Synthesis of isoprenoids and derivatives

Inventors

Gonzalez, Ramon • CLOMBURG, James M. • CHEONG, Seokjung

Assignees

William Marsh Rice University

Abstract

This disclosure generally relates to the use of enzyme combinations or recombinant microbes comprising same to make isoprenoid precursors, isoprenoids and derivatives thereof including prenylated aromatic compounds. Novel metabolic pathways exploiting Claisen, aldol, and acyloin condensations are used instead of the natural mevalonate (MVA) pathway or 1-deoxy-d-xylulose 5-phosphate (DXP) pathways for generating isoprenoid precursors such as isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), and geranyl pyrophosphate (GPP). These pathways have the potential for better carbon and or energy efficiency than native pathways. Both decarboxylative and non-carboxylative condensations are utilized, enabling product synthesis from a number of different starting compounds. These condensation reactions serve as a platform for the synthesis of isoprenoid precursors when utilized in combination with a variety of metabolic pathways and enzymes for carbon rearrangement and the addition/removal of functional groups. Isoprenoid alcohols are key intermediary products for the production of isoprenoid precursors in these novel synthetic metabolic pathways. These precursors can be modified to various isoprenoid products through prenyl transferase, terpene synthase, or terpene cyclases. The production of prenylated aromatic compounds is achieved through prenyl transfer of the hydrocarbon units of isoprenoid precursors to polyketides.

Core Innovation

This disclosure relates to the use of enzyme combinations or recombinant microbes comprising same to make isoprenoid precursors, isoprenoids and derivatives thereof including prenylated aromatic compounds through novel synthetic metabolic pathways instead of the natural mevalonate (MVA) pathway or 1-deoxy-d-xylulose 5-phosphate (DXP) pathways. The novel pathways exploit enzymes catalyzing Claisen, aldol, or acyloin condensation reactions, using both decarboxylative and non-decarboxylative condensations, to generate longer chain length intermediates from central carbon metabolites. Isoprenoid alcohols are described as key intermediary products that can be converted to isoprenoid precursors (e.g., IP, DMAP, IPP, DMAPP) through phosphorylation enzymes and further modified by prenyl transferase, terpene synthase, or terpene cyclases to make isoprenoids and derivatives.

The background identifies limitations in using the native MVA and DXP pathways, stating that synthesis of C5 building blocks by either pathway results in the inevitable loss of carbon from the starting intermediates and that both MVA and DXP pathways are energy (ATP) intensive with net consumption of three ATP equivalents. The disclosure states there exists a need for methods to overcome the inherently low carbon and energy efficiency of natural isoprenoid precursor synthesis pathways and that preferred pathways would diversify the range of products and provide a more carbon and energy efficient route.

One aspect is a CoA-dependent elongation platform based on the use of Claisen condensations, which accept functionalized acyl-CoAs as primers and extender units in a reverse beta-oxidation like pathway to form beta-keto acyl-CoA intermediates that can be step-wise reduced by hydroxyl-, dehydration- and reductase-type beta-reduction enzymes. Various carbon re-arrangement enzymes, such as acyl-CoA mutases, can be employed to modify carbon structure and branching, and termination by spontaneous or enzyme-catalyzed CoA removal, reduction and/or phosphorylation yields isoprenoid precursors (e.g., IPP, DMAPP, GPP) and downstream isoprenoids. The disclosure further describes combining these routes with polyketide pathways to produce prenylated aromatic compounds, including cannabinoids such as cannabigerolic acid, by prenyl transfer of the hydrocarbon units of isoprenoid precursors to aromatic polyketides.

Claims Coverage

Overview: Claim 1 recites eight inventive features relating to enzymatic steps and enzyme combinations for synthesis of isoprenoids.

Claim 1 covers a sequence of enzymatic features from thiolase condensation of acetyl-CoAs to formation of HMG-CoA, dehydration and decarboxylation to 3-methylcrotonyl-CoA, conversion to prenol, phosphorylation to DMAPP/IPP, condensation to GPP by GPP synthase, and conversion of GPP to an isoprenoid by a terpene synthase.

Stated Advantages

Documented Applications