dissolving 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar powder in 1L of distilled water, and adjusting the pH value to 7.0; sterilizing at 121 deg.C under high pressure for 15 min; cooling to 60 ℃, and adding kanamycin to ensure that the concentration of kanamycin in the system is 75 mug/mL; finally pouring the mixture into a culture dish, and naturally cooling.

Original MDFA fermentation medium: mixing sucrose 36g, DL-methionine 3.2g, L-asparagine 7.5g, and Fe (NH)₄)₂(SO₄)₂·6H₂0.16g of O, 20mL of the solution I, 40mL of the solution II, 144mL of the solution III and 8mL of the solution IV are dissolved in distilled water, the volume is adjusted to 1L by using the distilled water, and the pH value is adjusted to 7.4 to obtain the culture medium.

Modified MDFA fermentation medium: mixing sucrose 36g, L-asparagine 7.5g, and Fe (NH)₄)₂(SO₄)₂·6H₂O0.16g, solution I20 mL, solution II 40mL, solution III 144mL and solution IV 8mL were dissolved in distilled water and made up to 1L with distilled water, and the pH was adjusted to 7.4 to obtain a medium. Modified MDFA fermentation Medium and Only the original MDFA fermentation MediumOne difference is that it does not contain DL-form methionine.

Solution I was an aqueous solution containing 50% (50mg/100mL) glucose.

The solution II is an aqueous solution containing 50% (v/v) glycerol.

Solution III is prepared by mixing K₂HPO₄·3H₂O 128.61g、KH₂PO₄·H₂O 102g、NaSO₄·10H₂O 11.5g、MgSO₄·7H₂O 2.4g、ZnSO₄·7H₂O 0.2g、MnSO₄·4H₂O 0.2g、CuSO₄·5H₂O0.05 g and CaCl₂·2H₂0.5g of O was dissolved in 1L of distilled water.

The solution IV contains 2% (2mg/100mL) Fe (NH)₄)₂(SO₄)₂·6H₂An aqueous solution of O.

LB liquid medium: dissolving 10g of tryptone, 5g of yeast extract and 10g of sodium chloride in 1L of distilled water, and adjusting the pH value to 7.0; then autoclaved at 121 ℃ for 15 min.

The formulations of MM medium, IM medium and CM plates are shown in Table 1.

TABLE 1

Note: 20% (20mg/100mL) glucose aqueous solution, 0.01% (0.01mg/100mL) FeSO₄The aqueous solution, 1MMES (pH5.3) and kanamycin sulfate aqueous solution (0.2M) were each subjected to filter sterilization and stored at-20 ℃ for use. "-" means absent.

Example 1 obtaining of recombinant bacteria

Firstly, construction of recombinant plasmid pAg1-AcsamsoE

A schematic diagram of the construction of recombinant plasmid pAg1-AcsamsoE is shown in FIG. 1. The method comprises the following specific steps:

1. extracting total RNA of cephalospora acremonium CGMCC3.3795 by using Trizol reagent, and then carrying out reverse transcription to obtain cDNA.

2. Taking the cDNA obtained in the step 1 as a template, adopting a primer SAMSOE-F: 5' -ttaattaaATGTCTGCCAACGGCATCAAGGGCGT-3' (recognition site for restriction enzyme PacI underlined) and primer SAMSOE-R: 5' -ggcgcgc cTTAGAACTTGAGAGGCTTGGGCTGCTCC-3' (recognition sites for the restriction enzyme AscI are underlined) was subjected to PCR amplification to obtain a PCR amplification product of about 1201 bp.

3. And (3) connecting the PCR amplification product obtained in the step (2) with a vector pEASY-Blunt to obtain a recombinant plasmid pEASY-samsOE.

4. The recombinant plasmid pEASY-samsOE is double-digested by restriction enzymes PacI and AscI, and a DNA fragment samsOE of about 1201bp is recovered.

5. The plasmid pAg1-G418-P-T was double-digested with the restriction enzymes PacI and AscI, recovering a vector backbone of about 8600 bp.

6. And connecting the DNA fragment samsOE with a vector skeleton to obtain a recombinant plasmid pAg 1-AcsamsOE.

The recombinant plasmid pAg 1-AcsamseOE was sequenced. According to the sequencing result, the structure of the recombinant plasmid pAg1-AcsamsoE is described as follows: the small fragment between the restriction enzymes PacI and AscI of plasmid pAg1-G418-P-T was substituted into the DNA molecule shown in sequence 1 of the sequence listing. The recombinant plasmid pAg1-Acsamso expresses adenosylmethionine synthetase shown in sequence 2 in the sequence table.

The recombinant plasmid pAg1-AcsamsoE has an expression cassette A, the nucleotide sequence of the expression cassette A is shown as a sequence 4 in a sequence table, wherein the 1 st to 2093 th sites of the sequence 4 in the sequence table from the 5' end are promoters, the 2109 th to 3293 th sites are coding genes for coding adenosylmethionine synthetase, and the 3319 th to 3838 th sites are terminators.

II, obtaining recombinant agrobacterium

The recombinant plasmid pAg 1-AcsamsoOE is introduced into Agrobacterium tumefaciens AGL-1 to obtain recombinant Agrobacterium tumefaciens, which is named AGL-1/pAg 1-AcsamsoOE.

Thirdly, obtaining of transformant of quasi-AcsamsOE gene

1. Single colonies of AGL-1/pAg1-AcsamsoE were inoculated into 5mL of MM medium and cultured at 28 ℃ for 2 days at 200rpm to give culture broth 1.

2. After the step 1 is finished, taking the culture solution 1, and diluting the culture solution to OD by using an IM (instant messenger) culture medium_600nmThe OD was 0.15, and then cultured at 28 ℃ for 6 hours at 200rpm to obtain OD_600nmAbout 0.6 of inoculum 2.

3. Mixing 100 μ L of culture solution 2 and 100 μ L of spore suspension of Cephalosporium acremonium CGMCC3.3795 (spore concentration in the spore suspension is 1.0 × 10)⁷pieces/mL) were mixed and then spread evenly on CM plates (glassine paper already spread) and co-cultured for 3d at 25 ℃ upright.

4. After completion of step 3, the co-cultured cells on cellophane were transferred to a TSA-resistant plate and subjected to inverted culture at 28 ℃ for 5-7 days. The transformant capable of growing normally on the TSA-resistant plate is a transformant which is intended to transfer the AcsamsoE gene.

Identification of transformants which were transformed with the AcsamsoOE Gene

1. PCR amplification identification

Genomic DNA of a transformant that is pseudotransferred to the AcsamsOE gene was extracted and used as a template, and the DNA sequence of a transformant prepared from Neo-F: 5' -AGATCTTTAACGCTTACAATTTCC-3' and Neo-R: 5' -AGATCTGAATAGGAACTTCGGAAT-3' and then electrophoresed.

The genomic DNA of the transformant which was transformed with the AcsamsOE gene was replaced with water in the same manner as described above, and the other steps were carried out as a negative control.

According to the method, the genomic DNA of a transformant which is to be transferred with the Acsamsom OE gene is replaced by the genomic DNA of the Cephalosporium acremonium CGMCC3.3795, and other steps are the same and used as a control.

The experimental results are shown in A in FIG. 2 (M is DNA Marker, WT is Cephalosporium acremonium CGMCC3.3795, AcsamsoE is a transformant for transforming the AcsamsoOE gene, and NC is negative control). The result shows that the genome DNA of a transformant which is subjected to AcsamsoOE gene transfer is used as a template, and the primer pair is adopted to amplify to obtain a band of about 2 kb; the primer pair can not amplify to obtain a band of about 2kb by taking water or genome DNA of the cephalosporium acremonium CGMCC3.3795 as a template.

2. qRT-PCR identification

(1) Trizol reagent is adopted to respectively extract the transformant of the AcsamsoOE gene to be transferred and the total RNA of the Cephalosporium acremonium CGMCC3.3795, and then PrimeScriptTM RT kit is adopted to synthesize cDNA.

(2) After the step (1) is completed, performing qRT-PCR (β -actin gene as internal reference) on the AcsamsOE gene in each cDNA by using a thermal cycler Mastercycler, and then calculating the relative expression amount of the AcsamsOE gene by using a Pfaffl's method.

Reaction system: 12.5. mu.L of 2 XSSYBR Premix Ex Taq, 1. mu.L of the forward primer (10. mu. mol. L concentration)^-1) 1. mu.L of the downstream primer (10. mu. mol. L in concentration)^-1) 0.5. mu.L cDNA template and 10. mu.L ddH₂O。

Reaction procedure: 10min at 95 ℃; 95 ℃ for 5s, 58 ℃ for 30s, 72 ℃ for 15s, 40 cycles.

The primers for detecting the AcsamsOE gene are RTACsams-F: 5'-CTATGCCACCGACGAGACTC-3' and RTACsams-R: 5'-TGGGCGGAGATGACAACA-3', and the primers for detecting the β -actin gene are RTactin-F: 5'-AGTCCAAGCGTGGTATCC-3' and RTactin-R: 5'-TAGAAGGCAGGGGCGTTG-3'.

The experimental results are shown in B in FIG. 2 (WT is Cephalosporium acremonium CGMCC3.3795, and AcsamsoE is a transformant for transforming the AcsamsoOE gene). The result shows that the relative expression quantity of the Acsams gene in a transformant of the AcsamsoE gene is obviously increased compared with the initial strain of Acremonium acremonium CGMCC 3.3795.

Therefore, the transformant which is subjected to AcsamsoOE gene transformation and obtained in the third step is a positive transformant which is subjected to AcsamsoOE gene transformation. Three positive transformants which are transformed with the AcsamseOE gene are randomly selected and are sequentially named as a recombinant bacterium AcsamseOE-1, a recombinant bacterium AcsamseOE-2 and a recombinant bacterium AcsamseOE-3.

According to the method of the second to fourth steps, the recombinant plasmid pAg 1-AcsamseOE is replaced by the plasmid pAg1-G418-P-T, the other steps are the same, a transformant of the empty transfer vector is obtained, and the transformant is named as a recombinant bacterium pAg 1-G418-P-T.

Example 2 Effect of methionine on the growth of recombinant bacteria

The bacteria to be detected are recombinant bacteria AcsamseOE-1, recombinant bacteria AcsamseOE-2, recombinant bacteria AcsamseOE-3, recombinant bacteria pAg1-G418-P-T or Cephalosporium acremonium CGMCC 3.3795.

1. Spreading the bacterial liquid of the bacteria to be detected on an LPE plate, culturing at 28 deg.C for 7d, and adding sterile water to obtain a solution with a concentration of 3 × 10⁷And (4) one/mL of bacterial spore suspension to be detected.

2. mu.L of the spore suspension of the bacterium to be tested was spotted on a TSA plate containing no methionine or a TSA plate containing methionine, cultured at 28 ℃ for 7d, and then the growth of the colonies was observed.

The results of some experiments are shown in FIG. 3(WT is Cephalosporium acremonium CGMCC3.3795, and Acsamso is recombinant bacterium Acsamsoe OE-1, and methionine is not added, namely on a TSA plate without methionine, and methionine is added, namely on a TSA plate with methionine). The result shows that the recombinant bacterium AcsamseOE-1, the recombinant bacterium AcsamseOE-2, the recombinant bacterium AcsamseOE-3, the recombinant bacterium pAg1-G418-P-T and the Cephalosporium acremonium CGMCC3.3795 can normally grow under the conditions of no methionine addition and methionine addition, and the growth speeds are basically consistent. As can be seen, the overexpression of the Acsams gene in the Cephalosporium acremonium CGMCC3.3795 does not affect the growth of thalli.

Example 3 fermentation of recombinant bacterium AcsamsoE-1 and measurement of CPC production

The size of the triangular flask in this example was 250 mL.

The bacteria to be detected are recombinant bacteria AcsamseOE-1, recombinant bacteria pAg1-G418-P-T or Cephalosporium acremonium CGMCC 3.3795.

The culture medium to be tested is an original MDFA fermentation culture medium or an improved MDFA fermentation culture medium.

1. And (3) coating the bacterial liquid of the bacteria to be detected on an LPE flat plate, culturing for 7d at 28 ℃, and then adding sterile water to obtain the bacterial spore suspension to be detected.

2. Suspending the spore suspension (containing 3X 10) of the bacteria to be detected⁷Spores) into a triangular flask containing 40mL of original MDFA fermentation medium, and culturing at 28 ℃ and 220rpm for 48h to obtain seed liquid.

3. Taking 4mL of seed solution, inoculating into a triangular flask containing 40mL of culture medium to be detected (namely, the inoculation amount is 10% (v/v), culturing at 28 ℃ and 220rpm for 5d or 6d to obtain fermentation liquor.

4. Taking the fermentation liquor, centrifuging at 4 ℃ and 5000rpm for 10min, and collecting the supernatant and the precipitate.

And (4) drying and weighing the precipitate collected in the step (4) to obtain the biomass of the thalli.

And (4) taking the supernatant collected in the step (4), and carrying out biological activity detection, namely detecting the CPC yield. The experiment was repeated three times, each repetition of the steps as follows:

(1) inoculating the single clone of the bacillus subtilis to 3mL LB liquid culture medium, culturing at 37 ℃ and 220rpm for 2-3h to obtain a bacterial liquid 1. Then diluting the bacterial liquid 1 to OD by using LB liquid culture medium_600nmBacterial liquid 2 was obtained at 0.2.

(2) After the step (1) is finished, 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar powder are dissolved in 1L of distilled water, and the pH value is adjusted to be 7.0; sterilizing at 121 deg.C under high pressure for 15min, cooling to 50 deg.C, collecting 200mL culture medium, adding 1mL bacterial liquid 2 (volume ratio of 1:200) and 1mL penicillinase (volume ratio of 1:200), and mixing; and finally, pouring the mixture into a culture dish, and naturally cooling to obtain the detection flat plate.

(3) After completion of step (2), a punch (6 mm in diameter) was used to punch the wells, 40. mu.L of CPC-Zn aqueous solution (25. mu.g/mL, 50. mu.g/mL, 75. mu.g/mL, 100. mu.g/mL, 125. mu.g/mL or 150. mu.g/mL) was added to the wells, and the zone diameter was measured after culturing in an inverted state at 37 ℃ for 8 hours. And (4) obtaining a standard curve equation for measuring the CPC-Zn titer by taking the concentration of the CPC-Zn as the ordinate and the diameter of the inhibition zone as the abscissa.

The standard curve equation for CPC-Zn titer determination is as follows: y 0.7094x + 0.849; wherein y is log [ CPC-Zn, μ g/mL ], and x is zone diameter-pore diameter (cm).

(4) After completion of step (2), a punch (diameter: 6mm) was used to punch the wells, 40. mu.L of the supernatant collected in step 4 was added to the wells, and after culturing in an inverted state at 37 ℃ for 8 hours, the diameter of the zone of inhibition was measured. And (4) calculating the CPC yield (mu g/mL) of the bacteria to be detected in the culture medium to be detected according to the standard curve equation of the step (3).

The results of some experiments are shown in Table 2 (CPC yield of Cephalosporium acremonium CGMCC3.3795 is highest on 5 days of fermentation, and CPC yield of recombinant bacterium AcsamsoE-1 is highest on 6 days of fermentation, therefore, data of Cephalosporium acremonium CGMCC3.3795 in Table 2 is data on 5 days of fermentation, and data of recombinant bacterium AcsamsoE-1 is data on 6 days of fermentation) and FIG. 4(WT is Cephalosporium acremonium CGMCC3.3795, and AcsamsoE is recombinant bacterium AcsamsoE-1). The result shows that when the strain is cultured in the original MDFA fermentation culture medium (namely the culture medium contains methionine), the biomass of the cephalosporium acremonium CGMCC3.3795, the recombinant bacterium pAg1-G418-P-T and the recombinant bacterium AcsamsoE-1 has no significant difference; compared with the Cephalosporium acremonium CGMCC3.3795, the CPC yield of the recombinant strain AcsamseOE-1 is remarkably improved (about 1.9 times), and the CPC yield of the recombinant strain pAg1-G418-P-T has no remarkable difference. When the strain is cultured in an improved MDFA fermentation culture medium (namely the culture medium does not contain methionine), the biomass of the cephalosporium acremonium CGMCC3.3795, the recombinant bacterium pAg1-G418-P-T and the recombinant bacterium AcsamsoE-1 has no significant difference; compared with the Cephalosporium acremonium CGMCC3.3795, the CPC yield of the recombinant strain AcsamsoOE-1 is remarkably improved (about 3.6 times), and the CPC yield of the recombinant strain pAg1-G418-P-T has no remarkable difference.

The results show that the recombinant bacterium AcsamseOE-1 is used for producing CPC, so that the addition of methionine is omitted, and the yield of CPC is improved. The recombinant bacterium AcsamseOE-1 has important application value in the aspects of improving the yield of CPC, reducing the production cost and the like.

TABLE 2 Biomass and CPC yield of Acremonium acremonium CGMCC3.3795 fermented for 5 days and recombinant bacterium AcsamsoE-1 fermented for 6 days

Example 4 transcriptional analysis of CPC biosynthesis-related Gene of recombinant bacterium AcsamsoE-1 in fermentation Process

Both the pcbAB gene and cefEF gene have been shown to be involved in CPC biosynthesis (described in Pengjie Hu, Ying Wang, Jun Zhou, Yuanyuan Pan, Gang Liu. AcstuA, which codes and APSES transcription regulator, is involved in the infection, cephalosporism biosynthesis and cell wall integration of Acremonium chrysogenum. fungalgogenics and Biology 83(2015) 26-40.). Therefore, the level of CPC synthesizing ability of the cells can be evaluated by measuring the transcription levels of the pcbAB gene and cefEF gene of the cells during fermentation.

The size of the triangular flask in this example was 250 mL.

The bacteria to be detected are recombinant bacteria AcsamseOE-1 or Cephalosporium acremonium CGMCC 3.3795.

2. After completing step 1, the bacterial spore suspension (containing 3X 10) to be tested is added⁷Spores) into a triangular flask containing 40mL of original MDFA fermentation medium, and culturing at 28 ℃ and 220rpm for 48h to obtain seed liquid.

3. After completing step 2, 4mL of seed solution was inoculated into a flask containing 40mL of modified MDFA fermentation medium (i.e., inoculum size is 10% (v/v), and cultured at 28 ℃ and 220rpm for 2d, 3d, 4d, 5d or 6d to obtain a fermentation broth.

4. And (3) after the step 3 is finished, taking the fermentation liquor, centrifuging for 10min at 4 ℃ and 5000rpm, and collecting precipitates, namely the thalli.

5. After completion of step 4, total RNA of the cells was extracted using Trizol reagent, and then cDNA was synthesized using PrimeScriptTM RT kit.

6. After the step 5 is completed, qRT-PCR (β -actin gene as reference) is carried out on the target gene (pcbAB gene or cefEF gene) in each cDNA by using a thermal cycler Mastercycler, and then the relative expression amount of the target gene is calculated by using a Pfaffl's method.

The primers for detecting the pcbAB gene are RTpcbAB-F: 5'-ACCAGTCCGACGTGCAGAAT-3' and RTpcbAB-R: 5'-TCGGTGATATGGGCCATGTAG-3' are provided.

The primer for detecting the cefEF gene is RTcefEF-F: 5'-CCGTAACCACCAAGGGTATCT-3' and RTCefEF-R: 5'-CTCCTCGCTTCCGTTCTTGA-3' are provided.

The primers for detecting β -actin gene are RTactin-F: 5'-AGTCCAAGCGTGGTATCC-3' and RTactin-R: 5'-TAGAAGGCAGGGGCGTTG-3'.

The experimental results are shown in figure 5 (WT-is Acremonium cephamatum CGMCC3.3795, and AcsamseOE-is recombinant bacterium AcsamseOE-1). Results show that the relative expression quantity of the pcbAB gene in the recombinant strain AcsamsoE-1 is remarkably higher than that of an original strain (namely, Cephalosporium acremonium CGMCC 3.3795) in fermentation from 2d to 6 d; the relative expression quantity of the cefEF gene in the recombinant strain AcsamsoE-1 is obviously higher than that of the original strain (namely, Cephalosporium acremonium CGMCC 3.3795) in the fermentation steps from 2d to 5d

The result shows that the recombinant strain AcsamseOE-1 has stronger CPC synthesis capacity than the original strain (i.e. Cephalosporium acremonium CGMCC 3.3795).

<110> institute of microbiology of Chinese academy of sciences

<120> recombinant bacterium for producing cephalosporin C and application thereof

<160>4

<170>PatentIn version 3.5

<210>1

<211>1185

<212>DNA

<213> Acremonium chrysogenum.

<400>1

atgtctgcca acggcatcaa gggcgtgcag aagcacgagg gtagcttcct cttcacctcc 60

gagtccgtcg gcgagggcca cccggacaag atcgccgatc aggtctccga cgcaatcctc 120

gatgcatgcc tgaaggagga ccccctctcc aaggtggctt gcgagaccgc taccaagacg 180

ggcatgatca tggtcttcgg cgagatcacc acccgcgcca acctcgacta ccagaaggtg 240

gtccgtgact gcatcaagga cattgggtat gacgactcct ccaaggggtt cgattacaag 300

acctgcaacc tgctcgtcgc cattgagcag cagtcccccg atattgctca gggtctccac 360

tacgacaagg ccatcgagga gctcggtgcc ggtgaccagg gcatcatgtt cggctatgcc 420

accgacgaga ctcccgagct cttccctctc agccttctcc tcgcccacaa gctgaacgct 480

gccatgtcca aggctcgccg tgacggcacc ctgccctggc tgcgacccga caccaagacc 540

caggtcaccg tcgagtacac cgaggacaac ggcgctgtca tcccccagcg cgtccacact 600

gttgtcatct ccgcccagca ctccgaggac atcaccaccg agaagctccg tgaggagatc 660

aaggagaaga tcatcaaggc taccatcccc gccaagtacc tcgacgacga cgttgtctac 720

cacattcaac cctccggcaa gttcatcatc ggcggccctc agggtgacgc cggtctgacc 780

ggccgtaaga tcatcgtcga cacctacggt ggctggggtg cccacggtgg tggtgccttc 840

tccggcaagg acttctccaa ggtcgatcgc tcggccgcct acactgcccg ctgggtcgcc 900

aagtcgctgg ttgccgccgg cctcgcccgc cgtgccctcg tccagttctc gtacgccatt 960

ggtgttgccg agcccctctc catccacgtc gacacctacg gcacctccaa gaagacctct 1020

gccgagctcg ttgagatcat cgacaagaac ttcaacctcc gtcccggcgt cattgtccgt 1080

gccctcaacc tcgaccagcc catctacctc cagactgcca agaacggcca ctttggcacc 1140

aaccaggact tcagctggga gcagcccaag cctctcaagt tctaa 1185

<210>2

<211>394

<212>PRT

<213> Acremonium chrysogenum.

<400>2

Met Ser Ala Asn Gly Ile Lys Gly Val Gln Lys His Glu Gly Ser Phe

1 5 10 15

Leu Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp Lys Ile Ala

20 25 30

Asp Gln Val Ser Asp Ala Ile Leu Asp Ala Cys Leu Lys Glu Asp Pro

35 40 45

Leu Ser Lys Val Ala Cys Glu Thr Ala Thr Lys Thr Gly Met Ile Met

50 55 60

Val Phe Gly Glu Ile Thr Thr Arg Ala Asn Leu Asp Tyr Gln Lys Val

65 70 75 80

Val Arg Asp Cys Ile Lys Asp Ile Gly Tyr Asp Asp Ser Ser Lys Gly

85 90 95

Phe Asp Tyr Lys Thr Cys Asn Leu Leu Val Ala Ile Glu Gln Gln Ser

100 105 110

Pro Asp Ile Ala Gln Gly Leu His Tyr Asp Lys Ala Ile Glu Glu Leu

115 120 125

Gly Ala Gly Asp Gln Gly Ile Met Phe Gly Tyr Ala Thr Asp Glu Thr

130 135 140

Pro Glu Leu Phe Pro Leu Ser Leu Leu Leu Ala His Lys Leu Asn Ala

145 150 155 160

Ala Met Ser Lys Ala Arg Arg Asp Gly Thr Leu Pro Trp Leu Arg Pro

165 170 175

Asp Thr Lys Thr Gln Val Thr Val Glu Tyr Thr Glu Asp Asn Gly Ala

180 185 190

Val Ile Pro Gln Arg Val His Thr Val Val Ile Ser Ala Gln His Ser

195 200 205

Glu Asp Ile Thr Thr Glu Lys Leu Arg Glu Glu Ile Lys Glu Lys Ile

210 215 220

Ile Lys Ala Thr Ile Pro Ala Lys Tyr Leu Asp Asp Asp Val Val Tyr

225 230 235 240

His Ile Gln Pro Ser Gly Lys Phe Ile Ile Gly Gly Pro Gln Gly Asp

245 250 255

Ala Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Trp

260 265 270

Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Phe Ser Lys Val

275 280 285

Asp Arg Ser Ala Ala Tyr Thr Ala Arg Trp Val Ala Lys Ser Leu Val

290 295 300

Ala Ala Gly Leu Ala Arg Arg Ala Leu Val Gln Phe Ser Tyr Ala Ile

305 310 315 320

Gly Val Ala Glu Pro Leu Ser Ile His Val Asp Thr Tyr Gly Thr Ser

325 330 335

Lys Lys Thr Ser Ala Glu Leu Val Glu Ile Ile Asp Lys Asn Phe Asn

340 345 350

Leu Arg Pro Gly Val Ile Val Arg Ala Leu Asn Leu Asp Gln Pro Ile

355 360 365

Tyr Leu Gln Thr Ala Lys Asn Gly His Phe Gly Thr Asn Gln Asp Phe

370 375 380

Ser Trp Glu Gln Pro Lys Pro Leu Lys Phe

385 390

<210>3

<211>8626

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

caaattgacg cttagacaac ttaataacac attgcggacg tttttaatgt actggggtgg 60

tttttctttt caccagtgag acgggcaaca gcggcgccat tcgccattca ggctgcgcaa 120

ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 180

atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa 240

aacgacggcc agtgaattcg agctcggtac caaggcccgg gctggccacg gccgcctagg 300

cgcgcaagga tcctctagaa taggaacttc ggaataggaa cttcaaagcg tttccgaaaa 360

cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt cacgtcgcac 420

ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata gtgcgtgttt 480

atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag tctagtacct 540

cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca ctacccttta 600

gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat gctatcattt 660

cctttgatat tggatcatat gcatagtacc gaaaactagg ccagcagtag acacttggaa 720

tctaaacaga cattatcatc atcatgcaac atgcatgtac tgtctgatgt attaagagta 780

taggggtcaa caactagagt gtctgacagg tatatctagc agtttgattt gaccattctt 840

ttattaaaac actgtaaccc gcaacgaaga actcctccgc ttcaagtcaa gaatgcgcat 900

atgtgcgtaa aatcgacaag cccggcggca ttccaccgag cagggtattg cctttggcac 960

tttacgtgcg cagaggagcc tgaatgttga gtggaatgat tcagaagaac tcgtcaagaa 1020

ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc 1080

ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct 1140

gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt 1200

ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg 1260

gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt 1320

ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat 1380

gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg 1440

catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc 1500

ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag 1560

ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt 1620

cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca 1680

gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata 1740

gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcattttga 1800

agattgggtt cctgcgaagc tgtatcaata agttggatat ggaatgaatt ggagattgcc 1860

gtgcggaacg tgcagtggtg gtgtgggatg atgcgtgttg ttgggaagag tcgggactcg 1920

cgacagtgat ggggttacgg ttcgatgggg ttgagttggt gacggatcga gtggtgtcaa 1980

ctgtgttgct ggctggcagg ctgggaacga ggtgtttcca ggttggtgct tgactggcac 2040

tgctggggcg ctggggcgag catcatattc ctgccgccaa tcaggtagcg cttgcgaagg 2100

cggctggaaa ttgggggttt cggaatgtcg tcaagcggga accaatttga gtacccaatt 2160

cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact 2220

gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 2280

ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 2340

gcgaatggaa attgtaagcg ttaagatcta gaggatcccc cgactagttg gtaaaaacga 2400

gcaagcgagc tctagtgttg caaatacgag accataatac ctacctcggc catacaaata 2460

cgctggtgca cctctcaaca tcgaaacaca acaacccgac tcgcctgatc aagctggcta 2520

ttagacagcc aagcgattcc gtcacgtctt cccattgaac gagtctagaa cggaaagcaa 2580

aaaaagttat tgtatatcaa cctcagcttc ctacacccct aggctcgcat ccctaaacgc 2640

atccgactac aaaagtggcc gccgtcatgt ctgttccgag aaaaggcaac gcccaccacc 2700

gcctgaccat gtgaaataac ttttcgtaaa tatgagactt gagtgggata cgcgcgcagc 2760

tgagtagacg ttgcatcggt cgccaccgac tccgacatct tggcgttctt ttgcggaggg 2820

tggaaggatt tggttctggc cggttgcctt gtcttttcga acctgtcaac cttacattcg 2880

ggtatccggc ttcccgacga caacatacac tagtgcgcga tcgcggccgg ccggcgcgcc 2940

gtttaaacgg atttaaatta attaatgtcg actttgattg atctgggaag attattagcc 3000

atggccagca aaagtttaac gaagagccga gagccgatga tgcggggagt gcgtgaccgg 3060

agaattcaaa agcagcaata gcagcagaag cagtagaagc aacagctaag tcatctgctg 3120

gccgcccgag ggacgcgacg agaatgggac gaggtgagct gaggttgggt gggaaggttt 3180

tggaggaggg gcgaggggga agctcagagc tttcccccgg agctcgcggg gctgcggaag 3240

agctcaaagg caagacgaac gtgagcaggg cacttcactt actatagtag ttttttgaag 3300

aagagtgagc ttcgtggaat aataatcgac gcttttcgtt aacgacgacg gatgggttct 3360

cgggaggacg tggatggaag aaggaagaat ggagggaggg aggaggggat agaagcgaag 3420

ctgaaagggc tctcttattc acgcgccctt tccgccaggt gcgtaaggta cttggtaggt 3480

aaagtagttg ccaacattcg tctgatcatg gaatcacggt aacacgggct tgattatgaa 3540

cccccactaa aaccaaagtg gaaagaaatg gcgcattatt catacaggtg ttgggcgaaa 3600

atttccgtcc gctcacgaaa cctgctggta cgaaggaaat acatgcattt tccggtgatg 3660

ttccagtaag tgacgcagtt gatcttacct aaccaggtgg tcgctcacaa cggcgccaca 3720

gtgctccagc aacggtaccg catgagccga agggtcgggc tgttgttggc atagagttag 3780

tggaggtgaa tattttctat cagcctgtgc gtccttatac ccgcggctgg gcggagctgc 3840

atgctcgtat aagataattc ttgcttggta tgtacgacga ctcgtaccag gattaatact 3900

actactctgg tagtacctta attacctact agtagtattg aatccggata cagacgggag 3960

taatcgacgg agggtacgga tactctttgc tgttctattc cgtacttgcc cgtcgtcccc 4020

tcatccctca gccgccagct tagaaaccac tctgatctct acacagccgt atcggacgtt 4080

tacttttggg aaattgacat atctacagta cctatcccac cagggcccaa cacgcatgcc 4140

accgattacc atgctatgac ttcaccactt ctgttgacga ttcttctatt cacaaccgtc 4200

gtgatggcca ctttcaaaag gctttcgcct gcaggcatgg ctggatgcga tagtctgcgg 4260

ccgaacgtcc cggtgcagtt atcatcgagg tctgccagcg gcggacggac tgcctgtctg 4320

gcctttcccc tcttctaccc cccctcaccc gcgccgattt cgtgtttttt ccgtcatcgc 4380

cagcaacact gcgagcagca cgctgctgta gccggcattg gccgcacaga ccacggcctc 4440

cgacatccgc actgttgcct gaggcaccag tgttttggcc ttctggtgca ctaagatttt 4500

cgcggccacc gccactcgcg attgggttta gcgccaatgt cgttagaggc aagaggagcg 4560

gcggcggggc gtctctgaga tgagcactcc ctggcgcaca tcgccaactt gtctctggtt 4620

ggccagctgc gtccgggtcc cagtacgtac cgcccatgta ccctgccact gcagcccacc 4680

tgcccgcctt cccagcccag cagcaggctt cgaggtctcc ctggacactt gctgcaactt 4740

acgcgtgcga cgacgactaa ggtgaagggt gtccgcccgc tgctggcgtg cccgtgcaat 4800

gcgctgcgac cggcggcgtc cagcacccat cgcccagcct gccacccagc accaacaatg 4860

ccaccaaacc cacccagccc attcagtgca gcgccagcag gcaagttcag ctctcattct 4920

caggcgctct ttccccatcc atccgctctc ctctcctccc attcctcact ctctcctcgt 4980

gccctccccc aaacctcttc tcttttcacg accggactat tcttacctgg gcgcccgacc 5040

gccgaacctc tgccttctac gctgggtcga cctgcaggca tgcaagcttc gtgactccct 5100

taattctccg ctcatgatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt 5160

gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa taatcggata 5220

tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tttgttcatg ccaaccacag 5280

ggttccagat ccgacgagca aggcaagacc gagcgccttt gcgacgctca ccgggctggt 5340

tgccctcgcc gctgggctgg cggccgtcta tggccctgca aacgcgccag aaacgccgtc 5400

gaagccgtgt gcgagacacc gcggccgccg gcgttgtgga tacctcgcgg aaaacttggc 5460

cctcactgac agatgagggg cggacgttga cacttgaggg gccgactcac ccggcgcggc 5520

gttgacagat gaggggcagg ctcgatttcg gccggcgacg tggagctggc cagcctcgca 5580

aatcggcgaa aacgcctgat tttacgcgag tttcccacag atgatgtgga caagcctggg 5640

gataagtgcc ctgcggtatt gacacttgag gggcgcgact actgacagat gaggggcgcg 5700

atccttgaca cttgaggggc agagtgctga cagatgaggg gcgcacctat tgacatttga 5760

ggggctgtcc acaggcagaa aatccagcat ttgcaagggt ttccgcccgt ttttcggcca 5820

ccgctaacct gtcttttaac ctgcttttaa accaatattt ataaaccttg tttttaacca 5880

gggctgcgcc ctgtgcgcgt gaccgcgcac gccgaagggg ggtgcccccc cttctcgaac 5940

cctcccggcc cgctaacgcg ggcctcccat ccccccaggc gtacgccact ggagcacctc 6000

aaaaacacca tcatacacta aatcagtaag ttggcagcat cacccataat tgtggtttca 6060

aaatcggctc cgtcgatact atgttatacg ccaactttga aaacaacttt gaaaaagctg 6120

ttttctggta tttaaggttt tagaatgcaa ggaacagtga attggagttc gtcttgttat 6180

aattagcttc ttggggtatc tttaaatact gtagaaaaga ggaaggaaat aataaatggc 6240

taaaatgaga atatcaccgg aattgaaaaa actgatcgaa aaataccgct gcgtaaaaga 6300

tacggaagga atgtctcctg ctaaggtata taagctggtg ggagaaaatg aaaacctata 6360

tttaaaaatg acggacagcc ggtataaagg gaccacctat gatgtggaac gggaaaagga 6420

catgatgcta tggctggaag gaaagctgcc tgttccaaag gtcctgcact ttgaacggca 6480

tgatggctgg agcaatctgc tcatgagtga ggccgatggc gtcctttgct cggaagagta 6540

tgaagatgaa caaagccctg aaaagattat cgagctgtat gcggagtgca tcaggctctt 6600

tcactccatc gacatatcgg attgtcccta tacgaatagc ttagacagcc gcttagccga 6660

attggattac ttactgaata acgatctggc cgatgtggat tgcgaaaact gggaagaaga 6720

cactccattt aaagatccgc gcgagctgta tgatttttta aagacggaaa agcccgaaga 6780

ggaacttgtc ttttcccacg gcgacctggg agacagcaac atctttgtga aagatggcaa 6840

agtaagtggc tttattgatc ttgggagaag cggcagggcg gacaagtggt atgacattgc 6900

cttctgcgtc cggtcgatca gggaggatat cggggaagaa cagtatgtcg agctattttt 6960

tgacttactg gggatcaagc ctgattggga gaaaataaaa tattatattt tactggatga 7020

attgttttag tacctagatg tggcgcaacg atgccggcga caagcaggag cgcaccgact 7080

tcttccgcat caagtgtttt ggctctcagg ccgaggccca cggcaagtat ttgggcaagg 7140

ggtcgctggt attcgtgcag ggcaagattc ggaataccaa gtacgagaag gacggccaga 7200

cggtctacgg gaccgacttc attgccgata aggtggatta tctggacacc aaggcaccag 7260

gcgggtcaaa tcaggaataa gggcacattg ccccggcgtg agtcggggca atcccgcaag 7320

gagggtgaat gaatcggacg tttgaccgga aggcatacag gcaagaactg atcgacgcgg 7380

ggttttccgc cgaggatgcc gaaaccatcg caagccgcac cgtcatgcgt gcgccccgcg 7440

aaaccttcca gtccgtcggc tcgatggtcc agcaagctac ggccaagatc gagcgcgaca 7500

gcgtgcaact ggctccccct gccctgcccg cgccatcggc cgccgtggag cgttcgcgtc 7560

gtctcgaaca ggaggcggca ggtttggcga agtcgatgac catcgacacg cgaggaacta 7620

tgacgaccaa gaagcgaaaa accgccggcg aggacctggc aaaacaggtc agcgaggcca 7680

agcaggccgc gttgctgaaa cacacgaagc agcagatcaa ggaaatgcag ctttccttgt 7740

tcgatattgc gccgtggccg gacacgatgc gagcgatgcc aaacgacacg gcccgctctg 7800

ccctgttcac cacgcgcaac aagaaaatcc cgcgcgaggc gctgcaaaac aaggtcattt 7860

tccacgtcaa caaggacgtg aagatcacct acaccggcgt cgagctgcgg gccgacgatg 7920

acgaactggt gtggcagcag gtgttggagt acgcgaagcg cacccctatc ggcgagccga 7980

tcaccttcac gttctacgag ctttgccagg acctgggctg gtcgatcaat ggccggtatt 8040

acacgaaggc cgaggaatgc ctgtcgcgcc tacaggcgac ggcgatgggc ttcacgtccg 8100

accgcgttgg gcacctggaa tcggtgtcgc tgctgcaccg cttccgcgtc ctggaccgtg 8160

gcaagaaaac gtcccgttgc caggtcctga tcgacgagga aatcgtcgtg ctgtttgctg 8220

gcgaccacta cacgaaattc atatgggaga agtaccgcaa gctgtcgccg acggcccgac 8280

ggatgttcga ctatttcagc tcgcaccggg agccgtaccc gctcaagctg gaaaccttcc 8340

gcctcatgtg cggatcggat tccacccgcg tgaagaagtg gcgcgagcag gtcggcgaag 8400

cctgcgaaga gttgcgaggc agcggcctgg tggaacacgc ctgggtcaat gatgacctgg 8460

tgcattgcaa acgctagggc cttgtggggt cagttccggc tggatctgct ctcccgctga 8520

cgccgtcccg gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt 8580

cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaa 8626

<210>4

<211>3838

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>4

ggtcgcatct tccgtctcca agccgccagc ccgcgggtcc attcttatca ggccagcact 60

tttctcttct ccaaaccccc tcccgtgctc ctctctcact ccttaccctc ctctcctctc 120

gcctacctac ccctttctcg cggactctta ctctcgactt gaacggacga ccgcgacgtg 180

acttacccga cccacccaaa ccaccgtaac aaccacgacc caccgtccga cccgctaccc 240

acgacctgcg gcggccagcg tcgcgtaacg tgcccgtgcg gtcgtcgccc gcctgtggga 300

agtggaatca gcagcagcgt gcgcattcaa cgtcgttcac aggtccctct ggagcttcgg 360

acgacgaccc gacccttccg cccgtccacc cgacgtcacc gtcccatgta cccgccatgc 420

atgaccctgg gcctgcgtcg accggttggt ctctgttcaa ccgctacacg cggtccctca 480

cgagtagagt ctctgcgggg cggcggcgag gagaacggag attgctgtaa ccgcgatttg 540

ggttagcgct caccgccacc ggcgctttta gaatcacgtg gtcttccggt tttgtgacca 600

cggagtccgt tgtcacgcct acagcctccg gcaccagaca cgccggttac ggccgatgtc 660

gtcgcacgac gagcgtcaca acgaccgcta ctgccttttt tgtgctttag ccgcgcccac 720

tcccccccat cttctcccct ttccggtctg tccgtcaggc aggcggcgac cgtctggagc 780

tactattgac gtggccctgc aagccggcgt ctgatagcgt aggtcggtac ggacgtccgc 840

tttcggaaaa ctttcaccgg tagtgctgcc aacacttatc ttcttagcag ttgtcttcac 900

cacttcagta tcgtaccatt agccaccgta cgcacaaccc gggaccaccc tatccatgac 960

atctatacag ttaaagggtt ttcatttgca ggctatgccg acacatctct agtctcacca 1020

aagattcgac cgccgactcc ctactcccct gctgcccgtt catgccttat cttgtcgttt 1080

ctcataggca tgggaggcag ctaatgaggg cagacatagg cctaagttat gatgatcatc 1140

cattaattcc atgatggtct catcatcata attaggacca tgctcagcag catgtatggt 1200

tcgttcttaa tagaatatgc tcgtacgtcg aggcgggtcg gcgcccatat tcctgcgtgt 1260

ccgactatct tttataagtg gaggtgattg agatacggtt gttgtcgggc tgggaagccg 1320

agtacgccat ggcaacgacc tcgtgacacc gcggcaacac tcgctggtgg accaatccat 1380

tctagttgac gcagtgaatg accttgtagt ggccttttac gtacataaag gaagcatggt 1440

cgtccaaagc actcgcctgc ctttaaaagc gggttgtgga catacttatt acgcggtaaa 1500

gaaaggtgaa accaaaatca cccccaagta ttagttcggg cacaatggca ctaaggtact 1560

agtctgctta caaccgttga tgaaatggat ggttcatgga atgcgtggac cgcctttccc 1620

gcgcacttat tctctcggga aagtcgaagc gaagataggg gaggagggag ggaggtaaga 1680

aggaagaagg taggtgcagg agggctcttg ggtaggcagc agcaattgct tttcgcagct 1740

aataataagg tgcttcgagt gagaagaagt tttttgatga tatcattcac ttcacgggac 1800

gagtgcaagc agaacggaaa ctcgagaagg cgtcggggcg ctcgaggccc cctttcgaga 1860

ctcgaagggg gagcggggag gaggttttgg aagggtgggt tggagtcgag tggagcaggg 1920

taagagcagc gcagggagcc cgccggtcgt ctactgaatc gacaacgaag atgacgaaga 1980

cgacgataac gacgaaaact taagaggcca gtgcgtgagg ggcgtagtag ccgagagccg 2040

agaagcaatt tgaaaacgac cggtaccgat tattagaagg gtctagttag ttttcagctg 2100

taattaatat gtctgccaac ggcatcaagg gcgtgcagaa gcacgagggt agcttcctct 2160

tcacctccga gtccgtcggc gagggccacc cggacaagat cgccgatcag gtctccgacg 2220

caatcctcga tgcatgcctg aaggaggacc ccctctccaa ggtggcttgc gagaccgcta 2280

ccaagacggg catgatcatg gtcttcggcg agatcaccac ccgcgccaac ctcgactacc 2340

agaaggtggt ccgtgactgc atcaaggaca ttgggtatga cgactcctcc aaggggttcg 2400

attacaagac ctgcaacctg ctcgtcgcca ttgagcagca gtcccccgat attgctcagg 2460

gtctccacta cgacaaggcc atcgaggagc tcggtgccgg tgaccagggc atcatgttcg 2520

gctatgccac cgacgagact cccgagctct tccctctcag ccttctcctc gcccacaagc 2580

tgaacgctgc catgtccaag gctcgccgtg acggcaccct gccctggctg cgacccgaca 2640

ccaagaccca ggtcaccgtc gagtacaccg aggacaacgg cgctgtcatc ccccagcgcg 2700

tccacactgt tgtcatctcc gcccagcact ccgaggacat caccaccgag aagctccgtg 2760

aggagatcaa ggagaagatc atcaaggcta ccatccccgc caagtacctc gacgacgacg 2820

ttgtctacca cattcaaccc tccggcaagt tcatcatcgg cggccctcag ggtgacgccg 2880

gtctgaccgg ccgtaagatc atcgtcgaca cctacggtgg ctggggtgcc cacggtggtg 2940

gtgccttctc cggcaaggac ttctccaagg tcgatcgctc ggccgcctac actgcccgct 3000

gggtcgccaa gtcgctggtt gccgccggcc tcgcccgccg tgccctcgtc cagttctcgt 3060

acgccattgg tgttgccgag cccctctcca tccacgtcga cacctacggc acctccaaga 3120

agacctctgc cgagctcgtt gagatcatcg acaagaactt caacctccgt cccggcgtca 3180

ttgtccgtgc cctcaacctc gaccagccca tctacctcca gactgccaag aacggccact 3240

ttggcaccaa ccaggacttc agctgggagc agcccaagcc tctcaagttc taaccggccg 3300

gcgctagcgc gtgatcacca tacaacagca gcccttcggc ctatgggctt acattccaac3360

tgtccaagct tttctgttcc gttggccggt cttggtttag gaaggtggga ggcgttttct 3420

tgcggttcta cagcctcagc caccgctggc tacgttgcag atgagtcgac gcgcgcatag 3480

ggtgagttca gagtataaat gcttttcaat aaagtgtacc agtccgccac cacccgcaac 3540

ggaaaagagc cttgtctgta ctgccgccgg tgaaaacatc agcctacgca aatccctacg 3600

ctcggatccc cacatccttc gactccaact atatgttatt gaaaaaaacg aaaggcaaga 3660

tctgagcaag ttacccttct gcactgcctt agcgaaccga cagattatcg gtcgaactag 3720

tccgctcagc ccaacaacac aaagctacaa ctctccacgt ggtcgcataa acataccggc 3780

tccatccata ataccagagc ataaacgttg tgatctcgag cgaacgagca aaaatggt 3838

Claims

1. The application of the recombinant bacteria or the microbial inoculum containing the recombinant bacteria in the production of cephalosporin C;

the preparation method of the recombinant bacterium comprises the following steps: increasing the content of adenosylmethionine synthetase in the fermentation strain to obtain recombinant strain; the germination strain is cephalosporium acremonium; the amino acid sequence of the adenosylmethionine synthetase is shown as a sequence 2 in a sequence table; the method for improving the content and/or the activity of the adenosylmethionine synthetase in the starting bacteria comprises the steps of introducing nucleic acid molecules for coding the adenosylmethionine synthetase into the starting bacteria; the nucleic acid molecule for coding the adenosylmethionine synthetase is shown as a sequence 1 in a sequence table.

2. A method for producing cephalosporin C, comprising the step of fermentatively culturing the recombinant bacterium of claim 1 to obtain cephalosporin C;