Amyris, Inc.

Total Marketing Services

Kristy Michelle Hawkins

Adam Meadows

Annie Ening Tsong

Andrew Horwitz

Yoseph Tsegaye

Max Schubert

Timothy Stevens Gardner

Wayne Szeto

Tina Tipawan Mahatdejkul-Meadows

Lauren Barbara Pickens

Anna Tai

Adam Leon Meadows

Provided herein are methods of integrating one or more exogenous nucleic acids into one or more selected target sites of a host cell genome. in certain embodiments, the methods comprise contacting the host cell genome with one or more integration polynucleotides comprising an exogenous nucleic acid to be integrated into a genomic target site, a nuclease capable of causing a break at the genomic target site, and a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the population of cells, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker. in some embodiments, the methods further comprise selecting a host cell that expresses the selectable marker.

Methods for genomic integration

Provided herein are methods of integrating one or more exogenous nucleic acids into one or more selected target sites of a host cell genome. In certain embodiments, the methods comprise contacting the host cell genome with one or more integration polynucleotides comprising an exogenous nucleic acid to be integrated into a genomic target site, a nuclease capable of causing a break at the genomic target site, and a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the population of cells, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker. In some embodiments, the methods further comprise selecting a host cell that expresses the selectable marker.

Provided herein are compositions and methods for the heterologous production of acetyl-CoA-derived isoprenoids in a host cell. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding an acetaldehyde dehydrogenase, acetylating (ADA, E.C. 1.2.1.10) and an MEV pathway comprising an NADH-using HMG-CoA reductase. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding an ADA and an MEV pathway comprising an acetoacetyl-CoA synthase. In some embodiments, the genetically modified host cell further comprises one or more heterologous nucleotide sequences encoding a phosphoketolase and a phosphotransacetylase. In some embodiments, the genetically modified host cell further comprises a functional disruption of the native PDH-bypass. The compositions and methods described herein provide an energy-efficient yet redox balanced route for the heterologous production of acetyl-CoA-derived isoprenoids.

Production of acetyl-coenzyme a derived isoprenoids

Provided herein are compositions and methods for improved production of acetyl-CoA and acetyl-CoA derived compounds in a host cell. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding a phosphoketolase (PK), and a functional disruption of an endogenous enzyme that converts acetyl phosphate to acetate. In some embodiments, the host cell further comprises a heterologous nucleotide sequence encoding a phosphotransacetylase (PTA). In some embodiments, the enzyme that converts acetyl phosphate to acetate is a glycerol-1-phosphatase. In some embodiments, the glycerol-1-phosphatase is GPP1/RHR2. In some embodiments, the glycerol-1-phosphatase is GPP2/HOR2. The compositions and methods described herein provide an efficient route for the heterologous production of acetyl-CoA-derived compounds, including but not limited to, isoprenoids, polyketides, and fatty acids.

Use of phosphoketolase and phosphotransacetylase for production of acetyl-coenzyme A derived compounds

Production of acetyl-coenzyme A derived isoprenoids

Growing community of inventors

Emeryville, CA, United States of America

Kristy Michelle Hawkins