Genetically engineered
The present disclosure provides engineered Vibrio sp. organisms. The organisms can be engineered to have an altered Chromosome II so that the altered Chromosome II can be used in Vibrio sp. and other organisms for the cloning or amplification of nucleic acid molecules and for the expression and production of proteins and peptides in a Vibrio sp. organism. One or more genetic elements have been deleted from Chromosome II and/or relocated from Chromosome II to Chromosome I. The engineered Vibrio sp. organisms of the invention can also have signal sequences fused to proteins or peptides to be secreted from the cell. In some embodiments the engineered Vibrio sp. organisms can have sequences that enable them to retain viability after incubation at low temperatures.
1 . A Vibrio sp. organism comprising
a. a Chromosome I comprising essential genetic elements; and
b. an altered Chromosome II comprising at least one piece of exogenous DNA of greater than 10 kb in size that encodes at least one heterologous protein or peptide; and
c. wherein the organism comprises all cellular components functional for the replication and amplification of the altered Chromosome II, and the altered Chromosome II is replicated during cellular growth of the organism.
2 . The organism of claim 1 wherein Chromosome II comprises SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity to SEQ ID NO: 1.
3 . The organism of claim 2 wherein the at least one piece of exogenous DNA comprised on the altered Chromosome II is greater than 50 kb in size.
4 . The organism of claim 2 wherein the at least one piece of exogenous DNA comprised on the altered Chromosome II is greater than 100 kb in size.
5 . The organism of claim 1 further comprising an additional vector encoding at least one origin of replication and at least one selection marker.
6 . The organism of claim 5 comprising tetA/tetR or chloramphenicol resistance genes, an R6Kgamma origin of replication, and further comprising an RP4 oriT region.
7 . The organism of claim 1 wherein at least one essential genetic element removed from Chromosome II is comprised on Chromosome I.
8 . The organism of claim 1 wherein the Vibrio sp. is Vibrio natriegens.
9 . The organism of claim 1 wherein the altered Chromosome II comprises an inducible promoter.
10 . The organism of claim 9 wherein the inducible promoter is selected from the group consisting of: the IPTG inducible trc promoter, the temperature inducible lambda pR promoter, and arabinose inducible araBAD promoter.
11 . The organism of claim 1 further comprising that Chromosome I or Chromosome II comprise at least one deletion of a gene encoding a protein selected from the group consisting of: a recombinase, an endonuclease, a protease, and a restriction enzyme.
12 . The organism of claim 11 wherein the recombinase is recA and the endonuclease is Dns.