University of California

Amyris Biotechnologies, Inc.

Amyris, Inc.

Jack Newman

Jay D Keasling

Douglas J Pitera

Sydnor T Withers, Iii

Yasuo Yoshikuni

Seon-Won Kim

Artem Valentinovich Khlebnikov

Vincent J J Martin

Keith Kinkead Reiling

Neil Stephen Renninger

Rika Regentin

Christopher John Paddon

Vincent Martin

Larry Cameron Anthony

Farnaz F Nowroozi

Jeffrey Lance Kizer

Vincent Jj Martin

Brian F Pfleger

Neil Renninger

Vinent J J Martin

Jennifer Anthony

Methods for producing an isoprenoid are provided. A plurality of bacterial or fungal host cells is obtained. These cells comprise a heterologous nucleic acid encoding one or more enzymes of a mevalonate pathway for making isopentenyl pyrophosphate. Expression of the one or more enzymes is under control one or more heterologous transcriptional regulator. The mevalonate pathway comprises (i) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, (ii) an enzyme that converts HMG-CoA to mevalonate, (iii) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate, (iv) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate, and (v) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate. The host cells are cultured in a medium under conditions that are suboptimal as compared to conditions for the maximum growth rate. Temperature is maintained at a level below that which would provide for a maximum specific growth rate for the host cells.

Production of isoprenoids

Methods for producing an isoprenoid are provided. A plurality of bacterial or fungal host cells is obtained. These cells comprise a heterologous nucleic acid encoding one or more enzymes of a mevalonate pathway for making isopentenyl pyrophosphate. Expression of the one or more enzymes is under control of at least one heterologous transcriptional regulator. The mevalonate pathway comprises (i) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, (ii) an enzyme that converts HMG-CoA to mevalonate, (iii) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate, (iv) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate, and (v) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate. The host cells are cultured in a medium under conditions that are suboptimal as compared to conditions for the maximum growth rate. Temperature is maintained at a level below that which would provide for a maximum specific growth rate for the host cells.

Methods for synthesizing isopentenyl pyrophosphate are provided. A first method comprises introducing into a host microorganism a plurality of heterologous nucleic acid sequences, each coding for a different enzyme in the mevalonate pathway for producing isopentenyl pyrophosphate. A related method comprises introducing into a host microorganism an intermediate in the mevalonate pathway and at least one heterologous nucleic acid sequence, each sequence coding for an enzyme in the mevalonate pathway necessary for converting the intermediate into isopentenyl pyrophosphate. The invention also provides nucleic acid sequences, enzymes, expression vectors, and transformed host cells for carrying out the methods.

Host cells for production of isoprenoid compounds

The present invention provides genetically modified host cells and use of same for producing isoprenoid compounds.

Production of isoprenoids and isoprenoid precursors

The present invention provides methods of increasing production of an isoprenoid or an isoprenoid precursor in a host cell, the methods generally involving modulating the level of activity of a fatty acid biosynthetic pathway enzyme in the host cell and/or culturing the host cell in a culture medium comprising a fatty acid or a compound that can be metabolized in a cell or broken down in the medium to yield a fatty acid and/or culturing the host cell in a culture medium having increased osmolarity.

Methods for increasing isoprenoid and isoprenoid precursor production by modulating fatty acid levels

The present invention provides isolated, genetically modified host cells, where a host cell is genetically modified with a nucleic acid that includes a nucleotide sequence encoding a biosynthetic pathway enzyme. Synthesis of the enzyme in the host cell results in conversion of a substrate for the enzyme into a biosynthetic pathway intermediate, which intermediate is produced in an amount effective to inhibit growth of the genetically modified host cell. The present invention further provides compositions and kits comprising a subject genetically modified host cell. Subject host cells are useful for identifying a gene product having activity in a biosynthetic pathway. The present invention further provides methods of identifying a gene product having activity in a biosynthetic pathway.

Methods for identifying a biosynthetic pathway gene product

A fuel composition comprises at least a Cisoprenoid compound or its derivative and a conventional fuel additive. The Cisoprenoid compound or its derivative can be used as a fuel component or as a fuel additive in the fuel composition. The fuel composition may further comprise a conventional fuel component selected from a diesel fuel, jet fuel, kerosene or gasoline. Methods of making and using the fuel composition are also disclosed.

Fuel components, fuel compositions and methods of making and using same

The present invention provides a method of identifying an enzyme, the method generally involving contacting a sample containing an enzyme with a selected enzyme substrate, where the contacting provides for covalent binding of the substrate to an amino acid of the enzyme to form a covalently modified enzyme; and determining the amino acid sequence of at least a portion of the covalently modified enzyme, using any available peptide sequencing technology, such as tandem mass spectrometry. The present invention further provides methods of identifying a nucleic acid encoding an enzyme, the methods generally involving identifying an enzyme; and, based on the amino acid sequence of at least a portion of the enzyme, designing nucleic acid probes or primers that hybridize to the nucleic acid encoding the enzyme.

Method for identifying terpene synthase

The present invention provides methods of producing an isoprenoid or an isoprenoid precursor in a genetically modified host cell. The methods generally involve modulating the level of hydroxymethylglutaryl-CoA (HMG-CoA) in the cell, such that the level of HMG-CoA is not toxic to the cell and/or does not substantially inhibit cell growth, but is maintained at a level that provides for high-level production of mevalonate, IPP, and other downstream products of an isoprenoid or isoprenoid pathway, e.g., polyprenyl diphosphates and isoprenoid compounds. The present invention further provides genetically modified host cells that are suitable for use in a subject method. The present invention further provides recombinant nucleic acid constructs for use in generating a subject genetically modified host cell, including recombinant nucleic acid constructs comprising nucleotide sequences encoding one or more mevalonate pathway enzymes, and recombinant vectors (e.g., recombinant expression vectors) comprising same. The present invention further provides methods for identifying nucleic acids that encode HMG-CoA reductase (HMGR) variants that provide for relief of HMG-CoA accumulation-induced toxicity. The present invention farther provides methods for identifying agents that reduce intracellular accumulation of HMG-CoA.

Method for enhancing production of isoprenoid compounds

Isolated mevalonate pathway enzyme nucleic acids

The present invention provides methods for a robust production of isoprenoids via one or more biosynthetic pathways. The invention also provides nucleic acids, enzymes, expression vectors, and genetically modified host cells for carrying out the subject methods. The invention also provides fermentation methods for high productivity of isoprenoids from genetically modified host cells.

Growing community of inventors

Berkeley, CA, United States of America

Jack Newman