3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof

A technology of dehydrogenase and genetically engineered strains, which is applied to 3-sterone-Δ1-dehydrogenase and engineering bacteria and application fields to achieve the effects of improving utilization rate, mild reaction conditions, high economic and social benefits

Active Publication Date: 2011-08-31
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, another approach is to construct an engineered bacterium expressing 3-sterone-1-dehydrogenase gene (ksdD), but because the ksdD gene of bacterial origin all expresses membrane protein, it greatly limits the application of this protein in industry.

Method used

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  • 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof
  • 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof
  • 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 , Acquisition of 3-sterone-Δ1-dehydrogenase gene from Metarhizium anisopliae

[0052] 1.1. Construction of the gene library of Metarhizium anisopliae

[0053] Refer to the method provided by the manufacturer, use the fungal genome extraction kit, extract the genome of Metarhizium anisopliae, and send it to Introvigen to prepare a gene library that can cover the genome of Metarhizium anisopliae 10 times.

[0054] 1.2. Design of merger primers

[0055] Through the Metarhizium anisopliae EST tag library, the reported fumigatus gene cluster of Aspergillus fumigatus (Silvia Lodeiro. Protostadienol Biosynthesis and Metabolism in the Pathogenic Fungus Aspergillus fumigatus. Organic Letters, 11, 2009) and the acquired gene fragments of Metarhizium anisopliae , design the degenerate primer pair of ksdD gene, the sequence of the degenerate primer pair of ksdD gene is shown as primer pair 2.

[0056] 1.3. The PCR method was used to catch the fumagic acid gene cluster...

Embodiment 2

[0072] Example 2 , Construction of Pichia pastoris engineering bacteria KM71-ksdD and GS115-ksdD

[0073] 2.1. Construction of Pichia pastoris (P. pastoris) expression vector pPIC3.5K-ksdD

[0074] According to the sequence characteristics of the multiple cloning site of the pPIC3.5K plasmid, construct upstream primers and downstream primers, as shown in primer pair 1, respectively. Using the primer pair, perform PCR, wherein the PCR reaction conditions and reaction system are as follows:

[0075] The 50ul PCR reaction system is as follows:

[0076] 2×PCR mix 25ul

[0077] Upstream primer 1ul

[0078] Downstream primer 1ul

[0079] Template 0.5ul

[0080] Water 22.5ul

[0081] The reaction procedure is as follows:

[0082] Pre-denaturation at 94°C for 5 minutes;

[0083] Denaturation 94°C 30sec

[0084] Annealing 50℃ 30sec 30 cycles;

[0085]Extend at 72°C for 10 minutes;

[0086] Keep warm at 4°C.

[0087] The PCR product was recovered with a PCR cycle pure kit ...

Embodiment 3

[0104] Example 3 , Pichia pastoris fermentation culture and protein expression detection

[0105] 3.1. Fermentation and cultivation of Pichia pastoris

[0106] Inoculate the Pichia pastoris KM71-ksdD1 and GS115-ksdD1 strains obtained in step 2.3 into 30ml BMGY liquid medium with 1% inoculum size, culture at 28°C and 200rpm for 40-48 hours, and then centrifuge under sterile conditions (5000rmp, 5mim, 4°C), transfer the precipitate into 30ml BMMY liquid medium, shake culture at 28°C, 200rpm, add 0.5% methanol every 24 hours to induce culture, cultivate for 7 days, and use an empty host without ksdD as a blank control .

[0107] 3.2 Determination of growth curve

[0108] Inoculate the Pichia yeast KM71-ksdD1 and GS115-ksdD1 strains obtained in step 2.3 into 30ml of BMGY liquid medium with 1% inoculum, culture at 28°C with shaking at 200rpm, take samples every 2 hours, and measure the OD 600nm . To draw a growth curve see Image 6 .

[0109] 3.3. Electrophoretic detection ...

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Abstract

The invention provides a 3-ketosteroid-delta 1-dehydrogenase gene, cholesterol oxidase coded by the same, an expression vector containing a gene sequence of the 3-sterone-delta 1-dehydrogenase gene, a gene engineering recombinant strain containing the expression vector and a method for preparing 3-sterone-delta 1-dehydrogenase. The 3-sterone-delta 1-dehydrogenase provided by the invention is new 3-sterone-delta 1-dehydrogenase. The recombinant strain provided by the invention can be used for transforming 3-keto steroids, wherein the optimized recombinant strain can also be used for transforming androstane-4-alkenyl-3,17-diketone into boldenone, the expressed target protein is a soluble protein, the bottleneck of membrane protein in the traditional industrial production is broken through, and the recombinant strain has great significance in the industrial production of KSDD (3-ketosteroid -delta 1-dehydrogenase). Steroids are produced through microbial transformation, the production efficiency and the product quality of a steroid medicine production system are improved, and the production cost is reduced.

Description

technical field [0001] The invention relates to the field of genetic engineering, more specifically, to a 3-sterone-Δ1-dehydrogenase, engineering bacteria and applications. Background technique [0002] Many fungi, such as Aspergillus sp., Metarhizium sp., Fusarium sp., Neurospora sp., Neosartorya sp., etc., can produce steroidal antibiotics. Steroidal antibiotics include fusidic acid, fusidic acid and related analogues, wherein, structural formula I shows cyclopentane polyhydrophenantrene (Cyclopentanoperhydrophenantrene), cholesterol (Cholesterol), fusidic acid (Helvolic acid) and Fusidic acid respectively The structure of Fusidic acid. These steroidal antibiotics have a good inhibitory effect on bacteria, for example, fusidic acid has been used clinically for more than 30 years; however, for some fungi, steroidal antibiotics will be an important pathogenic virulence factor , for example, fumonic acid is a virulence factor for the insecticide of Metarhizium anisopliae. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/19C12P33/02C12R1/84
Inventor 魏东芝王风清陈苗苗林良才
Owner EAST CHINA UNIV OF SCI & TECH
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