Described herein are production cells, and methods for identifying, selecting, or culturing production cells comprising tyrosine auxotrophy selection marker system, based on a combination of sequence encoding a phenylalanine hydroxylase (PAH) which lacks a functional N-terminal regulatory domain, and a sequence encoding a GTP cyclohydrolase 1 (GCH1). Also described are methods of making a production cell and making a product with said production cell.

The invention provides a composition for use as a medicament, comprising cells of a recombinant microorganism capable of producing increased amounts of one or more of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT) and tryptamine (TRM) as compared to the non-recombinant microorganism from which it was derived. The composition finds use in preventing and/or treating TRM-; 5-HTP-, or 5-HT-related disorders of the central nerve system (CNS); enteric nervous system (ENS); gastro intestine (GI) and metabolism in a mammal, and may be orally administered to a mammal in need thereof. Additionally, a composition comprising cells of a recombinant microorganism capable of producing melatonin is provided for use as a medicament, such as for treatment of depression, dementia, cancer and sleep disorder.

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.

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.

A method of improving motor function and reducing dyskinesia in a subject suffering from a neurodegenerative disease or a disease where endogenous dopamine levels are reduced in the subject comprising administering an effective amount of a viral vector comprising a nucleic acid construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1), (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC), or any combination thereof to the subject.

The present invention provides a 5-methylfolate producing microorganism which a) has been modified to have a decreased expression and/or activity of a polypeptide having both dihydrofolate synthase activity and folylpolyglutamate synthetase activity compared to an otherwise identical microorganism (reference microorganism); b) has been (further) modified to express a heterologous polypeptide having only dihydrofolate synthase activity; c) has been (further) modified to have an increased expression level of at least one enzyme (such as at least two, at least three, at least four, at least five, at least six, at least seven or at least eight) enzymes involved in the biosynthesis of a 5-methylfolate compared to an otherwise identical microorganism (reference microorganism); and/or d) has been (further) modified to have a decreased expression and/or activity of a polypeptide having 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase activity compared to an otherwise identical microorganism (reference microorganism).

The invention relates to microbial cells and microbial cells for use as a medicament, the cells expressing a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase. The cells produce L-DOPA and dopamine.

The invention relates to the use of genetic constructs, expression cassettes and recombinant vectors comprising such constructs and cassettes for gene therapy and methods for treating neurodegenerative disorders, such as Parkinson's disease (PD). The constructs comprise 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). The second coding sequence is 3′ to the first coding sequence, and the first and second coding sequences are part of a single operon, wherein the genetic construct does not encode aromatic amino acid decarboxylase (AADC). The construct is delivered to the cerebrospinal fluid (CSF) of the subject.

The disclosure relates, in some aspects, to compositions and methods for treatment of central nervous system (CNS) diseases, for example Parkinson's disease (PD) and Gaucher disease. In some embodiments, the disclosure provides expression constructs comprising a transgene encoding one or more CNS disease-associated gene products and/or one or more an inhibitory nucleic acids targeting a CNS disease-associated gene or gene product. In some embodiments, the disclosure provides methods of treating CNS diseases by administering such expression constructs to a subject in need thereof.