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New strain for producing penicillium chrysogenum and ferment for preparing penicillin by using the strain

A technology for producing Penicillium chrysogenum and Vitiligo, applied in fermentation, application, fungi, etc., can solve problems such as troublesome work and failure to improve penicillin production capacity, so as to improve efficiency, increase cell production and penicillin synthesis, and increase production Effect

Inactive Publication Date: 2006-07-05
NORTH CHINA PHARMA COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sun Chuanbao and others transferred the vhb gene into the penicillin industrial production strain. When producing Penicillium chrysogenum P-9, a total of 53 transformants were obtained, but only one strain had the ability to produce penicillin, which not only did not improve the penicillin production capacity of the production strain, but also had to It can only grow in the medium containing high concentration of KCl, which brings a lot of trouble to the follow-up work

Method used

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  • New strain for producing penicillium chrysogenum and ferment for preparing penicillin by using the strain

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Effect test

Embodiment 1

[0017] Embodiment 1 gene cloning and the construction of gene transformation vector

[0018] The transformation vector pPIPKA of Penicillium chrysogenum was constructed according to the description of the Chinese invention patent application number 200410012139.4. The plasmid vector of pDH25 was purchased from Fungal Genetics Stock Center, University of Kansas Medical Center. The host bacteria Penicillium chrysogenum ATCC 28953 for gene transformation was purchased from the American Type Culture Collection. pGEM-T is a product of Promega, USA.

[0019] Molecular cloning techniques used in the present invention include separation and purification of DNA, DNA synthesis, polymerase chain reaction (PCR), restriction endonuclease digestion of DNA, connection of DNA, Escherichia coli transformation screening, sequence analysis, etc. is well known in the art and is fully described in Molecular Cloning, a laboratory manual by Sambrook et al.

[0020] According to the Oxygen Binding...

Embodiment 2

[0024] Embodiment 2 Penicillium chrysogenum transformation and positive clone screening

[0025] 1. Culture of microorganisms

[0026] Penicillium chrysogenum NCPC10086 was grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose). The chromosomal DNA of this strain was used to isolate the Pipns sequence. At the same time, the bacterium is also used as a host for cefE gene transformation. YPD medium is also used for the culture of protoplasts.

[0027] 2. Transformation of Penicillium chrysogenum

[0028] Using the Ca-PEG-mediated protoplast transformation method, the pBIVT vector was transformed into Penicillium chrysogenum according to the method of Wang Fuqiang et al. (Acta Mycophyta Sinica, 23:66-72, 2004). Positive clones were screened on YPD selective medium containing bleomycin.

[0029] 3. Detection of transformants

[0030] P. chrysogenum transformants were purified by subculturing twice on selective medium (1% yeast extract, 2% peptone, 2% glucose, 2ug / ml ...

Embodiment 3

[0031] Embodiment 3 shaking flask fermentation and target product detection

[0032] Penicillium chrysogenum ATCC 28953 was transformed with the transgene vector pBIVT described in Example 1, and 272 positive clones were obtained. Transgenic positive strains were used in 2×10 6 Conidia / ml inoculated seed medium (g / l): glucose, 30; lactose, 10; cotton cake powder, 10; yeast powder, 10; corn steep liquor, 10; (NH 4 ) 2 SO 4 , 2; KH 2 PO 4 , 1; CaCO 3 , 5. pH 5.8. 26°C, 48 hours. Seed culture was inoculated with 5% inoculum of fermentation medium (g / l): lactose, 120; cotton cake powder, 30; corn steep liquor, 20; (NH 4 ) 2 SO 4 , 5; KH 2 PO 4 ,1;K 2 SO 4 , 5; CaCO 3 , 10; pH 6.5, cultured at 26°C for 144 hours.

[0033] After the fermentation, the fermentation liquid was centrifuged with a centrifugal force of 8000 g for 15 minutes to remove the mycelium, and the fermentation supernatant was obtained for detection of penicillin yield. Assays were performed accord...

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Abstract

This invention describes a penicillium chrysogenum strain and its application id the production of penicillin. The strain described in this invention is penicillium chrysogenum CGMCC No.1248, and has simple cultivation conditions as well as a high yield. This invention also describes the process for producing penicillin from the said strain.

Description

Technical field: [0001] The invention relates to the field of penicillin production, in particular to a new Penicillium chrysogenum and the application of the bacterium in the production of penicillin. Background technique: [0002] β-lactam antibiotics are currently known antibiotics with the lowest toxicity, the largest variety, and the most widely used clinically. The most important ones are penicillin and cephalosporin C, which are used as raw materials to obtain intermediates through chemical or enzymatic reactions, and a series of semi-synthetic penicillins and semi-synthetic cephalosporins can be obtained. These intermediates mainly include 6-aminopenicillane acid (6-APA), 7-aminodesacetylcephalosporanic acid (7-ADCA) and chloromethylcephalexin ester (GCLE), 7-aminocephalosporanic acid (7-ACA). 6-APA, 7-ADCA, and GCLE are all produced from penicillin, and 7-ACA is produced from cephalosporin C. The acquisition of these intermediates has led to an increasing variety ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N15/80C12N15/31C12R1/82
Inventor 徐平任志红王富强丰玫玫张佳戴梦韦春玲穆岷贾茜贺建功
Owner NORTH CHINA PHARMA COMPANY
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