Methods, materials, synthetic hosts and reagents for the biosynthesis of hydrocarbons and derivatives thereof
Genetically engineered hosts and methods for their production and use in synthesizing hydrocarbons are provided.
1. A genetically engineered Cupriavidus host capable of producing hydrocarbons comprising one or more isoprene units, said genetically engineered Cupriavidus host comprising a genome-integrated synthetic operon comprising an exogenous nucleic acid sequence encoding a polypeptide having acetoacetyl-CoA C-acetyltransferase (AACT), an exogenous nucleic acid sequence encoding HMG-CoA reductase (HMGR) or hydroxymethylglutaryl-CoA synthase (HMGS) enzyme activity and an exogenous nucleic acid sequence encoding an isoprene synthase (IspS) enzyme
wherein the polypeptide having AACT enzyme activity that catalyzes the chemical reaction of 2 acetyl-CoA to CoA and acetoacetyl-CoA and has at least 90% sequence identity to SEQ ID NO:56 or is encoded by a nucleic acid sequence with at least 90% sequence identity to SEQ ID NO: 55, the polypeptide having HMGR enzyme activity that catalyzes the reaction of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) to mevalonic acid and has at least 90% sequence identity to SEQ ID NO:58, 60, 62, 64 or 66 or is encoded by a nucleic acid sequence with at least 90% sequence identity to SEQ ID NO:57, 59, 61, 63 or 65 or the polypeptide having HMGS enzyme activity that catalyzes the reaction of acetoacetyl-CoA to HMG-CoA and has at least 90% sequence identity to SEQ ID NO: 68, 70, 72 or 74 or is encoded by a nucleic acid sequence with at least 90% sequence identity to SEQ ID NO: 67, 69, 71 or 73, and wherein the IspS enzyme has at least 90% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 7, 8, 29 or 30 and catalyzes the reaction dimethylallyl pyrophosphate to isoprene and diphosphate,
wherein said genetically engineered Cupriavidus host produces the hydrocarbon comprising one or more isoprene units from a gas stream comprising at least one of natural gas, methanol, ethanol, non-volatile residue, caustic wash from cyclohexane oxidation processes, or waste stream, or derivative thereof, in an amount at least 60 times greater than a Cupriavidus host cell harbouring a plasmid expressing only IspS.
2. The genetically engineered host of claim 1 , wherein said hydrocarbon comprises said one or more isoprene units as depicted in Formula I
or a salt or derivative thereof.
3. The genetically engineered host of claim 1 further comprising one or more enzymes selected from mevalonate kinase (MVK), phosphomevalonate kinase (MPK), mevalonate diphosphate decarboxylase (MDD) and isopentenyl diphosphate isomerase (IDI).
4. The genetically engineered host of claim 1 wherein the genome-integrated synthetic operon encodes one or more Enterococcus faecalis enzymes.
5. The genetically engineered host of claim 1 further comprising one or more plasmids encoding one or more enzymes of the lower and/or upper MVA pathways.
6. The genetically engineered host of claim 1 wherein the genome integrated synthetic operon comprises an exogenous nucleic acid sequence encoding a polypeptide having AACT enzyme activity, an exogenous nucleic acid sequence encoding a polypeptide having HMGR enzyme activity, an exogenous nucleic acid sequence encoding a polypeptide having HMGS enzyme activity and an exogenous nucleic acid sequence encoding a polypeptide having IspS enzyme activity.