A method enables olefin compound production with a high productivity, and an enzyme is used in the method. Mutations involving amino acid substitution are introduced into various sites of diphosphomevalonate decarboxylase (MVD) to prepare multiple MVD variants. Then, these variants are evaluated in terms of catalytic activity for producing an olefin compound such as isoprene, and have found as a result that the catalytic activity is improved when serine at position 153 and threonine at position 209 are each substituted with a different amino acid. In addition, the catalytic activity of the variants is further improved when glycine at position 152 is further substituted with a different amino acid.

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.

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 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 present invention provides for a system for increasing the production of a compound using an artificial positive feedback loop (APFL). In some embodiments, the system diverts a compound produced in a first metabolic pathway into a second metabolic pathway in order to produce a compound of interest.

Mutations involving amino acid substitution were introduced into various sites of diphosphomevalonate decarboxylase (MVD), thus preparing a large number of MVD variants. Then, the variants were each evaluated in terms of a catalytic activity for production of olefin compounds such as isoprene. As a result, it was found that substitution of glycine at position with a different amino acid resulted in improvement in the catalytic activity. In addition, it was found that the MVD in which arginine at position and threonine at position in addition to the position were further substituted with different amino acids, respectively, also had the high catalytic activity.

This invention relates to the field of genetic engineering. Specifically, the invention relates to the construction of operons to produce biologically active agents. For example, operons may be constructed to produce agents that control the function of biochemical pathway proteins (e.g., protein phosphatases, kinases and/or proteases). Such agents may include inhibitors and modulators that may be used in studying or controlling phosphatase function associated with abnormalities in a phosphatase pathway or expression level. Fusion proteins, such as light activated protein phosphatases, may be genetically encoded and expressed as photoswitchable phosphatases. Systems are provided for use in controlling phosphatase function within living cells or in identifying small molecule inhibitors/activator/modulator molecules of protein phosphatases associated with cell signaling.

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.

A method is provided for biosynthetic production of cannabinoids in microorganisms from a carbon source precursor. This method describes the genetic modifications needed to engineer microorganisms to produce cannabinoids as well as a method for identifying and quantifying cannabinoids from fermentation broth. A system is also provided for tuning the method to produce different cannabinoids of interest by systematically modulating the enzymes encoded by the genetic modifications introduced in the microorganism.

A method is provided for biosynthetic production of cannabinoids in microorganisms from a carbon source precursor. This method describes the genetic modifications needed to engineer microorganisms to produce cannabinoids as well as a method for identifying and quantifying cannabinoids from fermentation broth. A system is also provided for tuning the method to produce different cannabinoids of interest by systematically modulating the enzymes encoded by the genetic modifications introduced in the microorganism.