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WO1999058649A1 - Techniques visant a modifier la production d'isopentenyl pyrophosphate, de dimethylallyl pyrophosphate et/ou d'isoprenoides - Google Patents

Techniques visant a modifier la production d'isopentenyl pyrophosphate, de dimethylallyl pyrophosphate et/ou d'isoprenoides Download PDF

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WO1999058649A1
WO1999058649A1 PCT/US1999/007041 US9907041W WO9958649A1 WO 1999058649 A1 WO1999058649 A1 WO 1999058649A1 US 9907041 W US9907041 W US 9907041W WO 9958649 A1 WO9958649 A1 WO 9958649A1
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acid sequence
nucleic acid
heterologous nucleic
cell
host cell
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PCT/US1999/007041
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Francis X. Cunningham
Dean Dellapenna
Charles P. Moehs
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University Of Maryland
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Priority to AU39645/99A priority Critical patent/AU3964599A/en
Priority to EP99922707A priority patent/EP1078045A1/fr
Publication of WO1999058649A1 publication Critical patent/WO1999058649A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)

Definitions

  • the present invention is directed to genes encoding deoxyxylulose-5- phosphate synthase (dxps), glyceraldehyde-3-phosphate dehydrogenase (gapd), and the lytB gene product, as well as vectors containing the same and hosts transformed with said vectors.
  • the present invention also provides methods for modifying the production of isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP) and/or an isoprenoid compound (i.e., a compound derived from IPP and/or DMAPP). Additionally, the present invention provides a method for screening for procaryotic and eukaryotic genes encoding enzymes that participate in the nonmevalonate pathway leading to IPP and DMAPP.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • an isoprenoid compound i.e., a compound derived from
  • IPP isopentenyl pyrophosphate
  • DMAPP allylic isomer dimethylallyl pyrophosphate
  • the C 40 skeleton of plant carotenoid pigments is assembled from two molecules of a C 20 compound, geranylgeranyl pyrophosphate (GGPP), that is itself assembled from 3 units of IPP and 1 unit of DMAPP, and that also serves as a precursor for many other branches of the isoprenoid pathway in plants.
  • GGPP geranylgeranyl pyrophosphate
  • Isoprenoids are formed from IPP and DMAPP in at least three different compartments of plants cells: the cytosol/endoplasmic reticulum, the mitochondria (and/or Golgi apparatus), and the plastids (McGarvey and Croteau, 1995).
  • the source of IPP and DMAPP for isoprenoid biosynthesis in these different compartments has long been a matter of some controversy and debate (see Bach, 1995; McGarvey and Croteau, 1995).
  • the well-known "classical” or acetate/mevalonate route to IPP and DMAPP proceeds from acetyl-CoA via 3- hydroxy-3-methylglutaryl-CoA (HMG-CoA) and mevalonic acid (MVA).
  • HMGR HMG-CoA reductase
  • coli genome the entire sequence for which has now been deposited in GenBank, does not identify any open reading frames with significant similarity to known bacterial, plant or mammalian HMG-CoA reductases. Nor can we discern an HMG-CoA reductase homologue in the genome sequence data base of the cyanobacterium Synechococystis 6803. Because ancestors of the cyanobacteria are the presumptive progenitors of plant chloroplasts, the non- mevalonate route to IPP in these photosynthetic procaryotes is likely to resemble that in plant chloroplasts.
  • DXP deoxyxylulose-5-phosphate
  • DXP also is a substrate leading to other essential compounds in these organisms, including thiamin and pyridoxal (see Lois et al., 1998). Therefore the biochemical reaction catalyzed by the DXP synthase enzyme is not dedicated or restricted to providing substrate for the pathway or pathways leading to IPP and/or DMAPP, and isoprenoids derived therefrom. Because the product of the reaction is shared with several other pathways, the DXP synthase enzyme would not seem to be an obvious candidate for a controlling step in the pathway leading to isoprenoids. An ability to modify isoprenoid production, enhancing production of desired compounds or reducing that of undesirable compounds, would be quite advantageous in many applications.
  • lytB is not an enzyme directly involved in the pathway leading to peptidogiycan but rather exerts some control on the production of the global regulator molecule guanosine 3',5'-bisphosphate (ppGpp) (Gustafson et al., 1993; Rodionov and Ishiguro, 1995).
  • ppGpp global regulator molecule guanosine 3',5'-bisphosphate
  • lytB may encode an enzyme that catalyzes one of the later reactions in the nonmevalonate pathway leading to IPP and DMAPP in certain bacteria and in plant chloroplasts.
  • lytB may encode an enzyme that catalyzes one of the later reactions in the nonmevalonate pathway leading to IPP and DMAPP in certain bacteria and in plant chloroplasts.
  • a subject of the present invention is an isolated nucleic acid sequence which encodes for a protein having DXP synthase enzyme activity, wherein the nucleic acid sequence is at least 85% identical to SEQ ID NO: 1 or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 85% identical to SEQ ID NO: 2.
  • Another subject of the present invention is an isolated nucleic acid sequence which encodes for a protein having GAP dehydrogenase enzyme activity, wherein the nucleic acid sequence is at least 85% identical to SEQ ID NO: 3 or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 85% identical to SEQ ID NO: 4.
  • a further subject of the present invention is an isolated nucleic acid sequence which encodes for a protein having LYTB activity, wherein the nucleic acid sequence is at least 85% identical to SEQ ID NO: 5 or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least
  • the invention also includes vectors which comprise any of the nucleic acid sequences listed above, and host cells transformed with such vectors.
  • Another subject of the present invention is a method of enhancing the production of IPP, DMAPP and/or an isoprenoid compound in a host ceil, comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence which encodes for a protein having DXP synthase, GAP dehydrogenase and/or LYTB activity, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence, thereby producing the protein.
  • Yet another subject of the present invention is a method of modifying the production of IPP, DMAPP and/or an isoprenoid compound in a host cell, comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence which encodes for a protein which modifies DXP synthase, GAP dehydrogenase and/or LYTB activity in the host cell, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence, thereby producing the protein.
  • the present invention also includes a method of expressing, in a host cell, a heterologous nucleic acid sequence which encodes for a protein having DXP synthase, GAP dehydrogenase and/or LYTB activity, the method comprising inserting into the host cell a vector comprising the heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence.
  • Another subject of the present invention is to provide a method for screening for eukaryotic genes which encode enzymes involved in isoprenoid biosynthesis and metabolism.
  • FIG 1 is an illustration showing that IPP and DMAPP serve as the central metabolites leading to an immense variety of different isoprenoid compounds in plants, bacteria, and other organisms.
  • the mevalonic acid pathway via acetyl-CoA in the cytosol of plants and in animals is well characterized.
  • a second route from pyruvate and GAP, not yet elucidated, is thought to operate in the chloroplasts of plants and algae, in cyanobacteria, and in many bacteria. Later reaction steps in the pathway remain to be determined and are therefore denoted with question marks.
  • the pathway is shown leading to a box containing IPP and DMAPP because it has not been established which of these two is the initial product of this pathway.
  • G3P glyceraldehyde-3-phosphate
  • G3PD GAP dehydrogenase
  • DXPS DXP synthase
  • IPP isopentenyl pyrophosphate
  • IPI IPP isomerase
  • DMAPP dimethylallyl pyrophosphate
  • FPP farnesyi pyrophosphate
  • GPP geranyl pyrophosphate GPP geranyl pyrophosphate
  • GGPP geranylgeranyi pyrophosphate.
  • Figure 2 illustrates a route to IPP/DMAPP from pyruvate and giyceraldehyde-3- phosphate (GAP), which is thought to operate in the chloroplasts of plants and algae, in cyanobacteria, and in many bacteria including E. coli. Later reaction steps in the pathway remain to be determined and are therefore denoted with question marks. The pathway is shown leading to a box containing IPP and DMAPP because it has not been established which of these two is the initial product of this pathway. Enzymes of interest are shown in bold white text in a black box.
  • GAP giyceraldehyde-3- phosphate
  • DMAPP dimethylallyl pyrophosphate
  • DXPS deoxyxylulose-5- phosphate synthase
  • DXPR deoxyxylulose-5-phosphate reductoisomerase
  • FPS farnesyl pyrophosphate synthase, GAPD, glyceraldehyde-3-phosphate dehydrogenase
  • GGPP geranylgeranyl pyrophosphate
  • IPI isopentenyl pyrophosphate isomerase
  • IPP isopentenyl pyrophosphate.
  • FIG. 3 illustrates that the C 40 carotenoid phytoene is derived by a head-to-head condensation of two molecules of the C 20 GGPP compound, which itself is assembled from 3 molecules of IPP and 1 molecule of DMAPP.
  • FPP farnesyl pyrophosphate
  • GPP geranyl pyrophosphate
  • PPPP prephytoene pyrophosphate.
  • Figure 4 is a schematic illustration and restriction map of plasmid pAC-LYC (Cunningham et al., 1994) which contains genes (crtE, crtB, and crtl) of Erwinia herbicola encoding all of the enzymes required for production of the pink-colored isoprenoid pigment iycopene from the colorless IPP and DMAPP compounds.
  • Cm chloramphenicol resistance gene.
  • Figure 5A is a cDNA sequence and Figure 5B is the predicted amino acid sequence of a putative DXP synthase isolated from a flower cDNA library of Tagetes erecta (SEQ ID NOS: 1 and 2). The cDNA is incorporated into the plasmid pMarDXPS.
  • Figure 6A is a cDNA sequence and Figure 6B is the predicted amino acid sequence of a chloroplast isoform of GAP dehydrogenase isolated from Arabidopsis thaliana (SEQ ID NOS: 3 and 4). The cDNA is incorporated into the plasmid pAtG3PD.
  • Figure 7A is a cDNA sequence and Figure 7B is the predicted amino acid sequence of a LYTB protein isolated from an Adonis palaestina flower cDNA library (SEQ ID NOS: 5 and 6). The sequence is of clone Ipi3 except that the 14 bp at the N- terminus were obtained from the slightly longer cDNA clone Ipi18. The cDNA is incorporated into the plasmid pApLYTB.
  • Figure 8 is an alignment of the predicted amino acid sequences of LYTB from Adonis palaestina, Synechocystis PCC6803 and E. coli.
  • the N-terminal extension of the Adonis polypeptide, relative to that of Synechocystis PCC6803, is predicted by the program ChloroP (Emanuelsson et al., 1999) to constitute a chloroplast transit peptide, serving to target the polypeptide to this organelle in plants. Black boxes with white letters are used where all three of the aligned residues are identical. Grey boxes with black letters are used where two of the three aligned residues are identical.
  • Figure 9 is a schematic representation of the mapping of an E. coli genomic fragment to ascertain which of the genes in this fragment will enhance or impair lycopene accumulation in E. coli.
  • Deletion mapping of an E. coli genomic fragment (the insert in plasmid pEc3.9 is essentially identical to GenBank U32768: 4879..8819) with genes encoding DXP synthase (orf620), FPP synthase (ispA) and the small subunit of exonuclease VII (xseB). Numbers to the right indicate relative lycopene accumulation per mL of liquid culture with plasmids in lycopene- accumulating E. coli strain TOP10.
  • the insert in pEc3.9 is oriented in the forward direction in the multicopy plasmid vector pBluescript SK-.
  • the EcoRI and Smal sites in the vector preceding the genomic fragment and a Kpn ⁇ site following it were used, along with ⁇ /del, Smal, and Sa/I sites in the genomic DNA, to construct the deletion subclones illustrated. Incomplete genes and open reading frames are not shown.
  • isoprenoid is intended to mean any member of the class of naturally occurring compounds whose carbon skeletons are composed, in part or entirely of isopentyl C 5 units.
  • the carbon skeleton is of an essential oil, a fragrance, a rubber, a carotenoid, or a therapeutic compound, such as paclitaxel.
  • GenBank public databases
  • a recent publication discusses the distribution of this gene in various bacteria.
  • a homologue in the plant Adonis palaestina reveals for the first time that this gene is present and expressed in a eucaryotic organism.
  • a recently deposited genomic DNA sequence for the green plant Arabidopsis thaliana (GenBank accession number AL035521 ) contains what appears to be a gene (the probable coding sequence is interrupted by several apparent introns) encoding LYTB in this organism.
  • the predicted sequence of this Arabidopsis LYTB is somewhat uncertain (a comparison of the Adonis sequence with that listed given in AL035521 for Arabidopsis suggests that several of the exon-intron junctions predicted for this gene in the GenBank record are incorrect), but sequence identity in a comparison with the Adonis sequence is ca. 80% or more.
  • Partial cDNA sequences in the data base of expressed sequence tags (dbEST) that predict peptides with sequence similarity to portions of the Adonis LYTB sequence indicate that homologues exist and mRNAs encoding LYTB are produced in several other plant species including rice (D45948), loblolly pine (AA556723) and soybean (AI437981 ). Both the Adonis and Arabidopsis predicted amino acid sequences are more than 60% identical to that predicted by the cyanobacterium Synechocystis PCC6803 gene, and the two plant and the cyanobacterial sequences are more than 30% identical to the predicted E. coli gene product. An alignment of the Adonis, Synechocystis PCC6803 and E. coli predicted amino acid sequences is shown in Figure 7. A number of regions and residues conserved in LYTB are indicated in this Figure.
  • LYTB activity is intended to mean the ability of LYTB to affect the production of IPP, DMAPP and/or isoprenoids in a host cell containing the lytB gene or DNA copy of the lytB mRNA. It has not yet been confirmed that the LYTB protein is, in fact, an enzyme. The precise role of the LYTB protein in affecting the
  • the present invention is directed to an isolated nucleic acid sequence which encodes for a protein having DXP synthase enzyme activity, wherein the nucleic acid sequence is at least 85% identical to SEQ ID NO: 1 or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 85% identical to SEQ ID NO: 2.
  • the nucleic acid sequence is at least 90%, at least 95% or completely identical to SEQ ID NO: 1
  • the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 90%, at least 95% or completely identical to SEQ ID NO: 2.
  • nucleic acid sequence which encodes for a protein having GAP dehydrogenase enzyme activity, wherein the nucleic acid sequence is at least 85% identical to SEQ ID NO: 3 or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 85% identical to SEQ ID NO: 4.
  • the nucleic acid sequence is at least 90%, at least 95% or completely identical to SEQ ID NO: 3, or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 90%, at least 95% or completely identical to SEQ ID NO: 4.
  • a further subject of the present invention is an isolated nucleic acid sequence which encodes for a protein having LYTB activity, wherein the nucleic acid sequence is at least 85% identical to SEQ ID NO: 5 or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 85% identical to SEQ ID NO: 6.
  • the nucleic acid sequence is at least 90%, at least 95% or completely identical to SEQ ID NO: 5, or the nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 90%, at least 95% or completely identical to SEQ ID NO: 6.
  • sequence similarity is measured using sequence analysis software, for example, the Sequence Analysis software package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705), MEGAIign (DNAStar, Inc., 1228 S. Park St., Madison, Wisconsin 53715), or MacVector (Oxford Molecular Group, 2105 S.
  • sequence analysis software for example, the Sequence Analysis software package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705), MEGAIign (DNAStar, Inc., 1228 S. Park St., Madison, Wisconsin 53715), or MacVector (Oxford Molecular Group, 2105 S.
  • Conservative (i.e. similar) substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid, glutamic acid, asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Substitutions may also be made on the basis of conserved hydrophobicity or hydrophilicity (see Kyte and Doolittle, J. Mol. Biol.
  • the length of comparison sequences is at least 50 nucleotides, more preferably at least 60 nucleotides, at least 75 nucleotides or at least 100 nucleotides. It is most preferred if comparison is made between the nucleic acid sequences encoding the protein coding regions necessary for protein activity. If comparison is made between amino acid sequences, preferably the length of comparison is at least 20 amino acids, more preferably at least 30 amino acids, at least 40 amino acids or at least 50 amino acids.
  • the present inventors have isolated eukaryotic genes encoding DXP synthase from Tagetes erecta (marigold), GAP dehydrogenase from Arabidopsis thaliana, and LYTB from Adonis palaestina. All were identified on the basis of an enhancement of lycopene accumulation in E. coli. The E. coli DXP synthase was also identified in this same way.
  • Suitable vectors according to the present invention comprise a gene encoding one or more of the above-identified enzymes involved in IPP, DMAPP and/or isoprenoid biosynthesis or metabolism, wherein the gene is operably linked to a suitable promoter.
  • suitable promoters for the vector can be constructed using techniques well known in the art (see, for example, Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
  • Suitable vectors for prokaryotic expression include pACYC184, pUC119, and pBR322 (available from New England BioLabs, Bevery, MA) and pTrcHis (Invitrogen) and pET28 (Novagen) and derivatives thereof.
  • the vectors of the present invention can additionally contain regulatory elements such as promoters, repressors, selectable markers such as antibiotic resistance genes, etc., the construction of which is very well known in the art.
  • One or more of the genes encoding the enzymes as described above when cloned alone or in combination into a suitable expression vector, can be used to overexpress these enzymes in a plant expression system or to inhibit the expression of these enzymes.
  • a vector containing one or more of the genes of the invention may be used to increase the amount of isoprenoids in an organism and thereby alter the nutritional or commercial value or pharmacology of the organism.
  • the present invention includes a method of enhancing the production of IPP, DMAPP and/or an isoprenoid in a host cell, relative to an untransformed host cell, the method comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence which encodes for a protein having DXP synthase, GAP dehydrogenase and/or LYTB activity, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence.
  • the invention also includes a method of modifying the production of IPP, DMAPP and/or an isoprenoid in a host cell, the method comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence which encodes for a protein which modifies DXP synthase, GAP dehydrogenase and/or LYTB activity in the host cell, relative to an untransformed host cell, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence.
  • the invention further includes a method of expressing, in a host cell, a heterologous nucleic acid sequence which encodes for a protein having DXP synthase, GAP dehydrogenase and/or LYTB activity, the method comprising inserting into the host cell a vector comprising the heterologous nucleic acid
  • heterologous nucleic acid sequence is operably linked to a promoter, and expressing the heterologous nucleic acid sequence.
  • the invention also includes a method of expressing, in a host cell, a heterologous nucleic acid sequence which encodes for a protein which modifies DXP synthase, GAP dehydrogenase and/or LYTB activity in the host cell, relative to an untransformed host cell, the method comprising inserting into the host cell a vector comprising the heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is operably linked to a promoter, and expressing the heterologous nucleic acid sequence.
  • the isoprenoid comprises a compound derived from at least one member selected from the group consisting of geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP).
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • the isoprenoid comprises at least one member selected from the group consisting of a diterpene, a carotenoid, an essential oil, a fragrance, an isoprene, a cytokinin, a rubber, a quinone, a sterol, a hopanoid, a triterpene, a steroid, a prenylated protein, a phytoalexin, a gibberellin, a tocopherol, a dolichol, a chlorophyll and a therapeutic compound.
  • the isoprenoid comprises at least one member selected from the group consisting of an essential oil, a fragrance, a rubber, a carotenoid and a therapeutic compound, such as paclitaxel.
  • the heterologous nucleic acid sequence may originate from a eukaryotic or procaryotic cell.
  • tissue from we intend to mean that the sequence information for the heterologous nucleic acid came from the eukaryotic or the procaryotic cell.
  • specific nucleic acid itself does not have to be from the organism.
  • the nucleic acid may come from the organism, or it may be synthetically produced using recombinant nucleic acid techniques known in the art.
  • the heterologous nucleic acid sequence comprises a nucleotide sequence for dxps, gapd and/or lytB. It is most preferred that the heterologous nucleic acid sequence comprises a nucleotide sequence which encodes a dxps, gapd and/or a lytB gene and is at least 85% identical, preferably at least 90%, at least 95% or completely identical, to SEQ ID NO: 1 , 3 and/or 5, respectively, or the
  • nucleic acid sequence encodes a protein which has an amino acid sequence which is at least 90%, at least 95% or completely identical to SEQ ID NO: 2, 4 and/or 6, respectively. Identity is determined as noted above.
  • modifying the production in the methods of the invention means that the amount of target compounds produced (e.g., IPP, DMAPP and/or isoprenoids) can be enhanced or reduced, as compared to an untransformed host cell.
  • target compounds produced e.g., IPP, DMAPP and/or isoprenoids
  • the production or the biochemical activity of the target compounds (or the enzymes which catalyze their formation) may be reduced or inhibited by a number of different approaches available to those skilled in the art, including but not limited to such methodologies or approaches as anti-sense (e.g., Gray et al., 1992), ribozymes (e.g., Wegener et al., 1994), co-suppression (e.g.
  • Host systems according to the present invention can comprise any organism that utilizes a nonmevalonate (i.e., via DXP) pathway for production of IPP and/or DMAPP.
  • Organisms which produce isoprenoids using IPP and/or DMAPP derived from a nonmevalonate pathway include plants, algae, certain bacteria, cyanobacteria and other photosynthetic bacteria. Transformation of these hosts with vectors according to the present invention can be done using standard techniques. See, for example, Sambrook et al., Molecular Cloning A Laboratory JVlanual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology. Greene Publishing and Wiley Interscience, New York, 1991.
  • transgenic organisms can be constructed which include the nucleic acid sequences of the present invention.
  • the incorporation of these sequences can allow the controlling of isoprenoid biosynthesis, content, or composition in the host cell.
  • transgenic systems can be constructed to incorporate sequences which allow for the overexpression of the various nucleic acid sequences of the present invention.
  • Transgenic systems can also be constructed to incorporate sequences which allow for the overexpression of the various nucleic acid sequences of the present invention.
  • Such systems may contain anti-sense expression of the nucleic acid sequences of the present invention. Such anti-sense expression would result in the accumulation of the substrates of the enzyme encoded by the sense strand.
  • the plasmid pAC-LYC (Cunningham et al., 1994 and 1996) contains genes encoding all of the enzymes required for the formation of lycopene from IPP and DMAPP (see Figure 3).
  • Cells of E.coli containing the plasmid pAC-LYC accumulate the carotenoid lycopene and thereby form colonies on solid growth medium that are pink in color (Cunningham et al., 1994).
  • cyanobacteria, or bacteria are introduced into the iycopene-accumulating E.
  • cDNAs and genes isolated with this screening methodology are: Tagetes erecta (marigold) cDNAs encoding a homologue of DXP synthase (the DNA sequence of the longest is given in Figure 4), an E.coli genomic clone containing the DXP synthase (and no other complete open reading: a genomic
  • the mechanism of enhancement of isoprenoid production by introduction of cDNAs encoding LYTB is not yet known.
  • the nonmevalonate pathway leading to IPP and/or DMAPP has not yet been elucidated and these cDNAs may encode an enzyme subsequent to DXP synthase in the pathway.
  • the enhancement of isoprenoid accumulation may involve a mechanism less direct (e.g., as for GAP dehydrogenase, involvement in biochemical reactions that utilize or supply the substrates GAP and pyruvate, or by exerting a regulatory influence on isoprenoid pathways).
  • a size-fractionated 2-3 kB cDNA library of A. thaliana in lambda ZAPII was obtained from the Arabidopsis Biological Resource Center at The Ohio State University (stock number CD4-15).
  • Transformants were spread on large (150 mm diameter) LB agar petri plates containing antibiotics to provide for selection of cDNA clones (ampiciliin) and maintenance of pAC-LYC (chloramphenicol). Approximately 10,000 colony forming units were spread on each plate. Petri plates were incubated at room temperature for 2 to 7 days to allow maximum color development. Plates were screened visually with the aid of an illuminated 3x magnifier and a low power stage-dissecting microscope for the rare deep pink colonies that could be observed in the background of paler pink colonies.
  • EXAMPLE 3 Enhancement of Carotenoid Accumulation in E. coli by cDNAs and genes encoding LYTB, DXP synthase, IPP isomerase and GAP Dehydrogenase Individually and in
  • a synechocystis PCC6803 lytB gene was less effective but gave a significant enhancement as well. See Table 1.
  • GAP dehydrogenase was slightly detrimental to pigment accumlation in liquid culture, in contrast to earlier observed enhancement for cultures grown on solid media. The influence of this gene on pigment accumulation may depend on the specific growth regimen.
  • a combination of DXP synthase with IPP isomerase was significantly more effective at enhancing pigment accumulation than was either of the individual cDNAs.
  • EXAMPLE 4 Enhancement and Reduction of Lycopene Accumulation in E. coli containing multiple copies of genes encoding DXP synthase and Farnesyl Pyrophosphate synthase. respRctiv ⁇ ly

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Abstract

Cette invention a trait à des techniques permettant d'augmenter ou de diminuer dans une cellule hôte les teneurs en isopentényl pyrophosphate (IPP), en diméthylallyl pyrophosphate (DMAPP) et/ou en isoprénoïdes. Elle concerne également des séquences nucléotidiques codant la désoxyxylulose-5-phosphate synthase (DXP), la glycéraldéhyde 3 phosphate déshydrogénase (GAP) et une protéine à activité de type lytB. Elle porte, de surcroît, sur des vecteurs renfermant ces mêmes séquences ainsi que sur des cellules hôtes transformées par ces vecteurs.
PCT/US1999/007041 1998-05-13 1999-05-13 Techniques visant a modifier la production d'isopentenyl pyrophosphate, de dimethylallyl pyrophosphate et/ou d'isoprenoides WO1999058649A1 (fr)

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AU39645/99A AU3964599A (en) 1998-05-13 1999-05-13 Methods of modifying the production of isopentenyl pyrophosphate, dimethylallyl pyrophosphate and/or isoprenoids
EP99922707A EP1078045A1 (fr) 1998-05-13 1999-05-13 Techniques visant a modifier la production d'isopentenyl pyrophosphate, de dimethylallyl pyrophosphate et/ou d'isoprenoides

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WO2000044912A1 (fr) * 1999-01-28 2000-08-03 Royal Holloway And Bedford New College Manipulation de l'expression d'un isoprenoide
WO2001094561A2 (fr) * 2000-06-05 2001-12-13 Adelbert Bacher Voie isoprenoide non mevalonate
WO2002012478A2 (fr) * 2000-08-07 2002-02-14 Monsanto Technology, Llc Genes de la voie methyl-d-erythritol phosphate
WO2002020733A2 (fr) * 2000-09-01 2002-03-14 E.I. Dupont De Nemours And Company Genes impliques dans la production de composes isoprenoides
DE10119905A1 (de) * 2001-04-23 2002-10-24 Jomaa Pharmaka Gmbh Inaktivierung von Genen des MEP-Wegs
EP1434867A2 (fr) * 2001-04-24 2004-07-07 E.I. Du Pont De Nemours And Company Genes impliques dans la production de compose isoprenoide
US6872815B1 (en) 2000-10-14 2005-03-29 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7067647B2 (en) 1999-04-15 2006-06-27 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US7112717B2 (en) 2002-03-19 2006-09-26 Monsanto Technology Llc Homogentisate prenyl transferase gene (HPT2) from arabidopsis and uses thereof
US7122331B1 (en) 1999-08-04 2006-10-17 Wolfgang Eisenreich Isoprenoid biosynthesis
US7161061B2 (en) 2001-05-09 2007-01-09 Monsanto Technology Llc Metabolite transporters
US7230165B2 (en) 2002-08-05 2007-06-12 Monsanto Technology Llc Tocopherol biosynthesis related genes and uses thereof
US7238855B2 (en) 2001-05-09 2007-07-03 Monsanto Technology Llc TyrA genes and uses thereof
US7244877B2 (en) 2001-08-17 2007-07-17 Monsanto Technology Llc Methyltransferase from cotton and uses thereof
US7262339B2 (en) 2001-10-25 2007-08-28 Monsanto Technology Llc Tocopherol methyltransferase tMT2 and uses thereof
US7297509B2 (en) 2001-04-11 2007-11-20 Adelbert Bacher Intermediates and enzymes of the non-mevalonate isoprenoid pathway
US10662415B2 (en) 2017-12-07 2020-05-26 Zymergen Inc. Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation
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Cited By (35)

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WO2000044912A1 (fr) * 1999-01-28 2000-08-03 Royal Holloway And Bedford New College Manipulation de l'expression d'un isoprenoide
US7067647B2 (en) 1999-04-15 2006-06-27 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US7265207B2 (en) 1999-04-15 2007-09-04 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7141718B2 (en) 1999-04-15 2006-11-28 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7122331B1 (en) 1999-08-04 2006-10-17 Wolfgang Eisenreich Isoprenoid biosynthesis
US7288367B2 (en) 2000-06-05 2007-10-30 Adelbert Bacher Non-mevalonate isoprenoid pathway
WO2001094561A3 (fr) * 2000-06-05 2002-05-30 Adelbert Bacher Voie isoprenoide non mevalonate
WO2001094561A2 (fr) * 2000-06-05 2001-12-13 Adelbert Bacher Voie isoprenoide non mevalonate
US6841717B2 (en) 2000-08-07 2005-01-11 Monsanto Technology, L.L.C. Methyl-D-erythritol phosphate pathway genes
WO2002012478A3 (fr) * 2000-08-07 2003-07-03 Monsanto Technology Llc Genes de la voie methyl-d-erythritol phosphate
WO2002012478A2 (fr) * 2000-08-07 2002-02-14 Monsanto Technology, Llc Genes de la voie methyl-d-erythritol phosphate
US7405343B2 (en) 2000-08-07 2008-07-29 Monsanto Technology Llc Methyl-D-erythritol phosphate pathway genes
WO2002020733A3 (fr) * 2000-09-01 2003-08-14 Du Pont Genes impliques dans la production de composes isoprenoides
US6660507B2 (en) 2000-09-01 2003-12-09 E. I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production
WO2002020733A2 (fr) * 2000-09-01 2002-03-14 E.I. Dupont De Nemours And Company Genes impliques dans la production de composes isoprenoides
US7056717B2 (en) 2000-09-01 2006-06-06 E. I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production
US8362324B2 (en) 2000-10-14 2013-01-29 Monsanto Technology Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US6872815B1 (en) 2000-10-14 2005-03-29 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7297509B2 (en) 2001-04-11 2007-11-20 Adelbert Bacher Intermediates and enzymes of the non-mevalonate isoprenoid pathway
DE10119905A1 (de) * 2001-04-23 2002-10-24 Jomaa Pharmaka Gmbh Inaktivierung von Genen des MEP-Wegs
US7875279B2 (en) 2001-04-23 2011-01-25 Bioagency Ag Inactivation of genes of the MEP pathway
EP1434867A4 (fr) * 2001-04-24 2005-11-30 Du Pont Genes impliques dans la production de compose isoprenoide
EP1434867A2 (fr) * 2001-04-24 2004-07-07 E.I. Du Pont De Nemours And Company Genes impliques dans la production de compose isoprenoide
US7238855B2 (en) 2001-05-09 2007-07-03 Monsanto Technology Llc TyrA genes and uses thereof
US7161061B2 (en) 2001-05-09 2007-01-09 Monsanto Technology Llc Metabolite transporters
US7244877B2 (en) 2001-08-17 2007-07-17 Monsanto Technology Llc Methyltransferase from cotton and uses thereof
US7553952B2 (en) 2001-08-17 2009-06-30 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequence identified in Cuphea and uses thereof
US7595382B2 (en) 2001-08-17 2009-09-29 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequences from Brassica and uses thereof
US7605244B2 (en) 2001-08-17 2009-10-20 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequence from Brassica and uses thereof
US7262339B2 (en) 2001-10-25 2007-08-28 Monsanto Technology Llc Tocopherol methyltransferase tMT2 and uses thereof
US7112717B2 (en) 2002-03-19 2006-09-26 Monsanto Technology Llc Homogentisate prenyl transferase gene (HPT2) from arabidopsis and uses thereof
US7230165B2 (en) 2002-08-05 2007-06-12 Monsanto Technology Llc Tocopherol biosynthesis related genes and uses thereof
US10662415B2 (en) 2017-12-07 2020-05-26 Zymergen Inc. Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation
US10696991B2 (en) 2017-12-21 2020-06-30 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone
US11193150B2 (en) 2017-12-21 2021-12-07 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone

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