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Production of isoprenoids

An isoprenoid and heterologous nucleic acid technology, which is applied in the field of isoprenoid preparation and can solve the problems of complex commercial preparation and the like

Inactive Publication Date: 2010-10-20
AMYRIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third, some of the toxic solvents required for the extraction of isoprenoids require special handling and handling procedures, thus complicating the commercial preparation of isoprenoids

Method used

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  • Production of isoprenoids
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  • Production of isoprenoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0245] This example describes methods for preparing vectors for targeted integration of nucleic acids encoding enzymes, including those of the MEV pathway, into specific chromosomal loci in Saccharomyces cerevisiae.

[0246] Genomic DNA was isolated from Saccharomyces cerevisiae strains Y002 and Y003 (CEN.PK2 background MATA or MATαura3-52trp1-289leu2-3, 112his3Δ1MAL2-8C SUC2) (van Dijken et al. (2000) Enzyme Microb.Technol.26:706- 714), Y007 (S288C background MATA trp1Δ63) (ATCC No. 200873), and EG 123 (MATA ura3trp 1leu2his4can1) (Michaelis & Herskowitz. (1988) Mol. Cell. Biol. 8:1309-1318). Strains were grown overnight in liquid medium (YPD medium) containing 1% yeast extract, 2% bacto-peptone and 2% dextrose. Cells were separated from 10 mL of liquid medium by centrifugation at 3,100 rpm, washing the cell pellet in 10 mL of ultrapure water, and re-centrifugation. Genomic DNA was extracted using the Y-DER Yeast DNA Extraction Kit (Pierce Biotechnologies, Rockford, IL) foll...

Embodiment 2

[0264] This example describes methods that can be used to prepare plasmids and DNA fragments in the embodiments provided herein.

[0265] DNA fragment GAL7 4至1021 -HPH-GAL1 1637至2587 Insertion into TOPO ZEROBlunt II cloning vector (Invitrogen, Carlsbad, CA) generated plasmid pAM584. DNA fragment GAL7 4至1021 -HPH-GAL1 1637至2587 ORF fragment (GAL7 nucleotide position 4 to 1021) comprising Saccharomyces cerevisiae GAL7 gene (GAL7 4至1021 ), the hygromycin resistance cassette (HPH), and the 3 untranslated region (UTR) fragments of the Saccharomyces cerevisiae GAL1 gene (GAL1 nucleotide positions 1637 to 2587). As shown in Table 6, DNA fragments were generated by PCR amplification. Figure 4 F shows the map, and SEQ ID NO: 9 is the DNA fragment GAL7 4 至1021 -HPH-GAL1 1637至2587 the nucleotide sequence.

[0266]

[0267]

[0268] Generation of DNA fragment GAL80 by PCR amplification -50至-1 -NatR-GAL80 1309至1358 . This DNA fragment contains the nourthricin resistance ...

Embodiment 3

[0273] This example describes the generation of a Saccharomyces cerevisiae strain that can be used in embodiments provided herein.

[0274] Saccharomyces cerevisiae strains CEN.PK2-1C Y002 and Y003 were prepared by replacing the ERG9 promoter with the Saccharomyces cerevisiae MET3 promoter and replacing the ADE1 ORF with the Candida glabrata LEU2 gene (CgLEU2) (MATA or MATα; ura3-52; trp 1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2) (van Dijken et al. (2000) Enzyme Microb. Technol. 26(9-10): 706-714 ) to introduce inducible MEV pathway genes. This is done by using primers 50-56-pw100-G (SEQ ID NO: 10) and 50-56-pw101-G (SEQ ID NO: 11) (which contain 45 base pairs) PCR amplified KanMX-P of vector pAM328 MET3 Region (SEQ ID NO: 6), this region contains P MET3A promoter preceded by a kanamycin resistance marker flanked by the promoter and terminator of the Kluyveromyces lactis Tef1 gene; using 40% w / w polyethylene glycol 3350 (Sigma-Aldrich, St.Louis, MO), 100 mM lithium acetate ...

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Abstract

Provided herein are methods for a robust production of isoprenoids via one or more biosynthetic pathways. Also provided herein are nucleic acids, enzymes, expression vectors, and genetically modified host cells for carrying out the subject methods. Also provided herein are fermentation methods for high productivity of isoprenoids from genetically modified host cells.

Description

[0001] prior related application [0002] This application claims priority to US Provisional Application No. 60 / 994,790, filed September 20, 2007, and US Provisional Application No. 61 / 049,350, filed April 30, 2008, all of which are hereby incorporated by reference in their entirety. field of invention [0003] The present invention provides compositions, methods, and the like for efficiently preparing isoprenoids. The invention also provides nucleic acids, enzymes, expression vectors and genetically modified host cells for performing the method. The present invention also provides fermentation methods for producing isoprenoids in high yields from genetically modified host cells. Background of the invention [0004] Isoprenoids are ubiquitous in nature. They constitute a diverse family of more than 40,000 different products, many of which are essential to living organisms. Isoprenoids can maintain cell fluidity, electron transport, and other metabolic functions. A large ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/32C12P5/02C12P23/00C12N15/52
CPCY02E50/17C12N1/32C12P23/00C12P5/007Y02E50/10
Inventor 鹤田宽子雅各布·R·勒尼汉R·里根廷
Owner AMYRIS INC