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Diterpene-Producing Unicellular Organism

a technology of unicellular organisms and diterpenes, applied in the field of molecular genetics of yeast, metabolically engineered yeast, can solve the problems of inability to efficiently and inexpensively produce diterpenes, limited natural sources, and inability to commercialize total syntheses, etc., to achieve the effect of increasing sterol metabolic flux

Inactive Publication Date: 2008-01-24
RICE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0077] In the specific embodiment wherein a plant nucleic acid sequence is utilized, a skilled artisan is aware that, for instance, such as is required by E. coli, a plastidyl targeting sequence is be identified and removed. If the targeting sequence does not occur, the structural products are vulnerable to incorporation into inclusion bodies. However, the significant surplus of precursor generated by the compositions and methods of the present invention allow use of the full length plant nucleic acid sequence, which is a significant advantage of the present invention. In particular, this is advantageous in the methods of the present invention wherein a diterpene synthase is identified by enzymatic activity in the compositions of the present invention.
[0078] Alternatively, a unicellular organism comprising an isolated polypeptide encoding an amino acid sequence of a GGPP synthase, the soluble form of HMG-CoA reductase, the diterpene synthase, a gene that confers an increase in sterol metabolic flux, the squalene synthase, the hexaprenylpyrophosphate synthetase and / or the prenyltransferase are within the scope of the present invention. Non-limiting examples of amino acid sequences are provided herein.
[0079] In other embodiments, a modification is made that decreases, downregulates, diminishes or removes biosynthetic pathways that compete for GGPP bioavailability. In another specific embodiment, a hexaprenylpyrophosphate synthetase is modified to increase FPP flux into the engineered GGPP biosynthesis pathway. An example of a hexaprenylpyrophosphate synthetase is COQ1 (GenBank Accession No. J05547; SEQ ID NO:401). Hexaprenylpyrophosphate is the committed step of pathways which produce dolichols and ubiquinones. In an additional specific embodiment, a prenyltransferase is modified. Prenyltransferases are well known in the art. In a specific embodiment, the site of inhibition is protein farnesyltransferase (such as STE14; GenBank Accession No. L15442 (SEQ ID NO:402) or GenBank Accession No. L07952 (SEQ ID NO:403)), protein geranylgeranyltransferase I alpha subunit (such as CDC43; GenBank Accession No. M31114; SEQ ID NO:404), protein geranylgeranyltransferase I beta subunit (such as RAM2; GenBank Accession No. M88584; SEQ ID NO:405), protein geranylgeranyltransferase II alpha subunit (such as BET2; GenBank Accession No. M26597; SEQ ID NO:406), protein geranylgeranyltransferase II beta subunit (such as BET4; GenBank Accession No. U14132; SEQ ID NO:407), or a combination thereof.
[0080] A skilled artisan is aware of sequence repositories, such as GenBank, to obtain nucleic acid and amino acid sequences utilized in the present invention. Examples of geranylgeranyl pyrophosphate synthase nucleic acid sequences for the present invention include the following: U31632 (SEQ ID NO:1); AF049658 (SEQ ID NO:2); AK025139 (SEQ ID NO:3); AB000835 (SEQ ID NO:4); AJ276129 (SEQ ID NO:5); AB034250 (SEQ ID NO:6); AB034249 (SEQ ID NO:7); AW132388 (SEQ ID NO:8); AW034766 (SEQ ID NO:9); AI496168 (SEQ ID NO:10); AF081514 (SEQ ID NO:11); AF020041 (SEQ ID NO:12); X98795 (SEQ ID NO:13); X92893 (SEQ ID NO:14); X80267 (SEQ ID NO:15); L37405 (SEQ ID NO:16); U15778 (SEQ ID NO:17); L40577 (SEQ ID NO:18); M87280 (SEQ ID NO:19); L25813 (SEQ ID NO:20); and AF049659 (SEQ ID NO:21). A skilled artisan is aware that sequences unrelated to geranylgeranyl pyrophosphate synthase in those sequences which comprise large regions of the genome of a particular organism are not within the scope of the invention. In a preferred embodiment, SEQ ID NO:1 is utilized as a geranylgeranyl pyrophosphate synthase nucleic acid sequence in the cell of the invention.
[0081] Examples of geranylgeranyl pyrophosphate synthase amino acid sequences for the present invention include the following: AAA83262.1 (SEQ ID NO:22); AAC05595.1 (SEQ ID NO:23); AAC05273.1 (SEQ ID NO:24); NP—043281.1 (SEQ ID NO:25); BAB18334.1 (SEQ ID NO:26); AAC68232.1 (SEQ ID NO:27); CAC12434.1 (SEQ ID NO:28); BAB02385.1 (SEQ ID NO:29); CAB94793.1 (SEQ ID NO:30); AAF38891.1 (SEQ ID NO:31); BAA23157.1 (SEQ ID NO:32); BAA19583.1 (SEQ ID NO:33); CAB89115.1 (SEQ ID NO:34); AAD12206.1 (SEQ ID NO:35); AAD08933.1 (SEQ ID NO:36); CAB80510 (SEQ ID NO:37); CAB80347.1 (SEQ ID NO:38); CAB38744.1 (SEQ ID NO:39); BAA16690.1 (SEQ ID NO:40); AAD38295.1 (SEQ ID NO:41); BAA86285.1 (SEQ ID NO:42); BAA86284.1 (SEQ ID NO:43); CAB53152.1 (SEQ ID NO:44); CAB56064.1 (SEQ ID NO:45); BAA77251 (SEQ ID NO:46); CAB16803.1 (SEQ ID NO:47); CAB37502.1 (SEQ ID NO:48); AAD16018.1 (SEQ ID NO:49); AAC77874.1 (SEQ ID NO:50); CAA17477.1 (SEQ ID NO:51); AAC06913.1 (SEQ ID NO:52); CAA67330.1 (SEQ ID NO:53); AAB67731.1 (SEQ ID NO:54); CAA63486.1 (SEQ ID NO:55); CAA56554.1 (SEQ I) NO:56); AAA96328.1 (SEQ ID NO:57); AAA91949.1 (SEQ ID NO:58); AAA86688.1 (SEQ ID NO:59); AAA81879.1 (SEQ ID NO:60); AAA81312.1 (SEQ ID NO:61); AAA32797.1 (SEQ ID NO:62); BAB01876 (SEQ ID NO:63); BAA23157 (SEQ ID NO:64); AAD43148 (SEQ ID NO:65); NP 043281 (SEQ ID NO:66); B18334 (SEQ ID NO:67); E81650 (SEQ ID NO:68); T36967 (SEQ ID NO:69); S76966 (SEQ ID NO:70); A72041 (SEQ ID NO:71); T02429 (SEQ ID NO:72); S74538 (SEQ ID NO:73); S71230 (SEQ ID NO:74); S71231 (SEQ ID NO:75); AAC05595 (SEQ ID NO:76); AAC05273 (SEQ ID NO:77); BAB02387 (SEQ ID NO:78); BAB01936 (SEQ ID NO:79); AAF39709 (SEQ ID NO:80); BAA23158 (SEQ ID NO:81); E70365 (SEQ ID NO:82); S49625 (SEQ ID NO:83); P34802 (SEQ ID NO:84); and P80042 (SEQ ID NO:85). In a preferred embodiment, SEQ ID NO:22 is utilized as a geranylgeranyl pyrophosphate synthase amino acid sequence in the cell.
[0082] One non-limiting example of a gene that confers an increase to sterol metabolic flux as compared to native sterol metabolic flux levels is the upc2-1 allele. The upc2-1 allele comprises a guanine to adenine transition in the open reading frame designated YDR213W on chromosome IV (Leak et al., 1999; incorporated by reference herein in its entirety). The nucleic acid sequence is known and / or obtained through GenBank Accession No. Z68194 (SEQ ID NO:399), and Leak et al. (1999) describe the mutations associated with the upc2-1 allele. Incorporation of the upc2-1 allele conferred an increase in sterol metabolic flux as compared to native sterol metabolic flux levels, and thus, demonstrates that other such genes that confer the same biological activity, e.g., increase sterol metabolic flux levels, are expected to increase production in vivo of a diterpene and a diterpene precursor.

Problems solved by technology

However, natural sources are limited and commercial-scale total syntheses are usually impractical.
Therefore, an alternative source for the efficient and inexpensive production of diterpenes is lacking in the art.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Methods and Materials

[0133] Restriction enzymes, DNA polymerase I large (Klenow) fragment, T4 DNA polymerase, calf intestinal alkaline phosphatase, single-stranded binding protein, and M13K07 helper phage were purchased from New England BioLabs. Ligation reactions utilized Fast-Link DNA ligation kit purchased from Epicentre Technologies. Expand high fidelity polymerase kit used for PCR was purchased from Boehringer-Mannheim. Pfu polymerase and pGEM-T vector kit were purchased from Promega. pT7Blue T-vector was purchased from Novagen. Ex-Taq was purchased from Panvera. Zymolyase 100T was purchased from Seikagaku Corporation. Media ingredients were purchased from Fisher Biotech. Reagent chemicals were purchased from Sigma Chemical Company. Organic solvents were purchased from EM Science.

[0134] All E. coli cultures were cultivated in sterile Luria broth, LB. E. coli selective plates, LB-amp, were impregnated with ampicillin (250×stock: 25 mg / mL, filter sterilized) to a final concentr...

example 2

Production of GGPP in De Novosterol Biosynthesis

[0136] The amount of endogenous GGPP available in yeast having a native sterol biosynthetic pathway was established. FIG. 2 illustrates the main intermediates of de novo sterol biosynthesis in native yeast. FIG. 3 illustrates the terpene sub-classes and the sterol intermediates that serves as their biosynthetic precursors. Diterpenes are synthesized from (e.g., diterpene precursor) the metabolic intermediate geranylgeranyl pyrophosphate (GGPP).

[0137] Wild type yeast JBY575 (Alani et al., 1987) was transformed with a vector comprising a nucleic acid sequence encoding A. grandis abietadiene synthase (Mende et al., 1997). The culture media contained a polyaromatic resin that indiscriminately adsorbed molecules onto its surface. The induced culture was filtered and extracted to remove the diterpene product, and about 0.01 mg / L abietadiene (extrapolated from the internal standard longifolene at known concentrations) was observed by GC and...

example 3

Generation of GGPP-Synthesizing Yeast

[0138] Investigation of increasing GGPP biosynthesis included heterologous expression of the S. cerevisiae geranylgeranyl diphosphate synthase (BTS1) under transcriptional control of the inducible GAL1 promoter (Jiang et al., 1993).

[0139] The BTS1 nucleic acid sequence was isolated from λ phage received from ATCC using standard methods. Phage DNA containing S. cerevisiae BTS1 was digested with Xho1 and Kpn1 to release a 7 kb DNA fragment that subsequent to purification was ligated into pBluescript (II) KS+ digested with the same two enzymes. Propagation in DH5αyielded pEH1.0. Excess sequence was removed from the insert of pEH1.0 to yield pEH1.1.

[0140] The native promoter of BTS1 was removed by installing a Sal1 site immediately upstream of the start codon by site-directed mutagenesis employing the oligonucleotide sequence GP5S: 5′-TATCTTGGCCTCCATGTCGACTCCAGACTCGTAAAC-3′ (SEQ ID NO:408) and standard methodologies known in the art. The resulting...

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Abstract

The present invention is directed to a unicellular organism system, such as a yeast, for producing geranylgeranyl pyrophosphate and a diterpene in vivo. The yeast cell preferably comprises an inducible nucleic acid sequence encoding geranylgeranyl pyrophosphate synthase, an inducible nucleic acid sequence encoding a soluble form of HMG-CoA reductase, a nucleic acid sequence of an allele that confers an increase in sterol metabolic flux and, in the diterpene-producing cell, a diterpene synthase.

Description

FIELD OF THE INVENTION [0001] The present invention is directed to the fields of molecular biology, yeast molecular genetics and organic chemistry. More specifically, the present invention is directed to metabolically engineered yeast which produce diterpenes and diterpene precursors in vivo. BACKGROUND OF THE INVENTION [0002] Metabolic engineering employs recombinant DNA technology to restructure metabolic networks of microorganisms leading to improved production and yields of natural products (Bailey, 1991) This method alters a synchronous series of transformations, defined as a pathway, to produce metabolites. Such pathway manipulations require an awareness of inherent complex regulation and a comprehensive understanding of the discrete enzymatic transformations involved. Metabolic engineering recently emerged in response to efforts made towards improving cellular function by modifying and / or introducing specific biochemical processes (Stephanopoulos, 1996). [0003] Examples of th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P5/02C12N9/10C12P9/00
CPCC12N9/1085C12Y205/01029C12P9/00C12P5/007
Inventor MATSUDA, SEIICHI P.T.HART, ELIZABETH A.
Owner RICE UNIV
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