Optimized microbial cells for production of melatonin and other compounds
AssigneeDanmarks Tekniske Universitet
InventorsHao Luo, Jochen Förster
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Abstract
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.
Claims21
A variant of E. coli GTP cyclohydrolase I (GCH1) having at least 94% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16 and comprising one or more mutations, wherein,
- in an E. coli cell comprising a pterin-4α-carbinolamine dehydratase (PCD) and at least one of a tryptophan hydroxylase (TPH), a tyrosine hydroxylase (TH) and a phenylalanine hydroxylase (PheH), the variant provides for an increased hydroxylation activity of at least one of the TPH, TH and PheH as compared to native E. coli GCH1,
- wherein at least one of the one or more mutations is in an amino acid residue selected from the group consisting of D97, M99, T101, V102, A125, K129, N170, V179, T196, T198, S199, L200, S207, H212, E213, F214, L215 and H221, and
- wherein the mutation in N170 is N170K, N170D or N170L;
- the mutation in T198 is T198I, T198V or T198L;
- the mutation in V179 is V179A;
- the mutation in H212 is H212R or H212K;
- and the mutation in H221 is H221R or H221K.
A variant of E. coli GTP cyclohydrolase I (GCH1) having at least 98% sequence identity to native E. coli GCH1 having the sequence of SEQ ID NO:16,
- wherein the variant comprises a mutation in amino acid residue T198, and
- wherein, in an E. coli cell comprising a PCD and at least one of a TPH, a TH and a PheH, the variant provides for an increased hydroxylation activity of at least one of the TPH, TH and PheH as compared to native E. coli GCH1.
A variant of E. coli GCH1 consisting of SEQ ID NO:16 except for a T198I mutation.
Dependencies are inferred from the claim text.