There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.

There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.

There are provided amino acid derivatives of formula V and VI as defined herein which are pyrrolysine analogs for use in bioconjugation processes.

Disclosed herein are methods for producing tulipalin A (-methylene--butyrolactone), recombinant cells or organisms for producing tulipalin A, enzymes needed for producing tulipalin A, and nucleic acids for expression of those enzymes.

A method for producing 2,4-dihydroxybutyrate (DHB) or L-threonine using a microbial metabolic pathway is disclosed, by expressing the metabolic pathway in a microbial production strain which was previously modified with respect to its natural wild type form by introducing at least one of the genes necessary for the expression of those enzymes used for the enzymatic conversions into the production strain.