Genetically engineered Vibrio sp. and uses thereof
Inventors
Weinstock, Matthew T • Wilson, Christopher M.
Assignees
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Publication Number
US-11203761-B2
Publication Date
2021-12-21
Expiration Date
Abstract
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.
Core Innovation
The invention provides a cold tolerant Vibrio natriegens organism that comprises an exogenous sequence encoding one or more enzymes from a reactive oxygen species detoxification system. The organism is able to generate new colonies after being stored at a temperature of 4°C or less for at least 14 days. The reactive oxygen species detoxification system addresses reactive oxygen species including singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals.
The reactive oxygen species detoxification system includes heterologous nucleotide sequences encoding one or more alkyl hydroperoxide reductase gene(s), a catalase gene, and/or a glutathione S-transferase gene. Specific enzyme examples include an alkyl hydroperoxide reductase operon, catalase genes katG and katE, and glutathione S-transferase gstA. The detoxification enzymes are encoded by an exogenous sequence and are operable in Vibrio natriegens under an exogenous promoter.
In some embodiments, the reactive oxygen species detoxification genetic elements are located on Chromosome I or Chromosome II. The organism can form new colonies after storage at about 4°C for at least 19 days, with examples described for improving viability after low-temperature storage.
Claims Coverage
The partial claim set includes one independent claim and multiple dependent claims, centered on cold tolerance in Vibrio natriegens by introducing exogenous reactive oxygen species detoxification enzymes and meeting storage-based colony-forming criteria. The inventive features primarily cover the detoxification system composition and, in narrower forms, the specific enzyme genes, operon source, genetic location, and minimum storage performance threshold.
Cold tolerant Vibrio natriegens with exogenous ROS detoxification enzymes and 4°C colony formation
A cold tolerant Vibrio natriegens organism comprising an exogenous sequence encoding one or more enzymes from a reactive oxygen species detoxification system and able to generate new colonies after being stored at a temperature of 4°C or less for at least 14 days.
ROS detoxification enzyme set under an exogenous promoter
The organism comprising a heterologous nucleotide sequence encoding one or more of alkyl hydroperoxide reductase gene(s), a catalase gene, and/or a glutathione S-transferase gene, each under an exogenous promoter and operable in Vibrio natriegens.
Alkyl hydroperoxide reductase operon as E. coli ahpCF
The organism in which the alkyl hydroperoxide reductase operon is the E. coli ahpCF alkyl hydroperoxide reductase operon.
Catalase gene selection as katG or katE
The organism in which the catalase gene is katG or katE.
Location of ROS detoxification genetic elements on ChI or ChII
The organism in which one or more of the alkyl hydroperoxide reductase operon, the catalase gene, and the glutathione S-transferase gene are located on ChI or ChII.
4°C storage threshold for new colony formation as at least 19 days
The organism specifying that it can form new colonies after storage at about 4°C for at least 19 days.
Across the independent claim and dependents, the main inventive concept is cold tolerance in Vibrio natriegens enabled by an exogenous reactive oxygen species detoxification system, with further narrowing by specifying the detoxification enzyme gene set and promoter context, particular gene/operon identities, optional chromosome location, and an explicit improved storage criterion.
Stated Advantages
Enables the cold tolerant Vibrio natriegens organism to generate new colonies after storage at 4°C or less for at least 14 days.
Provides improved cold-storage survivability with new colony formation after storage at about 4°C for at least 19 days.
Documented Applications
Cold storage use case for Vibrio natriegens where the organism is stored at 4°C or less and is able to generate new colonies after at least 14 days, and in narrower embodiments at least 19 days at about 4°C.
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