This application describes methods, including non-naturally occurring methods, for biosynthesizing 3-hydroxy-3-methylglutaryl-coA and intermediates thereof, as well as non-naturally occurring hosts for producing 3-hydroxy-3-methylglutaryl-coA. This application also describes methods, including non-naturally occurring methods, for biosynthesizing isoprene and intermediates thereof, as well as non-naturally occurring hosts for producing isoprene.

Describes is a fermentation method for producing a hydrocarbon compound comprising the culturing of an organism in a liquid fermentation medium, wherein said organism produces a desired hydrocarbon compound by an enzymatic pathway, said enzymatic pathway comprising an intermediate which evaporates into the gaseous phase and wherein said intermediate is recovered from the gaseous phase and is reintroduced into the liquid fermentation medium.

The present invention provides for a method to identify a second or mutant phosphomevalonate decarboxylase (PMD) with a higher PMD activity compared to a first PMD, comprising (a) culturing a medium comprising a first host cell expressing the first PMD and a second host cell expressing the second or mutant PMD wherein the first and second host cells have their respective PMD enzymatic activities coupled to the growth rates of the host cells, and (b) identifying the second host cell that has a higher growth rate than the first host cell, thereby identifying the second or mutant PMD having a higher PMD activity.

This application describes methods, including non-naturally occurring methods, for biosynthesizing unsaturated pentahydrocarbons, such as isoprene and intermediates thereof, via the mevalonate pathway, as well as non-naturally occurring hosts for producing isoprene.

The invention features methods for producing isoprene from cultured cells. The invention also provides compositions that include these cultured cells.

A method which enables olefin compound production with a high productivity and an enzyme used in the method, a mutation involving amino acid substitution has been introduced into various sites of diphosphomevalonate decarboxylase (MVD), thus preparing a large number of MVD variants. Next, the result of evaluating the variants for the catalytic activity related to the production of olefin compounds such as isoprene has revealed that MVD whose threonine at position 209 is substituted with a different amino acid has the catalytic activity, and that MVD whose arginine at position 74 is further substituted with a different amino acid in addition to position 209 has the catalytic activity at higher levels.

Described are HIV kinase variants showing an improved activity in converting 3-hydroxyisovalerate (HIV) into 3-phosphonoxyisovalerate (PIV), methods for the production of PIV using such enzyme variants as well as methods for the production isobutene in a subsequent reaction.

The invention provides a method for producing a terpene or a precursor thereof by microbial fermentation. Typically, the method involves culturing a recombinant bacterium in the presence of a gaseous substrate whereby the bacterium produces a terpene or a precursor thereof, such as mevalonic acid, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, geranyl pyrophosphate, farnesyl pyrophosphate, and/or farnesene. The bacterium may comprise one or more exogenous enzymes, such as enzymes in mevalonate, DXS, or terpene biosynthesis pathways.

The invention relates to microbial terpene production. Known methods for microbial production of terpenes are mostly based on the direct conversion of sugars. Therefore alternative substrates, in particular alternative carbon sources, for use in microbial terpene production were desirable. The invention relates to a methylotrophic bacterium containing recombinant DNA coding for at least one polypeptide with enzymatic activity for heterologous expression in said bacterium, wherein said at least one polypeptide with enzymatic activity is selected from the group consisting an enzyme of a heterologous mevalonate pathway, a heterologous terpene synthase and optionally a heterologous synthase of a prenyl diphosphate precursor. The invention further relates in particular to a method for de novo microbial synthesis of sesquiterpenes or diterpenes from methanol and/or ethanol.

The present invention provides for a genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD) (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol, (c) optionally the genetically modified host cell does not express, or has a decreased expression of one or more of NudB, phosphomevalonate kinase (PMK), and/or PMD, and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound, such as isoprene.