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Manufacturing method for 7-dehydrocholesterol and vitamin D3

A technology of dehydrocholesterol and manufacturing method, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, recombinant DNA technology, etc., and can solve the problems of adverse effects on the growth of host organisms and reduced reducing activity

Active Publication Date: 2017-08-18
KYOWA HAKKO BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, the reduction or loss of 7DHC reducing activity is considered to adversely affect the growth of its host organisms

Method used

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  • Manufacturing method for 7-dehydrocholesterol and vitamin D3
  • Manufacturing method for 7-dehydrocholesterol and vitamin D3
  • Manufacturing method for 7-dehydrocholesterol and vitamin D3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Preparation of clathomycetes microorganisms that lost 7DHC reducing activity

[0133] In the reference example, it is known that a large amount of cholesterol is accumulated Aurantiochytrium limacumum ATCCMYA-1381 (hereinafter referred to as MYA-1381) was deprived of 7DHC reducing activity by the following method.

[0134] Genomic DNA of MYA-1381 was prepared by a conventional method. Each DNA fragment was amplified by performing PCR using the genomic DNA as a template and DNA having the base sequence shown in "Primer pair" in Table 2 as a primer pair.

[0135] Table 2.

[0136]

[0137] In addition, using the drug resistance gene expression cassette (manufactured by Gene Bridges) as a template and DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 11 and 12 as a primer pair, PCR was performed to detect the hygromycin resistance gene (sequence No. 5) for amplification.

[0138] The three types of DNA fragments amplified above were used as a...

Embodiment 2

[0148] Manufacture of 7DHC

[0149] MYA-1381 (parent strain) and MYA-1381Δ7DHCR were cultured in the same manner as in Reference Example using a liquid medium (12% glucose, 1% yeast extract, 1% peptone, 50% artificial seawater). After the cultivation, the bacterial cells were collected and analyzed by gas chromatography in the same manner as in the reference example. The results are shown in Table 4 and Table 5.

[0150] In MYA-1381Δ7DHCR, cholesterol produced in the parent strain was not produced, and 7DHC was produced instead.

[0151] Table 4.

[0152] Cyticomycetes Growth (OD660nm) Cholesterol (mg / L) MYA-1381 54±9.3 435±41

[0153] table 5.

[0154] Cyticomycetes Growth (OD660nm) 7DHC (mg / L) MYA-1381Δ7DHCR 66±6.8 325±37

[0155] This result is substantially higher than that reported so far for budding yeast. This is considered to be due to the utilization of the clathromycin-like microorganisms that originally produce...

Embodiment 3

[0158] Manufacture of Vitamin D3

[0159] The 7DHC produced in Example 2 was irradiated with UV at 300 nm and treated at 100° C. for 30 minutes. After this treatment, analysis was performed by gas chromatography in the same manner as in the reference example.

[0160] As a result, it was confirmed that 7DHC was converted into vitamin D3.

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PUM

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Abstract

The present invention provides a method for manufacturing 7-dehydrocholesterol, the method being characterized by deleting, replacing or adding at least one base in a gene that is present on the chromosomal DNA in a parent strain and that codes a protein having 7-dehydrocholesterol reduction activity, whereby a labyrinthulean micro-organism that has decreased 7-dehydrocholesterol reduction activity in comparison to the parent strain or no 7-dehydrocholesterol reduction activity, and that produces 7-dehydrocholesterol is cultured in a culture medium, 7-dehydrocholesterol is generated in the culture, accumulated and then harvested from the culture medium. Also provided is a method for manufacturing vitamin D3, the method being characterized in that UV light is shone onto 7-dehydrocholesterol manufactured by the aforementioned manufacturing method.

Description

technical field [0001] The present invention relates to a method for producing 7-dehydrocholesterol (hereinafter, also referred to as 7DHC) using microorganisms of the class Lavirinchura, and a method for producing vitamin D3 characterized in that 7DHC produced by the production method is irradiated with ultraviolet rays . Background technique [0002] Vitamin D3 is a vitamin involved in the metabolism of calcium and phosphorus, maintenance of homeostasis, bone formation, etc., and is produced from cholesterol in the human body. However, it is produced in less than the required amount, so vitamin D3 needs to be obtained through food, medicines or supplements. [0003] Vitamin D3 contained in medicines or supplements is mainly produced by irradiating ultraviolet rays to 7DHC, which is produced by chemical conversion of cholesterol obtained from wool. However, due to the problems of BSE and zoonotic infections, animal-derived raw materials tend to be avoided intentionally as...

Claims

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Application Information

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IPC IPC(8): C12P33/00C07C401/00C12N15/00C12N15/09
CPCA61P3/02A61P19/08C07C401/00C07C2601/14C07C2602/24C07J9/00C12P33/00
Inventor 氏原哲朗三桥敏
Owner KYOWA HAKKO BIO CO LTD
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