An L-methionine-producing microorganism into which a metZ gene is introduced and a method of producing L-methionine using the same.

The present disclosure provides genetically modified host cells that produce a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. The present disclosure provides methods of synthesizing a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative.

Described herein are prenyltransferases including non-natural variants thereof having at least one amino acid substitution as compared to its corresponding natural or unmodified prenyltransferases and that are capable of at least two-fold greater rate of formation of cannabinoids such as cannabigerolic acid, cannabigerovarinic acid, cannabigerorcinic acid, and cannabigerol, as compared to a wild type control. Prenyltransferase variants also accept different hydrophobic substrates (e.g., “donor” molecules), compared to wild type controls, to create different minor and novel cannabinoids. Prenyltransferase variants also demonstrated regioselectivity to desired cannabinoid isomers such as CBGA (3-GOLA), 3-GDVA, 3-GOSA, and CBG (2-GOL). The prenyltransferase variants can be used to form prenylated aromatic compounds, and can be expressed in an engineered microbe having a pathway to such compounds, which include 3-GOLA, 3-GDVA, 3-GOSA, and CBG. 3-GOLA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered microorganisms. The present invention describes engineered microorganisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These microorganisms can dephosphorylate N-acetylglucosamine-6-phosphate to N-acetylglucosamine and convert the N-acetylglucosamine to N-acetylmannosamine. These microorganisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetylglucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

The process and system led to the identification of prenyltransferase genes from elicitor-treated peanut hairy roots. One of the prenyltransferases, AhR4DT-1 catalyzes a key reaction involved in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, while another, AhR3′DT-1, was able to add the prenyl group to C-3′ of resveratrol. Each of these prenyltransferases has a high specificity for stilbenoid substrates, and their subcellular location in the plastid was confirmed by fluorescence microscopy. Structure analysis of the prenylated stilbenoids suggest that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut.

The present disclosure relates to the production of cannabinoids in either recombinant microorganism or in cell-free systems using a combination of enzymes, including but not limited to a PKS enzyme, a npgA enzyme, a cs-OLAS-1, a pp-DVAS-1, a cs-HEX-1 and/or Butiryl synthase.

Methods for recombinant production of steviol glycoside and compositions containing steviol glycosides are provided by this invention.

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

The present disclosure relates to a non-naturally occurring microorganism that includes an endogenous genetic deletion that eliminates the expression of at least a pyruvate kinase, where the genetically modified prokaryotic microorganism is capable of producing 3-deoxy-D-arabino-heptulosonate-7-phosphate.

The present disclosure relates to recombinant prenyltransferase enzymes with increased thermostability and activity and the use of these enzymes in compositions and methods for biosynthesis involving prenylation reactions, including compositions and methods for the preparation of cannabinoids.