Provided herein are biocatalysts and systems thereof for pterin-dependent enzymes and pathways and methods of making and using the same.

Provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding TH is linked to the nucleotide sequence encoding CH1 such that they encode a fusion protein TH-CH1. Also provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding AADC is linked to the nucleotide sequence encoding TH such that they encode a fusion protein AADC-TH or TH-AADC. Further provided is a viral vector comprising such nucleotide sequences and its use in the treatment and/or prevention of Parkinson's disease.

Provided herein are biocatalysts and systems thereof for pterin-dependent enzymes and pathways and methods of making and using the same. Provided herein in some embodiments are biocatalysts having a pterin source and a pterin-dependent enzymatic pathway biologically coupled to the pterin source. Tetrahydrobiopterin (referred to herein as BH4 or BH 4) can be the pterin source. The BH4 can be synthesized by a tetrahydrobiopterin synthesis pathway. The tetrahydrobiopterin synthesis pathway can include a GTP cyclohydrase; a pyruvoyl tetrahydropterin synthase; a sepiapterin reductase, and/or any combination thereof. The biocatalyst can further contain a pterin-dependent enzymatic pathway. The pterin-dependent enzymatic pathway can be amino acid mono-oxygenase, phenylalanine hydroxylase, tryptophan hydroxylase, tyrosine hydroxylase, nitric oxide synthase, alkylglycerol monooxygenase, and/or any combination thereof.

A method of demethylizing an opioid to a nor-opioid is provided. The method comprises contacting an opioid with at least one enzyme. Contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid. A method of converting a nor-opioid to a nal-opioid is provided. The method comprises contacting a nor-opioid with at least one enzyme. Contacting the nor-opioid with the at least one enzyme converts the nor-opioid to a nal-opioid.

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors.

A gene sequence construct used for the treatment of central nervous system diseases: by means of the construction of an auto-processing expression vector, tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), and so on may be simultaneously expressed; proteins are connected by means of an auto-processing unit (APU); the use of a viral vector to introduce the construct into a target cell may ultimately result in the high-efficiency expression of tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), and so on having independent functions, being used in the prevention or treatment of Parkinson's disease, Alzheimer's disease and other neurodegenerative diseases.

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors.

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors.

A method of demethylizing an opioid to a nor-opioid is provided. The method comprises contacting an opioid with at least one enzyme. Contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid. A method of converting a nor-opioid to a nal-opioid is provided. The method comprises contacting a nor-opioid with at least one enzyme. Contacting the nor-opioid with the at least one enzyme converts the nor-opioid to a nal-opioid.

A genetic construct comprises a promoter operably linked to a first coding sequence, which encodes tyrosine hydroxylase (TH), and a second coding sequence, which encodes GTP cyclohydrolase 1 (GCH1), wherein the second coding sequence is 3 to the first coding sequence, and the first and second coding sequences are part of a single operon. The genetic construct does not encode aromatic amino acid decarboxylase (AADC).