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Nucleic acids encoding modified cytochrome P450 enzymes and methods of use thereof

A cytochrome and nucleic acid technology, applied in the field of genetically modified host cells encoding nucleic acids, can solve problems such as high cost, limited availability of natural sources of natural products, and low yield of natural products.

Inactive Publication Date: 2008-10-08
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Extraction of natural products from natural sources is limited by the availability of natural sources; synthetic or semi-synthetic production of natural products may suffer from low yields and / or high costs
These manufacturing issues and limitations of the natural source can limit the commercial and clinical development of this product

Method used

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  • Nucleic acids encoding modified cytochrome P450 enzymes and methods of use thereof
  • Nucleic acids encoding modified cytochrome P450 enzymes and methods of use thereof
  • Nucleic acids encoding modified cytochrome P450 enzymes and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0268] Example 1 : Production of 8-Hydroxy-delta-cadinene in Escherichia coli

[0269] This example describes the use of natural high levels of P450 (up to 30 mg L) involved in biosynthetic pathways -1 ) to produce substrates produced in vivo. Delta-cadinene-8-hydroxylase (CadH) is a plant-derived membrane-bound P450 that hydroxylates the sesquiterpene delta-cadinene (cad) in the biosynthesis of the plant defense compound gossypol to 8-hydroxyl - delta-junpinene (CadOH). Figure 1 schematically depicts the biosynthesis of CadOH in E. coli. The terpene synthase CadS in E. coli produces a substrate (Cad) from endogenous farnesyl pyrophosphate (FPP). Cad is further hydroxylated to the product (CadOH) through the action of CadH and its redox partner (CPR).

[0270] The CadH expression vector includes the CadH gene and the gene encoding the cytochrome P450 reductase (CPR) redox partner of C. tropicalis. This construct was co-transformed into E. coli with a compatible expressi...

Embodiment 2

[0289] Example 2 : Oxidation of amorphadiene by amorphadiene oxidase (AMO)

[0290] This example describes the in vivo (eg, in living cells in in vitro culture) oxidation of amorphadiene by amorphadiene oxidase (AMO), also known as CYP71AV1, isolated from Artemisia annua. Various constructs containing the nucleotide sequence encoding the AMO were generated and tested to optimize the yield of the oxidation product. Figure 22 Various AMO constructs are schematically depicted. (1) nAMO, isolated from the natural AMO sequence of Artemisia annua. (2) sAMO, based on a codon-optimized synthetic AMO gene expressed in Escherichia coli. (3) A13-AMO, a synthetic AMO gene in which the wild-type transmembrane domain is replaced by the A13 N-terminal sequence of Candida tropicalis. (4) A17-AMO, a synthetic AMO gene in which the wild-type transmembrane domain is replaced by the A17 N-terminal sequence of Candida tropicalis. (5) Bov-AMO, a synthetic AMO gene in which the wild-type tran...

Embodiment 3

[0293] Example 3 : Substrate Oxidation in Cells Expressing the Complete Mevalonate Pathway

[0294] Substrate oxidation was also performed in cells expressing the complete mevalonate pathway starting with acetyl-CoA. The following example of CadOH generation utilizes 3 plasmids: (1) pMevT containing AtoB, HMGR and HMGS, (2) pMBIS (contains DNA encoding MK, PMK, PMD, IDI (IPP isomerase) and IspA (FPP synthase) nucleotide sequence) and (3) expression vectors containing CadH, CPR and CadS. Cells were cultured at 20°C in TB glycerol supplemented with heme supplement, delta-aminolevulinic acid. The CadOH titer produced by the cells was as high as 60mg / L. data see Figure 32 .

[0295] In the second example, the following 2 plasmids were used to produce artemisinic acid: (1) containing the encoding MevT (AtoB, HMGR and HMGS) (see Figure 35A and B), expression vectors of the nucleotide sequences of the MBIS (MK, PMK, PMD, IDI, and IspA) and ADS operons, and (2) nucleosides en...

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Abstract

The present invention provides nucleic acids comprising nucleotide sequences encoding modified cytochrome P450 enzymes; as well as recombinant vectors and host cells comprising the nucleic acids. The present invention further provides methods of producing a functionalized compound in a host cell genetically modified with a nucleic acid comprising nucleotide sequences encoding a modified cytochrome P450 enzyme.

Description

[0001] cross reference [0002] This application claims priority to U.S. Provisional Patent Application No. 60 / 724,525, filed October 7, 2005, and U.S. Provisional Patent Application No. 60 / 762,700, filed January 27, 2006, which are hereby incorporated by reference in their entirety. field of invention [0003] The invention relates to the field of isoprenoid compound production, in particular to host cells genetically modified with nucleic acid encoding isoprenoid precursor modifying enzymes. Background of the invention [0004] Isopentenoids constitute a large and diverse class of natural products that share the same biosynthetic source, isopentenyl diphosphate (IPP), a metabolic precursor. Isopentenoid compounds are also known as "terpenes" or "terpenoids". More than 40,000 isopentenoids have been described. As can be seen by definition, isopentenoids consist of so-called isoprene (C5) units. The number of C atoms present in isopentenoids is generally divisible by 5 (C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N1/20C12N15/00C07H21/04
Inventor M·C·-Y·常R·伊切斯D·-K·罗吉国靖雄J·D·基斯林
Owner RGT UNIV OF CALIFORNIA
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