Continuous bioproduction by decoupling growth and production
The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression and nucleotide production. The invention generally relates to systems and processes that are suitable or comprise a two stage production process, in which the growth of the bacterial host cell is spatiotemporally separated from the production of the protein or nucleic acid of interest. Accordingly, the present invention relates to a system and a process for use in continuous production of a protein of interest or a nucleotide of interest by a bacterial host cell.
1 . A continuous fermentation process for the production of a protein of interest or a polynucleotide of interest by a bacterial host cell, wherein the bacterial host cell comprises a nucleic acid encoding a protein from a phage which inhibits growth of said bacterial host cell, said nucleic acid under the control of a first inducible promoter, wherein said protein from phage which inhibits growth of said bacterial host cell (i) inhibits bacterial host cell RNA polymerase, (ii) inhibits host cell DNA replication, or (iii) causes host cell transcriptional shut-off, wherein said first inducible promoter promotes expression of said nucleic acid, comprising
(a) culturing said bacterial host cell in an uninduced state with respect to the nucleic acid encoding the protein from the phage which inhibits growth of said bacterial host cell in a seed reactor;
(b) transferring at least an amount of the bacterial host cells obtained in (a) from said seed reactor to a production reactor; and
(c) culturing said bacterial host cells in said production reactor in an induced state with respect to the nucleic acid encoding the protein from the phage which inhibits growth of said bacterial host cells;
wherein the seed reactor and production reactor is configured as an independent continuous fermentor and wherein the seed reactor and production reactor are connected with each other, wherein (a), (b), and (c) occur simultaneously during at least a portion of the continuous fermentation process, and wherein the seed reactor and the production reactor are configured as linked chemostats or turbidostats.
2 . The process of claim 1 , wherein (a) is for biomass production.
3 . The process of claim 1 , wherein in (c) growth of said bacterial host cells is inhibited by culturing said bacterial host cells in an induced state with respect to the nucleotide sequence encoding a protein from a phage which inhibits growth of said bacterial host cells, while said polynucleotide of interest or said protein of interest is produced.
4 . The process of claim 1 , wherein the bacterial host cell comprises the polynucleotide of interest or a nucleotide encoding the protein of interest.
5 . The process of claim 1 , wherein said polynucleotide of interest or a polynucleotide encoding said protein of interest is under the control of a second inducible promoter or under the control of a constitutive promoter.
6 . The process of claim 5 , wherein said second inducible promoter or said constitutive promoter is recognized by an RNA polymerase which is heterologous for said bacterial host cell, said heterologous RNA polymerase is encoded by a nucleotide sequence comprised by said bacterial host cell.
7 . The process of claim 1 , wherein said bacterial host cell has a non-functional arabinose operon.
8 . The process of claim 1 , wherein said bacterial host cell is E. coli.
9 . The process of claim 1 , wherein (a) further comprises culturing said bacterial host cells in an uninduced state with respect to the nucleotide sequence encoding a protein from a phage which inhibits growth of said bacterial host cells in culture medium in a production reactor for biomass production.
10 . The process of claim 1 , further comprising (d) harvesting said protein of interest or said polynucleotide of interest.
11 . The process of claim 1 , wherein the bacterial host cells in the at least one production reactor is genetically stable for at least about 5 days.
12 . The process of claim 1 , wherein the process is operated for at least about 5 days.
13 . The process of claim 1 , wherein said protein from a phage which inhibits growth of said bacterial host cell has at least 80% sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and 17.
14 . The process of claim 13 , wherein said protein has at least 90% sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and 17.
15 . The process of claim 1 , wherein said protein from a phage which inhibits growth of said bacterial host cell is selected from the group consisting of Gp2, Nun, GP0.7, Gp6, Gp8, A*, YkzG Epsilon-Subunit, Bacillus phage SPO1 GP40, Staphylococcus phage G1 GP67, Thermus thermophilus phage P23-45 GP39, Enterobacteria phage PhiEco32 GP79, Xanthomonas oryzae bacteriophage Xp10 P7 protein, Enterobacteria phage T4 Alc protein, Enterobacteria phage T4 Asia protein, and Gp5.7.