Metabolic engineering for microbial production of terpenoid products
Inventors
KUMARAN, Ajikumar Parayil • LIM, Chin-Giaw • GHOSH, SOUVIK • Pirie, Christopher • Donald, Jason • Love, Aaron • NAN, Hong • TSENG, Hsien-Chung • Santos, Christine Nicole S. • PHILIPPE, Ryan
Assignees
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Publication Number
US-12227783-B2
Publication Date
2025-02-18
Expiration Date
Abstract
In various aspects and embodiments, the invention relates to bacterial strains and methods for making terpene and terpenoid products. The invention provides bacterial strains with improved carbon flux through the MEP pathway, to thereby increase terpene and/or terpenoid product yield by fermentation with carbon sources such as glucose.
Core Innovation
The disclosed subject matter describes microbial metabolic engineering for production of a terpene or terpenoid. A bacterial strain produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol phosphate pathway (MEP), and converts the IPP and DMAPP to a terpene or terpenoid through a downstream synthesis pathway. The approach is directed to increasing carbon flow through the MEP pathway from glucose so that greater than 15% of carbon entering glycolysis becomes MEP carbon.
The invention limits accumulation of upstream MEP intermediates while increasing MEP carbon utilization. The bacterial strain overexpresses 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) such that expression levels of Dxr and IspE limit accumulation of each of 1-deoxy-D-xylulose (DOX) and 2-C-methyl-D-erythritol (ME) in the culture media to 1 g/L or less. This is paired with balancing downstream steps by overexpressing 1-hydroxy-2-methyl-2-E-butenyl 4-diphosphocytidyl-2-C-methyl-D-erythritol? synthase (IspG) and 1-hydroxy-2-methyl-2-(E) 4-diphosphate reductase (IspH) such that expression levels of IspG and IspH limit accumulation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) in the culture media.
The document further describes tuning of competing pathways and use of Fe—S cluster biogenesis control to support MEP pathway flux. It addresses reduction of a competitor in the ubiquinone synthesis pathway via IspB translation tuning and includes modifications related to isc operon regulation using iscR/ryhB-related modifications that affect Fe—S cluster proteins and IspG/IspH. Downstream bottleneck shifting is described through overexpression and/or enzyme engineering of dxr/ispE/ispG/ispH, including ispG variant improvements and ispE/dxr/ispG/ispH orthologs.
Claims Coverage
The document provides one independent claim that defines a method for producing a terpene or terpenoid with a defined carbon-flux constraint and specific enzyme overexpression that limits accumulation of multiple MEP intermediates. Additional dependent claims refine culturing conditions and further strain modifications tied to the same MEP pathway control logic.
Terpene or terpenoid production via MEP upstream IPP/DMAPP conversion and downstream synthesis
providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to a terpene or terpenoid through a downstream synthesis pathway; and culturing the bacterial strain in culture media to produce the terpene or terpenoid
High MEP carbon flux from glycolysis
culturing the bacterial strain in culture media to produce the terpene or terpenoid, wherein greater than 15% of carbon entering glycolysis becomes MEP carbon
Limiting DOX and ME accumulation by Dxr and IspE overexpression
wherein the bacterial strain overexpresses 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), such that expression levels of Dxr and IspE in the bacterial strain limits accumulation of each of 1-deoxy-D-xylulose (DOX) and 2-C-methyl-D-erythritol (ME) in the culture media to 1 g/L or less
Limiting MEcPP accumulation by IspG and IspH overexpression
wherein the bacterial strain further overexpresses 1-hydroxy-2-methyl-2-E-butenyl 4-diphosphocytidyl-2-C-methyl-D-erythritol? synthase (IspG) and 1-hydroxy-2-methyl-2-(E) 4-diphosphate reductase (IspH), such that expression levels of IspG and IspH in the bacterial cell limits accumulation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) in the culture media
Across the independent claim, the inventive combination centers on producing terpene/terpenoid from IPP/DMAPP generated via the upstream MEP pathway while enforcing a carbon-flux criterion (>15% of carbon entering glycolysis becomes MEP carbon). It further requires targeted overexpression of Dxr and IspE to limit DOX and ME accumulation to 1 g/L or less and overexpression of IspG and IspH to limit MEcPP accumulation in the culture media.
Stated Advantages
Increases terpene or terpenoid production while enforcing MEP pathway carbon flux from glycolysis.
Limits accumulation of upstream MEP intermediates DOX and ME in the culture media to 1 g/L or less.
Limits accumulation of the downstream MEP intermediate MEcPP in the culture media.
Documented Applications
Production of a terpene or terpenoid using a bacterial strain that produces IPP and DMAPP through the upstream methylerythritol phosphate pathway (MEP) and converts them through a downstream synthesis pathway.
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