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Improved production of isoprenoids

A kind of technology from isopentenyl pyrophosphate and methylglutaryl, applied in the field of molecular biology

Inactive Publication Date: 2009-05-13
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several strain improvement studies have been performed to obtain high producers of astaxanthin derived from P. rhodozyma, however, for the past decade, these efforts have been limited to the use of conventional methods of mutagenesis and protoplast fusion

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Isolation of mRNA of P.rhodozyma and construction of a cDNA library

[0084] To construct the cDNA library of P. rhodozyma, total RNA was isolated by phenol extraction immediately after cell disruption and the mRNA of P. rhodozyma ATCC96594 was purified using an mRNA preparation kit (Clontech).

[0085] First, cells of the ATCC 96594 strain in 2-day cultures in 10 ml of YPD medium were harvested by centrifugation (1500×g, 10 minutes) and washed once with extraction buffer (10 mM sodium citrate / HCl (pH 6.2), containing 0.7 M HCl). After being suspended in the extraction buffer of 2.5ml, use a French press homogenizer (Ohtake Works Corp., Japan) at 1500kgf / cm 2 Cells were disrupted and immediately mixed with twice the volume of isogen (Nippon gene) according to the manufacturer's recommendation. In this step, 400 μg of total RNA was recovered.

[0086] Total RNA was then purified using the mRNA Isolation Kit (Clontech) as described by the manufacturer. Final...

Embodiment 2

[0088] Example 2 Cloning of part of the hmc (3-hydroxy-3-methylglutaryl-CoA synthase) gene of P. rhodozyma

[0089] To clone part of the hmc gene of P. rhodozyma, a degenerate PCR method was used. Based on the consensus sequences from known HMG-CoA synthase genes with other species, the nucleotide sequences of two mixed primers were designed and synthesized as shown in Table 1.

[0090] Table 1

[0091] Primer sequences for cloning hmc gene

[0092] Hmgsl GGNAARTAYACNATHGGNNYTNGGNCA (sense primer) (SEQ ID NO: 11)

[0093] Hmgs3 TANARNSNNSWNGTRTACATR TINCC (antisense primer) (SEQ ID NO: 12) CN=A, C, G or T, R=A or G, Y=C or T, H=A, T or C, S=C or G , W=A or T)

[0094] After 25 cycles of PCR reaction at 95° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 15 seconds using ExTaq (Takara Shuzo) as a DNA polymerase and the cDNA library obtained in Example 1 as a template, the reaction mixture was Perform agarose gel electrophoresis. PCR bands of the desired length we...

Embodiment 3

[0095] Example 3 Isolation of Genomic DNA of P. rhodozyma

[0096] To isolate the genomic DNA of P. rhodozyma, the QIAGEN genomic kit was used according to the manufacturer's protocol.

[0097] First, cells of P. rhodozyma ATCC 96594 strain in 100 ml YPD medium overnight culture were harvested by centrifugation (1500×g, 10 minutes) and washed once with TE buffer (10 mM Tris / HCl (pH 8.0), containing ImM EDTA). After suspending in 8ml Y1 buffer of QIAGEN genome kit, lysozyme was added at a concentration of 2mg / ml to enzymatically break down the cells and the reaction mixture was incubated at 30°C for 90 minutes, followed by the next step of extraction. Finally, 20 μg of genomic DNA was obtained.

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Abstract

The present invention relates to an isolated DNA sequence encoding an enzyme in the mevalonate pathway or the pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. Vectors and plasmids including such DNA, are also set forth. The invention also includes host cells transformed by such DNAs, or vectors or plasmids containing such DNAs. A process for the production of isoprenoids and carotenoids using such transformed host cells is also provided.

Description

[0001] This application is a divisional application of a Chinese invention application (name of invention: improved production of isoprenoids, filing date: May 6, 1999; application number 99106367.8). technical field [0002] The present invention relates to the molecular biology of preparing isoprenoids and their useful biomaterials. Background technique [0003] Ascoxanthin is known to be distributed in various organisms such as animals (eg, birds such as flamingos and scarlet ibis, and fish such as rainbow trout and salmon), algae, and microorganisms. It has also been recognized that astaxanthin possesses strong antioxidant properties against oxygen free radicals, which should be applicable for pharmaceutical use to prevent some diseases of living cells such as cancer. In addition, from the viewpoint of industrial application, the demand for astaxanthin used as a coloring agent is rapidly increasing especially in the industry of farmed fish such as salmon, because astaxan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/64C12P23/00C07H21/04C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12N9/02C12N9/10C12N9/12C12N9/88C12P7/04
CPCC12N9/1205C12N9/88C12P23/00C12N9/0004C12N9/1085C12N15/52
Inventor 星野立夫小岛一行世户口丰
Owner DSM IP ASSETS BV
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