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Steroid 11 beta-hydroxylase in curvularia lunata and coding gene and application thereof

A technology for encoding and transgenic cell lines, applied in the field of steroid 11β-hydroxylase and its encoding gene and application

Active Publication Date: 2018-12-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after years of research, scholars have not successfully excavated the 11-position β-hydroxylation P450 of the most critical substrate in the synthesis process of hydrocortisone, so the successful excavation of this protein is of great significance to the study of hydrocortisone synthesis

Method used

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  • Steroid 11 beta-hydroxylase in curvularia lunata and coding gene and application thereof
  • Steroid 11 beta-hydroxylase in curvularia lunata and coding gene and application thereof
  • Steroid 11 beta-hydroxylase in curvularia lunata and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1, Cloning and expression of Curvularia lunae 11-position β-hydroxylase

[0086] Gene cloning and expression are divided into the following 3 steps:

[0087] 1. Extraction of total RNA from Curvularia lunae

[0088] First, Curvularia lunae AS3.4381 (ATCC12017) was cultured on a plate for several days, and a certain number of spores were harvested and inserted into 50mL potato-dextrose (PDA) medium, and cultivated overnight until a large number of bacteria were synthesized; then , centrifuge to collect the mycelia of Curvularia lunata, wash with potassium phosphate buffer (PBS), and finally resuspend with 50 mL of buffer and add the substrate hydrocortisone 21-acetate ( RSA) was induced for 2 h, and samples were taken for RNA extraction.

[0089] RNA extraction method:

[0090] (1) Add 0.5mm grinding beads (to fill the bottom of the conical tube) and 1mL Trizol into a 2.0mL screw-cap tube (RNase free), and then add liquid nitrogen quick-frozen mycelia (about 1...

Embodiment 2

[0116] Embodiment 2, the construction of Saccharomyces cerevisiae engineering bacteria HC101

[0117] The starting bacterium Saccharomyces cerevisiae BY4742 was cultured overnight in selection medium. The composition of the liquid screening medium is as follows: SD-Trp (Beijing Fanjinuo (Functional Genomics) Technology Co., Ltd.), 2% glucose, 0.005% His., 0.01% Leu., 0.01% Ura. (each percent sign represents g / 100mL). Take 1ml (OD about 0.6-1.0) into 1.5ml EP tubes, centrifuge at 10000g at 4°C for 1min, discard the supernatant, wash the precipitate with sterile water (4°C), centrifuge under the same conditions, and discard the supernatant. Add 1ml of treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5), add DTT only when the treatment solution is used) to the bacterial cells, and place it at 25°C for 20min. Centrifuge, discard the supernatant, add 1ml of 1M sorbitol (0.22μm aqueous membrane membrane sterilization) to resuspend the bacteria, centrifug...

Embodiment 3

[0118] Example 3, Saccharomyces cerevisiae engineering bacteria HC101 catalyzes the synthesis of hydrocortisone and 14α-hydroxycortisol

[0119] Shake flask fermentation catalysis: in solid selection medium (recipe: solid yeast selection medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage sign means g / 100mL) Activate the HC101 yeast strain in the medium, and prepare it in the corresponding liquid selection medium (recipe: liquid yeast selection medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu; each percentage sign represents g / 100mL) Seed solution (30°C, 250rpm, 16h), inoculate 1mL of the inoculum into three bottles of 500mL Erlenmeyer flasks containing 100mL of YPD liquid medium, culture at 30°C, 250rpm for 2 days, collect yeast cells at 5000rpm, and buffer with PBS The solution was washed and finally resuspended in a 250mL Erlenmeyer flask containing 30mL PBS, and the substrate RSA with a final concentration of 170mg / L was added to carry out ...

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Abstract

The invention discloses a steroid 11 beta-hydroxylase in curvularia lunata and a coding gene and an application thereof. The steroid 11 beta-hydroxylase is a protein (CL3-CYP021 protein) shown in SEQID No.1. In addition, a protein set composed of the steroid 11 beta-hydroxylase and the protein (CL3-CPR protein) shown in SEQ ID No.2 is further provided. Catalyzed synthesis of hydrocortisone (HC) and 14 alpha-hydroxylated cortisol is conducted by expressing CL3-CYP021 protein (or CL3-CYP021 and CL3-CPR protein) in a heterologous microorganism. The original filamentous fungus is replaced yeast for biocatalytic fermentation to produce HC. The advantages of shortening the fermentation time, simplifying the fermentation condition and separating and extracting steps and reducing the production cost can be achieved. The steroid 11 beta-hydroxylase has significant importance on replacement of the traditional hydrocortisone biocatalytic fermentation production mode.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to steroid 11β-hydroxylase in Curvularia lunae and its encoding gene and application. Background technique [0002] Hydrocortisone (HC) chemical name is 11β, 17α, 21-trihydroxypregn-4-ene-3,20-dione, which is an adrenal glucocorticoid drug and plays an important role in hormone drugs , whose structure is as figure 1 shown in . HC can affect glucose metabolism, and has antiviral, anti-inflammatory, anti-allergic and anti-shock effects [Dumas, B., et al., Hydrocortisone made in yeast: metabolic engineering turns a unicellular microorganism into a drug-synthesizing factory. Biotechnol J, 2006.1 (3):299-307.]. It is mainly used for the treatment of diseases caused by adrenal insufficiency and congenital adrenal hyperplasia. It can also be used for the treatment of inflammatory and allergic diseases such as bronchial asthma, rheumatoid arthritis, gout, and rheumatic fever. It can b...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N1/19C12P33/08C12P33/06
CPCC12N9/0073C12P33/06C12P33/08
Inventor 张学礼陈晶樊飞宇
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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