The present disclosure relates to a method for producing L-tryptophan through the enhancement of prephenate dehydratase (PheA) activity.

The present disclosure provides methods for preparing β-substituted tryptophan compounds. The methods include: combining i) an unsubstituted indole or a substituted indole, ii) a β-substituted serine, and iii) a tryptophan synthase β-subunit (i.e., a TrpB); and maintaining the resulting mixture under conditions sufficient to form the β-substituted tryptophan. The TrpB contains at least one amino acid mutation which promotes formation of an amino-acrylate intermediate. New TrpB variants and new β-substituted tryptophan analogs are also described.

Host cells that are engineered to produce benzylisoquinoline alkaloid (BIAs) precursors, such as norcoclaurine (NC) and norlaudanosoline (NL), are provided. The host cells may have one or more engineered modifications selected from: a feedback inhibition alleviating mutation in a enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene, an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods of producing a BIA of interest or a precursor thereof using the host cells and compositions, e.g., kits, systems etc., that find use in methods of the invention.

The present disclosure provides a variant tyrosine hydroxylase that provides for increased production of L-DOPA in a host cell that expresses the tyrosine hydroxylase. The present disclosure provides nucleic acids encoding the variant tyrosine hydroxylase, and host cells genetically modified with the nucleic acids. The present disclosure provides methods of making L-DOPA in a host cell. The present disclosure provides methods of making a benzylisoquinoline alkaloid (BIA), or a BIA precursor. The present disclosure provides methods of detecting L-DOPA level in a cell. The present disclosure provides methods of identifying tyrosine hydroxylase variants that provide for increased L-DOPA production; and methods of identifying gene products that provide for increased tyrosine production.

The present invention describes the use of thio-phosphate in the metabolic engineering of E. coli. Thio-phosphate can be used to increase the metabolic flux in important synthetic pathways to enhance the production of bioproducts. The pathways impacted include the following: fatty acid synthesis, isoprenoid syntheses, Vit K2 synthesis, ribonucleotide synthesis, and the synthesis of phosphoribosyl pyrophosphate (PRPP) derivatives like 5-aminoimidazole-4-carboxamide (AICA riboside), histidine, and tryptophan. Thus, thio-phosphate can be used to assist in the production of these molecules and/or their derivatives. Enhanced production of AICA in Bacillus megaterium is also demonstrated.

Biomass (e.g., plant biomass, animal biomass, microbial, and municipal waste biomass) is processed to produce useful products, such as food products and amino acids.

5-hydroxytryptophan (5-HTP), a precursor of serotonin, is produced in a microbial host cell. A modified bacterial phenylalanine 4-hydroxylase (P4H) catalyzes the tryptophan 5-hydroxylation reaction. Optionally the host cell includes a cofactor regeneration mechanism, allowing continuous production of 5-HTP without supplementation of exogenous cofactors.

Provided are a microorganism producing an L-amino acid or a precursor thereof, and a method of producing an L-amino acid or a precursor thereof using the microorganism.

Provided are a cAMP receptor protein variant, a microorganism including the same, and a method of producing an L-amino acid using the same.

Disclosed herein are methods that may be used for the synthesis of benzylisoquinoline alkaloids (“BIAs”) such as alkaloid morphinan. The methods disclosed can be used to produce thebaine, oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone, neopinone, and/or buprenorphine. Compositions and organisms useful for the synthesis of BIAs, including thebaine synthesis polypeptides, purine permeases, and polynucleotides encoding the same, are provided.