Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof

A technology of cytochrome and nucleic acid molecules, applied in the direction of oxidoreductase, cells modified by introducing foreign genetic material, enzymes, etc., can solve the problem that the synthesis method is difficult to realize in large quantities

Inactive Publication Date: 2015-12-02
THE UNIV OF BRITISH COLUMBIA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although chemical methods to produce santalol and other sesquiterpenoids in sandalwood oil have been attempted, the highly complex structures of these compounds make economically viable synthetic methods for their preparation difficult to achieve in large quantities

Method used

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  • Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof
  • Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof
  • Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0505] Cloning and Sequencing of Sandalwood cDNA

[0506] In this example, RNA was extracted from wood samples of sandalwood trees, after which cDNA was generated and sequenced.

[0507] A. Isolation and Extraction of Sandalwood RNA

[0508] Several 25 mm holes were drilled in the lower stem of a mature sandalwood tree growing on land managed by the Forest Products Commission of Western Australia. Collect wood samples from the heartwood-sapwood transition zone and freeze them immediately in liquid nitrogen. RNA was extracted from 10 g of tissue using a protocol modified from Kolosova et al., (2004) BioTechniques 36:821-824. After precipitation with LiCl, RNA was stored at -80°C until cDNA synthesis.

[0509] B. Generation of Sandalwood cDNA Library

[0510] Total RNA (1.4 μg) of sandalwood xylem was reverse transcribed with SuperScriptIII reverse transcriptase (Invitrogen) at 42° C. for 1 hour using SMART-Creator kit (Clontech; SEQ ID NO: 20) with pDNR-LIB vector. The lig...

Embodiment 2

[0514] Identification of nucleic acids encoding sandalwood cytochrome P450 polypeptides

[0515] The assembled sequence (from Example 1) was compared with a set of known plant P450-encoding genes from CYP76 family P450 proteins by using BLASTx search (blast.ncbi.nlm.nih.gov; Altschul et al. (1990) JMolBiol 215:403-410). Comparisons were performed to identify cytochrome P450-encoding genes.

[0516] Table 4 below provides a summary of the seven isolated sequences identified in the BLASTx search (blast.ncbi.nlm.nih.gov; Altschul et al. (1990) JMolBiol 215:403-410), including the isolated sequence, lowest E value, P450 Matching gene IDs, CYP450 families, and number of reads in the database. The E-value (Expected Value) describes the number of matches expected to occur at random with a given score. In general, the smaller the E value, the greater the possibility of a significant match.

[0517]

[0518] Transcripts from this family were most abundant in the EST database and ...

Embodiment 3

[0520] Isolation of cDNA encoding cytochrome P450

[0521] Group 1 and Group 2 cDNA molecules of the CYP76 family identified in Example 2 (numbers 1 to 5 in the table above) were selected for cDNA isolation.

[0522] A. Cloning of CYP76 family members

[0523] Amplified by polymerase chain reaction (PCR) using gene-specific primers designed according to the ORFs of Group 1 and Group 2 (shown in Table 5 below) and using Phusion Hot Start II DNA polymerase (ThermoScientific) as per Example Full-length cDNA molecule of sandalwood cDNA (shown in SEQ ID NO: 1) prepared as described in 1. The PCR conditions are as follows:

[0524] 98°C for 3 minutes;

[0525] 2 cycles: 98°C for 10 seconds, Tm-2°C for 20 seconds, 72°C for 30 seconds;

[0526] 30 cycles: 98°C for 10 seconds, Tm for 20 seconds, 72°C for 30 seconds;

[0527] Final extension: 7 minutes at 72°C.

[0528] The Tm of the isogroup 1 is 55°C, and the Tm of the isogroup 2 is 52°C. The PCR product was gel purified and cl...

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Abstract

The invention provides cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and / or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells.

Description

[0001] related application [0002] 要求于2012年11月1日提交的题为“CYTOCHROMEP450ANDCYTOCHROMEP450REDUCTASEPOLYPEPTIDES,ENCODINGNUCLEICACIDMOLECULESANDUSESTHEREOF”的美国临时申请序列号61 / 796,129和于2013年5月31日提交的题为“CYTOCHROMEP450ANDCYTOCHROMEP450REDUCTASEPOLYPEPTIDES,ENCODINGNUCLEICACIDMOLECULESANDUSESTHEREOF”的美国临时申请序列号61 Priority of / 956,086. The subject matter of each of the applications cited above is incorporated by reference in its entirety. [0003] Sequence listings provided electronically are incorporated by reference [0004] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The size of the electronic file is 301 kilobytes, and the title is 229SEQPC1.txt. technical field [0005] Provided are cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase and cytochrome P450 reductase, nucleic acid molecules encoding P450 santalene oxidase, cytochrome P450 bergamotene oxidase and cytochrome P450 red...

Claims

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Application Information

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IPC IPC(8): C12N15/53A01H5/00C11B9/00C12N1/19C12N1/21C12N5/10C12N9/02C12N9/96C12P7/02
CPCC12N9/0042C11B9/00C12P5/007Y02P20/52C12N9/0071C12Y106/02004C12Y114/00C12P7/02C12P15/00
Inventor 卡尔·约尔格·伯尔曼马里亚·路易莎·迪亚兹沙韦杰西·莫尼奥迪斯
Owner THE UNIV OF BRITISH COLUMBIA
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