Biosynthesis of isopentenyl pyrophosphate

An isolated DNA fragment coding for a fraction of the enzymes in the mevalonate pathway for producing isopentenyl pyrophosphate comprising the nucleic acid sequences comprised of:

• a) a DNA fragment coding for an enzyme capable of condensing two molecules of acetyl-CoA to acetoacetyl-CoA;
• b) a DNA fragment coding for an enzyme capable of condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• and
• c) a DNA fragment coding for an enzyme capable of converting HMG-CoA to mevalonate.

An isolated DNA fragment coding for a fraction of the enzymes in the mevalonate pathway for producing isopentenyl pyrophosphate comprising the nucleic acid sequences comprised of:

• a) a DNA fragment coding for an enzyme capable of phosphorylating mevalonate to mevalonate 5-phosphate;
• b) a DNA fragment coding for an enzyme capable of converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• c) a DNA fragment coding for an enzyme capable of converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.

A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, wherein the method comprises:

• culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising one or more nucleic acids heterologous to the host microorganism, wherein the one or more nucleic acids comprises nucleotide sequences encoding two or more enzymes in the mevalonate pathway, and wherein the host microorganism is a prokaryote that does not nornally synthesize IPP through the mevalonate pathway;
• wherein the mevalonate pathway comprises:
• (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) converting HMG-CoA to mevalonate;
• (d) phosphorylating mevalonate to mevalonate 5-phosphate;
• (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the two or more enzymes, wherein said production of said two or more enzymes results in production of IPP.

A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, wherein the method comprises:

• culturing a transfonned host prokaryote microorganism that does not normally synthesize IPP through the mevalonate pathway in a suitable medium, the transformed host microorganism comprising a single extrachromosomal expression vector heterologous to the host microorganism that comprises the nucleotide sequence set forth in SEQ ID NO 7 or a fragment thereof encoding the enzymes in a mevalonate pathway;
• wherein the mevalonate pathway comprises:
• (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) converting HMG-CoA to mevalonate;
• (d) phosphorylating mevalonate to mevalonate 5-phosphate;
• (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the enzymes, wherein said production of said enzymes results in production of IPP.

A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, wherein the method comprises:

• culturing a transfonned host prokaryote microorganism that does not normally synthesize IPP through the mevalonate pathway in a suitable medium, the transformed host microorganism comprising two extrachromosomal expression vectors, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO 8, and the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO 9 or a fragment thereof, which sequences from the two vectors are heterologous to the host microorganism and encode the enzymes in a mevalonate pathway;
• wherein the mevalonate pathway comprises:
• (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) converting HMG-CoA to mevalonate;
• (d) phosphorylating mevalonate to mevalonate 5-phosphate;
• (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate,
• said culturing providing for production of the enzymes, wherein said production of said enzymes results in production of IPP.

A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, the method comprising:

• culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising one or more nucleic acids heterologous to the host microorganism, wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway, wherein the one or more nucleic acids comprises nucleotide sequences encoding two or more enzymes selected from:
• a) an enzyme capable of condensing two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme is from Ralstonia, Saccharomyces, or Escherichia coli;
• b) an enzyme capable of condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, wherein said enzyme is from Blattella or Saccharomyces;
• c) an enzyme capable of converting HMG-CoA to mevalonate, wherein said enzyme is from Sulfolobus, Haloferax, or Saccharomyces;
• d) a Saccharomyces enzyme capable of phosphorylating mevalonate to mevalonate 5-phosphate;
• e) a Saccharomyces enzyme capable of converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• f) a Saccharomyces enzyme capable of converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate,
• said culturing providing for production of the enzymes, wherein said production of said twvo or more enzymes results in production of IPP.

A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, the method comprising:

• culturing a transformed host microorganism in a suitable medium, the transfonned host microorganism comprising one or more nucleic acids heterologous to the host microorganism, wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway, wherein the one or more heterologous nucleic acids comprises nucleotide sequences encoding two or more enzymes selected from:
• a) an enzyme capable of condensing two molecules of acetyl-CoA to acetoacetyl-CoA encoded by the nucleotide sequence of SEQ ID NO 1;
• b) an enzyme capable of condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, encoded by the nucleotide sequence of SEQ ID NO 2;
• c) an enzyme capable of converting HMG-CoA to mevalonate encoded by the nucleotide sequence of SEQ ID NO 3;
• d) a Saccharomyces enzyme capable of phosphorylating mevalonate to mevalonate 5-phosphate encoded by the nucleotide sequence of SEQ ID NO 4;
• e) a Saccharomyces enzyme capable of converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate encoded by the nucleotide sequence of SEQ ID NO 5;
• and
• f) a Sacchaio77ijces enzyme capable of converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate encoded by the nucleotide sequence of SEQ ID NO 6,
• said culturing providing for production of the enzymes, wherein said production of said two or more enzymes results in production of IPP.

A method for synthesizing isopentenyl pyrophosphate (IPP) via a mevalonate pathway in a host microorganism, wherein the method comprises:

• culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising at least two operons heterologous to the host microorganism, wherein each of said two operons comprises nucleotide sequences encoding enzymes in the mevalonate pathway, and wherein the host microorganism is a prokaryote that does not normally synthesize IPP through the mevalonate pathway;
• wherein the mevalonate pathway comprises:
• (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) converting HMG-CoA to mevalonate;
• (d) phosphorylating mevalonate to mevalonate 5-phosphate;
• (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate, said culturing providing for production of the enzymes,
• wherein said production of said two or more enzymes results in production of IPP.

A method for synthesizing dimethylallyl pyrophosphate (DMAPP) via a mevalonate pathway in a host microorganism, wherein the method comprises:

• culturing a transformed host microorganism in a suitable medium, the transformed host microorganism comprising one or more nucleic acids heterologous to the host microorganism, wherein the one or more nucleic acids comprises nucleotide sequences encoding two or more enzymes in the mevalonate pathway, and wherein the host microorganism is a prokaryote that does not normally synthesize isopententyl pyrophosphate through the mevalonate pathway;
• wherein the mevalonate pathway comprises:
• (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) converting HMG-CoA to mevalonate;
• (d) phosphorylating mevalonate to mevalonate 5-phosphate;
• (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate,
• said culturing providing for production of the two or more enzymes,
• wherein said production of said two or more enzymes results in production of DMAPP.