A genetically engineered bacterium and genetically stable method for the production of metabolites, peptides, and-or recombinant proteins
OwnerHidde De Jong, Johannes Geiselmann +2inventors
InventorsHidde De Jong, Johannes Geiselmann, Thibault Clavier, Célia Boyat
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Abstract
The invention concerns a genetically engineered bacterium for producing at least one metabolite, peptide or recombinant protein comprising a gene encoding a recombinant protein of interest or at least one gene encoding an enzyme involved in the production of a peptide or metabolite of interest. The invention is further directed to methods using the bacterium for producing at least one metabolite, peptide or recombinant protein.
Claims17
A genetically engineered bacterium for producing at least one metabolite, peptide or recombinant protein comprising a gene encoding a recombinant protein of interest or at least one gene encoding an enzyme involved in the production of a peptide or metabolite of interest, and further comprising genes encoding the ββ′ subunits of an RNA polymerase operably linked to an first inducible promoter, the natural promoter of said genes being replaced by said first inducible promoter, characterized in that:
- either it further comprises a gene encoding the α subunit of the RNA polymerase having the following features listed below:
- said gene being operably linked to a second inducible promoter, the natural promoter of the gene being replaced by said second inducible promoter,
- said gene being inserted by homologous recombination into a locus constituted of genes that are non-essential for the growth of the bacteria or into a non-coding sequence and said locus
- being a gene sequence whose probability of being the subject of spontaneous natural homologous recombination is less than 30%, preferably less than 20%, even more preferably less than 10%,
- said gene encoding the α subunit of the RNA polymerase being positioned at a maximum distance of 500,000 nucleotides from the replication origin, and
- said gene being oriented in the opposite direction of the upstream gene of the promoter of the gene coding for the α subunit,
- and the natural gene encoding the α subunit of the RNA polymerase is deleted from its natural location.
A method for producing at least one metabolite, peptide or recombinant protein of interest comprising the steps of
- a. culturing bacteria comprising:
- (i) a gene encoding said recombinant protein or at least one gene encoding an enzyme involved in the production of said peptide or metabolite,
- (ii) genes encoding the ββ′ subunits of bacterial RNA polymerase operably linked to a first inducible promoter, the natural promoter of said genes being replaced by said first inducible promoter, and
- (iii) a gene encoding the α subunit of bacterial RNA polymerase having the following features listed below:
- said gene being operably linked to a second inducible promoter, the natural promoter of the gene being replaced by said second inducible promoter,
- said gene being inserted by homologous recombination into a locus constituted of genes that are non-essential for the growth of the bacteria or into a non-coding sequence and said locus being a gene sequence whose probability of being the subject of spontaneous natural homologous recombination is less than 30%, preferably less than 20%, even more preferably less than 10%,
- said gene encoding the α subunit of the RNA polymerase being positioned at a maximum distance of 500,000 nucleotides from the replication origin, and
- said gene being oriented in the opposite direction of the upstream gene of the promoter of the gene coding for the α subunit,
- and the natural gene encoding the α subunit of the RNA polymerase is deleted from its natural location, in a first culture medium inducing the expression of the genes encoding the ββ′ subunits and further inducing the expression of the gene encoding the α subunit, thereby inducing bacterial growth;
- b. culturing said bacteria in a second culture medium inhibiting the expression of the genes encoding the ββ′ subunits and further inhibiting the expression of the gene encoding the α subunit, thereby producing said metabolite, peptide or recombinant protein while inhibiting bacterial growth;
- c. optionally iterating steps a. and b. successively;
- and
- d. optionally recovering said metabolite, peptide or recombinant protein produced by said bacteria.
(canceled)
Dependencies are inferred from the claim text.