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Microbial bacterial strain for producing sphingolipid alkali

A strain, the technology of Pichia pastoris, applied in the direction of microorganisms, methods based on microorganisms, treatment of microorganisms with electricity/wave energy, etc., can solve problems such as limitations

Inactive Publication Date: 2009-05-20
科兹莫弗姆有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This suggests that the activities of synthetase and long-chain base acetyl-CoA acetyltransferase are limited in the synthesis of acetylsphingosine in low-yielding bacteria

Method used

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  • Microbial bacterial strain for producing sphingolipid alkali
  • Microbial bacterial strain for producing sphingolipid alkali
  • Microbial bacterial strain for producing sphingolipid alkali

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment A

[0031] Preparation Example A: Preparation of Tetraacetylphytosphingosine Standards

[0032] A mixture of 1.0 g (2.8 mmol) phytosphingosine hydrochloride (Sigma), 2.7 ml (28 mmol) acetic anhydride, 2.1 ml (15 mmol) triethylamine and 10 ml pure chloroform was heated at reflux and stirred for 8 hours. After cooling to room temperature, the mixture was washed with a saturated aqueous solution of sodium bicarbonate until a pH of 7 was reached. The organic layer was then heated at 50 °C in MgSO 4 Dry over, filter and evaporate in vacuo. The residue was purified by column chromatography using a Merck: Lobor Fertich Saule GroBec (440-37) Lichroprep Si60 (40-60 μm) column. A mixture of dichloromethane and methanol (25:1) was used as eluent (aspiration speed of 10 ml / min). The obtained product is a white solid with a yield of 80% and a melting point of 41-43°C. The purity of the TAPS thus formed was in the range of CDCl 3 The purity was estimated to be 96% as determined by NMR (pro...

preparation Embodiment B

[0034] Preparation Example B: TAPS agar medium slant

[0035] 122 g of Nemoutex (Diastatische Produkten B.V.; Leiden, The Netherlands) were dissolved in 1 liter of water and sterilized at 110° C. for 60 minutes. The suspension was incubated overnight at room temperature and filtered to remove solid particles. The pH balance was at 6.4. 10 g / l agar (Bacto) was added and the solution was sterilized at 120°C for 30 minutes.

preparation Embodiment C

[0036] Preparation Example C: TAPS medium (for test tube culture and shake flask culture)

[0037] compound Quantity (g / l) KH-phthalic acid

NaCl

MgSO 4· 7H 2 o

CaCl 2· 2H 2 o

NH 4 Cl

K H 2 PO 4

(NH 4 ) 2 Fe(SO 4 ) 2

ZnSO 4

CuSO 4

MnSO 4

h 3 BO 3

Sodium molybdate

K1

Inositol

Niacin

Ca-D-panthotenate

Vitamin B1

p-amino benzoate vitamin

Vitamin B6

biotin

Yeast extract (Difco) 20

0.06

0.88

0.20

4.83

1.0

0.027

0.005

0.0075

0.0006

0.0006

0.0006

0.00015

0.059

0.003

0.003

0.003

0.002

0.0003

0.00001

1.0

[0038] In shake flasks and culture tubes, glucose was added to a final concentration of 33 and 7 g / l, respectively. After the ingredients were dissolved, the pH was adjusted to 5.4.

[0039] Add 30ml of culture medium into a 100ml Erlenmeyer flask (without baffle), and sterilize at 110°C for 30 m...

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Abstract

The invention relates to a microbial strain which can produce sphingosine, dihydrosphingosine, phytosphingosine and / or the derivatives thereof with improved level. In addition, the invention further discloses a method which is based on induced mutation or other selection technologies, and the strain is prepared accordingly. As an example, a mutant strain of pichia pastris is provided; and compared with the wild-type strain, compounds produced by the mutant strain are higher by about 50 percent.

Description

[0001] The present invention relates to methods for improving sphingolipid-producing microbial strains using mutagenesis and selection techniques. Background of the invention [0002] The term "sphingolipids" refers to a group of lipids derived from sphingosine. Sphingolipids are commonly found in cell membranes of animals, plants, and microorganisms. The actual function of sphingolipids in the human body is unknown, but this group of compounds is known to be involved in the transmission of electrical signals in the nervous system and in the stabilization of cell membranes. Glycosphingosines have also been suggested to play a role in the immune system: specific glycosphingosines function as receptors for bacterial toxins and also as receptors for bacteria and viruses. [0003] Sphingolipids contain sphingosine, dihydrosphingosine, or phytosphingosine, which acts as a base to form an amide bond with a fatty acid. Sphingosine or phytosphingosine bases can be used as starting m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N13/00C12P7/02C12R1/84
Inventor L·D·布尔I·V·D·维尔特
Owner 科兹莫弗姆有限公司
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