Steroidal 11β-hydroxylase from Curvularia lunae and its coding gene and application

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

Active Publication Date: 2021-09-17
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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  • Steroidal 11β-hydroxylase from Curvularia lunae and its coding gene and application
  • Steroidal 11β-hydroxylase from Curvularia lunae and its coding gene and application
  • Steroidal 11β-hydroxylase from Curvularia lunae and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1. Cloning and expression of β-hydroxylase at position 11 of Campylobacter crescentus

[0086] The cloning and expression of the gene is divided into the following three steps:

[0087] 1. Extraction of total RNA from Campylobacter crescentus

[0088] First, Campylobacter crescentus AS3.4381 (ATCC12017) was cultured on a plate for several days, and a certain number of spores were collected and inserted into 50 mL potato-glucose (PDA) medium, and cultured overnight to synthesize a large number of cells; then , centrifuged to collect the mycelium of Campylobacter crescentus, washed with potassium phosphate buffer (PBS), and finally resuspended with 50 mL of buffer and added the substrate hydrocortisone 21-acetate with a final concentration of 170 mg / L ( 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 1 mL of Trizol to a 2.0 mL ...

Embodiment 2

[0116] Example 2. Construction of Saccharomyces cerevisiae engineering strain HC101

[0117] The starting bacterium Saccharomyces cerevisiae BY4742 was grown overnight in selection medium. The composition of the liquid screening medium is as follows: SD-Trp (Beijing Pankino (Functional Genome) Technology Co., Ltd.), 2% glucose, 0.005% His., 0.01% Leu., 0.01% Ura. 100mL). Take 1ml (OD about 0.6-1.0) into 1.5ml EP tubes, centrifuge at 10000g at 4°C for 1 min, discard the supernatant, wash the precipitate with sterile water (4°C), centrifuge under the same conditions, and discard the supernatant. 1 ml of treatment solution (10 mM LiAc; 10 mM DTT; 0.6 M sorbitol; 10 mM Tris-HCl (pH 7.5) was added to the bacterial cells, and DTT was added only when the treatment solution was used), and placed at 25°C for 20 min. Centrifuge, discard the supernatant, add 1ml of 1M sorbitol (0.22μm aqueous membrane sterilization) to the cells to resuspend, centrifuge, discard the supernatant (resusp...

Embodiment 3

[0118] Example 3. Catalytic synthesis of hydrocortisone and 14α-hydroxycortisol by Saccharomyces cerevisiae engineering bacteria HC101

[0119] Shake flask fermentation catalysis: in solid selective medium (recipe: solid yeast screening medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage sign represents g / 100mL) Activated HC101 yeast strain in the corresponding liquid selection medium (recipe: liquid yeast screening medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu; each percentage sign indicates g / 100mL) The seed solution (30°C, 250rpm, 16h) was inoculated into three 500mL conical flasks containing 100mL YPD liquid medium with an inoculum volume of 1mL, 30°C, 250rpm shaking culture for 2 days, 5000rpm to collect yeast cells, and buffered with PBS The solution was washed and finally resuspended in a 250 mL Erlenmeyer flask containing 30 mL of PBS, and the substrate RSA with a final concentration of 170 mg / L was added to conduct a catalytic react...

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Abstract

The invention discloses a steroid 11β-hydroxylase from Curvularia lunae, its coding gene and application. The steroid 11β-hydroxylase provided by the present invention is the protein shown in SEQ ID No.1 (ie CL3-CYP021 protein). In addition, the present invention also provides a complete set of proteins consisting of steroid 11β-hydroxylase and the protein shown in SEQ ID No.2 (ie CL3-CPR protein). The present invention expresses CL3-CYP021 protein (or CL3-CYP021 and CL3-CPR protein) in heterologous microorganisms to catalyze the synthesis of hydrocortisone (HC) and 14α-hydroxycortisol. The present invention replaces the original filamentous fungus with yeast for biocatalytic fermentation to produce HC, which has the advantages of shortening fermentation time, simplifying fermentation conditions and separation and extraction steps, and reducing production costs, and is important for replacing traditional hydrocortisone biocatalytic fermentation production methods significance.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to steroid 11β-hydroxylase in Campylobacter crescentus and its encoding gene and application. Background technique [0002] Hydrocortisone (Hydrocortisone, HC) chemical name is 11β, 17α, 21-trihydroxypregn-4-ene-3,20-dione, is an adrenal glucocorticoid drug, occupies an important position in hormone drugs , whose structure is as figure 1 shown in. HC can affect glucose metabolism and has anti-viral, anti-inflammatory, anti-allergic and anti-shock effects [Dumas, B., et al., Hydrocortisone made in yeast: metabolic engineering turns a unicellularmicroorganism 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 bronchial asthma, rheumatoid arthritis, gout, rheumatic fever and other inflammatory and allergic d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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