Synthesis of isoprenoids and derivatives

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.

1 . A recombinant microorganism for the production of an isoprenoid(s) or an isoprenoid derivative(s), said microorganism comprising:
 a) one or more alcohol(s) selected from prenol, isoprenol, or both;   b) enzymes catalyzing conversion of said alcohol(s) to dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) comprising: a) an alcohol kinase (EC 2.7.1.-) plus a phosphate kinase (EC 2.7.4.-), orb) an alcohol diphosphokinase (EC 2.7.6.-), plus optionally c) an IPP isomerase (5.3.3.2);   c) a GPP synthase catalyzing conversion of said DMAPP and IPP to geranyl diphosphate (GPP); and   d) one or more enzyme(s) selected from a group comprising farnesyl diphosphate synthase, geranylgeranyl-diphosphate synthase, prenyl transferase, terpene synthase, terpene cyclase, tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase, cannabichromenic acid synthase, tetrahydrocannabivarinic acid synthase, cannabidivarinic acid synthase, and cannabichrovarinic acid synthase catalyzing conversion of said GPP to an isoprenoid(s) or an isoprenoid derivative(s).   
     
     
         2 . The recombinant microorganism of  claim 1 , wherein said microorganism comprises an aromatic prenyltransferase or a 4-hydroxybenzoate geranyltransferase catalyzing a prenyl transfer from said GPP or said isoprenoid to an aromatic polyketide(s) forming a prenylated aromatic compound(s). 
     
     
         3 . The recombinant microorganism of  claim 2 , wherein said prenylated aromatic compound(s) is a cannabinoid(s). 
     
     
         4 . The recombinant microorganism of  claim 1 , wherein said recombinant organism is grown in a culture medium and said isoprenoid(s) or isoprenoid derivative(s) is isolated from said culture medium or said recombinant microorganism or both. 
     
     
         5 . The recombinant microorganism of  claim 1 , wherein said isoprenoid(s) is selected from hemiterpenoid(s), monoterpenoid(s), sesquiterpenoid(s), diterpenoid(s), sesterterpenoid(s), triterpenoid(s), tetraterpenoid(s), polyterpenoid(s), or a derivative(s) thereof. 
     
     
         6 . The recombinant microorganism of  claim 1 , that further comprises reduced expression of gene(s) encoding one or more fermentation enzymes leading to reduced production of one or more of lactate, acetate, ethanol or succinate. 
     
     
         7 . The recombinant microorganism of  claim 1 , that is a bacteria or yeast cell. 
     
     
         8 . The recombinant microorganism of  claim 1 , that is an  Escherichia coli  cell. 
     
     
         9 . A recombinant microorganism for the production of one or more cannabinoid(s), said microorganism comprising:
 a) one or more alcoholics(s) selected from prenol and isoprenol;   b) enzymes catalyzing conversion of said alcohol(s) to DMAPP or IPP using one or more enzyme(s) selected from i) an alcohol kinase (EC 2.7.1.-) plus a phosphate kinase (EC 2.7.4.-), or ii) an alcohol diphosphokinase (EC 2.7.6.-);   c) an aromatic prenyltransferase or a 4-hydroxybenzoate geranyltransferase catalyzing a prenyl transfer from said DMAPP or IPP to an aromatic polyketide to form one or more cannabinoid(s);   d) optionally one or more enzymes selected from the group comprising tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase, cannabichromenic acid synthase, tetrahydrocannabivarinic acid synthase, cannabidivarinic acid synthase, and cannabichrovarinic acid synthase catalyzing conversion of said cannabinoid(s) to another cannabinoid(s).   
     
     
         10 . A recombinant microorganism for the production of a cannabinoid, said microorganism comprising:
 a) an alcohol selected from prenol, isoprenol, or both;   b) enzymes catalyzing conversion of said alcohol to DMAPP and IPP comprising a) an alcohol kinase (EC 2.7.1.-) plus a phosphate kinase (EC 2.7.4.-), or b) an alcohol diphosphokinase (EC 2.7.6.-), plus optionally c) an 1PP isomerase (5.3.3.2);   c) a GPP synthase catalyzing conversion of said DMAPP and IPP to GPP;   d) an aromatic prenyltransferase or a 4-hydroxybenzoate geranyltransferase catalyzing a prenyl transfer from said GPP to an aromatic polyketide to form a cannabinoid;   e) optionally one or more enzymes selected from the group comprising tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase, cannabichromenic acid synthase, tetrahydrocannabivarinic acid synthase, cannabidivarinic acid synthase, and cannabichrovarinic acid synthase catalyzing conversion of said cannabinoid to another cannabinoid.   
     
     
         11 . The recombinant microorganism of  claim 9  or  10 . wherein said aromatic polyketide is selected from olivetolic acid, olivetol, divarinolic acid or divarinol. 
     
     
         12 . The recombinant microorganism of  claim 9  or  10 , that is a bacteria or yeast cell. 
     
     
         13 . The recombinant microorganism of  claim 9  or  10 , that is an  Escherichia coli  cell. 
     
     
         14 . The recombinant microorganism of  claim 9  or  10 , that further comprises reduced expression of gene(s) encoding one or more fermentation enzymes leading to reduced production of one or more of lactate, acetate, ethanol or succinate. 
     
     
         15 . The recombinant microorganism of  claim 9  or  10 , wherein said recombinant organism is grown in a culture medium and said cannabinoid(s) is isolated from said culture medium or said recombinant microorganism or both.