Fermentation process
Inventors
Gabant, Philippe • El Bakkoury, Mohamed • Van Melderen, Laurence
Assignees
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Publication Number
US-11932672-B2
Publication Date
2024-03-19
Expiration Date
Abstract
Some embodiments relate to a method for producing a product of interest with a microbial host using an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid molecule is controlled.
Core Innovation
The disclosed invention provides a method for producing a product of interest with a microbial host using an auto-replicative extra-chromosomal nucleic acid molecule. The extra-chromosomal nucleic acid comprises a first nucleic acid sequence whose genetic activity confers immunity or resistance to a bacteriocin, and a second nucleic acid sequence coding for the product of interest. The first and second nucleic acid sequences are genetically controlled as part of culturing the transformed microbial host.
During culturing, the first nucleic acid sequence is expressed to a level that confers a selective advantage to the microbial host during the culturing, thereby maintaining the auto-replicative extra-chromosomal molecule in the growing microbial population. In parallel, the second nucleic acid sequence is genetically controlled to produce the product of interest. This enables maintaining extra-chromosomal presence via immunity or resistance while production proceeds from the second sequence.
During at least a portion of the culturing, conditions are such that the first nucleic acid sequence does not exhibit the genetic activity, thereby reducing the energetic burden for the microbial host cell during production of the product of interest. The approach is antibiotic-free, using bacteriocin immunity/resistance selection rather than antibiotic selection, while genetic activity control reduces energy burden without losing the selection functionality for the extra-chromosomal molecule.
The description emphasizes promoter control of the immunity or resistance sequence, including weak constitutive and inducible promoters such as P24/proC and P24 LacO to tune genetic activity. Exemplary immunity modulator sequences and promoter arrangements are discussed in connection with bacteriocins and resistance factors, including McbG for B17, MccE/C-terminal MccE for C7, and Cvi for ColV/colicin-V, to support maintaining selective advantage and subsequently turning off or reducing genetic activity during production.
Claims Coverage
The document includes one independent claim, supported by multiple dependent claims that refine the extra-chromosomal form, promoter/regulation for the immunity sequence, independent control relationships, specific bacteriocin/immunity pairs, and narrowed product categories. The independent claim centers on antibiotic-free production using bacteriocin immunity/resistance on an auto-replicative extra-chromosomal nucleic acid with genetic activity turned off during at least a portion of culturing to reduce energetic burden.
Auto-replicative extra-chromosomal immunity/resistance plus product sequence
A method for producing a product of interest with a microbial host using a microbial host comprising an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers immunity or resistance to a bacteriocin to the microbial host, and a second nucleic acid sequence coding for the product of interest.
Selective advantage expression maintains extra-chromosomal presence
Culturing the transformed microbial host under conditions allowing the transformed microbial host to express the first nucleic acid sequence to a given level so that the genetic activity of the first nucleic acid sequence confers a selective advantage during the culturing, thereby maintaining the auto-replicative extra-chromosomal molecule in the growing microbial population, while simultaneously genetically controlling the second nucleic acid sequence to produce the product of interest.
Genetic activity turned off reduces energetic burden during at least a portion of culturing
During at least a portion of the culturing, conditions are such that the first nucleic acid sequence does not exhibit the genetic activity, thereby reducing the energetic burden for the microbial host cell during the production of the product of interest.
Overall, the claim coverage is directed to maintaining an auto-replicative extra-chromosomal molecule via bacteriocin immunity/resistance selective advantage during growth, while reducing the host energetic burden by turning off or preventing genetic activity of the immunity/resistance sequence during at least a portion of culturing, without stopping control of the product-producing sequence.
Stated Advantages
Reducing the energetic burden for the microbial host cell during production of the product of interest by making the first nucleic acid sequence not exhibit genetic activity during at least a portion of culturing.
Maintaining the auto-replicative extra-chromosomal molecule in the growing microbial population by expressing the first nucleic acid sequence to a level that confers a selective advantage during culturing.
Documented Applications
Producing a product of interest with a microbial host using an antibiotic-free fermentation/production approach based on auto-replicative extra-chromosomal nucleic acid carrying bacteriocin immunity/resistance and a product-coding sequence.
Application to industrially useful molecules selected from carbohydrates, lipids, organic molecules, nutrients, biofuels, or precursors thereof, and pharmaceutical/biopharmaceutical products or precursors thereof, including mixtures of two or more as narrowed by dependent claim information.
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