University of California

Amyris, Inc.

Amyris Biotechnologies, Inc.

Keith Kinkead Reiling

Jack Newman

Neil Stephen Renninger

Rika Regentin

Christopher John Paddon

Jay D Keasling

Vincent J J Martin

Douglas J Pitera

Sydnor T Withers, Iii

Neil Renninger

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.

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 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

Oakland, CA, United States of America

Keith Kinkead Reiling