Metabolic engineering of E. coli for the biosynthesis of cannabinoid
Inventors
AYAKAR, Sonal R. • PAWAR, Sandip V. • HALLAM, Steven J. • Hossain, Sazzad • YADAV, Vikramaditya G. • ROY, Protiva R. • SRIVASTAVA, Sarvesh K.
Assignees
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Publication Number
US-12077802-B2
Publication Date
2024-09-03
Expiration Date
Abstract
Provided herein are methods and compositions for producing terpenoids in a host cell. In some cases, the terpenoids are cannabinoids.
Core Innovation
The invention relates to expression cassettes and host cells for metabolic engineering to produce terpenoids and cannabinoids by increasing flux through the mevalonate-independent pathway. A heterologous promoter is operably linked to a nucleic acid encoding a bifunctional ispDF enzyme that has 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase activity and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity.
The bifunctional ispDF enzyme comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and is used in an expression cassette to drive terpenoid and cannabinoid biosynthesis. Additional expression cassettes can encode enzymes of the mevalonate-independent pathway, including dxs, idi, and ispE.
The engineered host cell further includes a heterologous-promoter-driven nucleic acid encoding a terpenoid synthase, including isoprene synthase, and downstream pathway enzymes leading to lycopene/monoterpene production and cannabinoid synthases. Cannabinoid synthases may include CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase, including truncated variants defined by deleting all or part of a signal peptide.
Claims Coverage
The independent claims (1 and 20) are directed to two inventive features: an expression cassette encoding a bifunctional ispDF enzyme with specific sequence identity and dual catalytic activities, and a host cell carrying this cassette together with an additional terpenoid synthase expression cassette. Across the dependent claims, inventive features are refined by promoter type, additional pathway enzymes, specific terpenoid and cannabinoid synthase identities, optional truncation of cannabinoid synthases, comparative MEP-pathway flux, and negative constraints on particular heterologous isp genes.
Expression cassette encoding bifunctional ispDF with dual activity and defined sequence identity
An expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a bifunctional ispDF enzyme, wherein the bifunctional ispDF enzyme comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the bifunctional ispDF enzyme has 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase activity and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity.
Host cell with bifunctional ispDF cassette and terpenoid synthase cassette
A host cell comprising an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a bifunctional ispDF enzyme, wherein the bifunctional ispDF enzyme comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and wherein the bifunctional ispDF enzyme has 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase activity and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity; and an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a terpenoid synthase.
Overall claim coverage is anchored on a bifunctional ispDF expression cassette defined by amino-acid identity of at least 85% to SEQ ID NOs. 1-3 and dual activities, and is combined in host cells with a heterologous-promoter-driven terpenoid synthase cassette, including options for downstream cannabinoid synthase expression and structural truncation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
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