Biosynthesis of 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 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 mevainnate pathway comprises:
• (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) an enzyme that condenses acctoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) an enzyme that converts HMG-CoA to mevalonate;
• (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;
• (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) an enzyme that converts 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 transformed 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 nueleotide sequence set forth in SEQ ID NO 9 or a fragment thereof, which sequences or fragments thereof from the two vectors are heterologous to the host microorganism and encode the enzymes in a mevalonate pathway;
• wherein the mevalonate pathway comprises:
• (a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA;
• (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) an enzyme that converts HMG-CoA to mevalonate;
• (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;
• (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) an enzyme that converts 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 that condenses two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme is from Ralstonia, Saccharomyces, or Escherichia coli, wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of the IPP;
• b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, wherein said enzyme is from Blattella or Saccharomyces;
• c) an enzyme that converts HMG-CoA to mevalonate, wherein said enzyme is from Sulfolobus, Haloferax, or Saccharomyces;
• d) a Saccharomyces enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;
• e) a Saccharomyces enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• f) a Saccharomyces enzyme that converts 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 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 heterologous nucleic acids comprises nucleotide sequences encoding two or more enzymes selected from:
• a) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA encoded by the nucleotide sequence of SEQ ID NO 1, wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of the IPP;
• b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, encoded by the nucleotide sequence of SEQ ID NO 2;
• c) an enzyme that converts HMG-CoA to mevalonate encoded by the nucleotide sequence of SEQ ID NO 3;
• d) a Saccharomyces enzyme that phosphorylates mevalonate to mevalonate 5-phosphate encoded by the nucleotide sequence of SEQ ID NO 4;
• e) a Saccharomyces enzyme that converts a mevalonate 5-phosphate to mevalonate 5-pyrophosphate encoded by the nucleotide sequence of SEQ ID NO 5;
• and
• f) a Saccharomyces enzyme that converts 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) an enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA, wherein said enzyme that condenses two molecules of acetyl-CoA to acetoacetyl-CoA is present as the first step in the synthesis of IPP;
• (b) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA;
• (c) an enzyme that converts HMG-CoA to mevalonate;
• (d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;
• (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate;
• and
• (f) an enzyme that converts 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.