Metabolic engineering for microbial production of terpenoid products
AssigneeManus Bio Inc.
InventorsAjikumar Parayil KUMARAN, Ryan LIM, Jason DONALD, Hsien-Chung TSENG, Christine Nicole S. SANTOS, Ryan PHILIPPE
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Abstract
The invention relates to methods and bacterial strains for making terpene and terpenoid products, the bacterial strains having improved carbon pull through the MEP pathway and to a downstream recombinant synthesis pathway.
Claims136
A method for production of a terpene or terpenoid product, comprising:
- providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol phosphate pathway (MEP pathway) and converts the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis pathway;
- wherein IspG and IspH are overexpressed in the bacterial strain such that IspG activity and IspH activity are balanced to provide increased carbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, while preventing accumulation of HMBPP at an amount that feeds back and reduces MEP pathway flux and terpene or terpenoid productivity, the strain optionally comprising one or more genetic modifications that enhance supply and/or transfer of electrons through the MEP pathway and/or to terpene and terpenoid products, and culturing the bacterial strain to produce the terpene or terpenoid product.
A method for draining excess carbon flux from the MEP pathway in a bacterial cell, comprising:
- providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol phosphate pathway (MEP pathway) and optionally converts the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis pathway;
- wherein the bacterial strain overexpresses one or more MEP pathway genes and overexpresses PgpB and/or NudB,
- culturing the bacterial strain to produce MEP carbon.
A method for making an industrial or consumer product, comprising, incorporating the terpene or terpenoid made according to the method of any one of claims 1 to 100 into said industrial or consumer product.
A bacterial strain having one or more genetic modifications that increase production of products from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) precursors;
- the bacterial strain producing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an MEP pathway, and the MEP pathway comprising one or more overexpressed genes;
- the strain converting the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis pathway;
- wherein said genetic modifications comprise:
- (a) recombinant or modified IspG and IspH enzymes, the IspG and IspH enzymes having balanced expression or activity to prevent accumulation of HMBPP intermediate,
- (b) a recombinant or modified gene encoding an enzyme that enhances supply and/or transfer of electrons through the MEP pathway and/or to terpene or terpenoid products, which is optionally an overexpression of a YdbK gene and optionally with a non-native fdx and/or fldA homolog,
- (c) an inactivation or deletion, or reduced expression or activity, of aceE or aceE enzyme complex, and optionally
- (d) a recombinant or modified idi gene to tune activity for higher terpene or terpenoid production.
Dependencies are inferred from the claim text.