Materials and methods for extended continuous flow fermentation of reduced genome bacteria
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Publication Number
US-11142745-B2
Publication Date
2021-10-12
Expiration Date
Abstract
Methods and compositions for long term continuous flow fermentation using a two vessel continuous culture fermentation apparatus are described.
Core Innovation
The invention relates to a continuous fermentation process for the production of a biological product by culturing a population of reduced genome Escherichia coli bacteria cells. The cells comprise an inducible expression vector encoding a biological product and the vector is operated in at least two successive fermentors configured as independent continuous flow chemostats.
A first fermentor cultivates the cells under uninduced conditions, and a second successive continuous fermentor produces the biological product under conditions comprising culture medium including an inducer. Culture medium from the first fermentor is used to inoculate the second fermentor, and the cells are cultured under suitable conditions to produce the biological product for a period of at least two weeks, after which the biological product is recovered from the second fermentor.
The reduced genome E. coli are genetically engineered to lack transposable genetic elements, and the document further describes extended long-term operation using hardware and feed-system modifications associated with the two-vessel continuous flow chemostat approach. It includes multi-stage fermentation with an uninduced seed vessel and an inducer-driven production vessel, as well as culture-medium handling and inline monitoring enabling stable long-duration expression.
The document reports that reduced-genome strains maintain high productivity for weeks without culture collapse compared to conventional strains, and it provides examples of biological product production including CRM197, rEPA, and human gelsolin using the described continuous process with reduced-genome E. coli lacking transposable elements and associated long-duration operational and monitoring arrangements.
Claims Coverage
The claim set centers on one independent claim covering a continuous, multi-stage fermentation using reduced-genome E. coli in at least two successive independent continuous flow chemostats, with a first uninduced fermentor and a second inducer-producing fermentor that is inoculated by transferring culture medium. Dependent claims refine multiple inventive features including specific reduced-genome cells, feed architecture, gravimetric feeding hardware, and a quantitative culture stabilization threshold based on OD600.
Continuous multi-stage fermentation with uninduced-to-induced chemostats
A continuous fermentation process for producing a biological product by culturing reduced genome Escherichia coli cells with an inducible expression vector in at least two successive fermentors configured as independent continuous flow chemostats, where a first fermentor runs under uninduced conditions and a second successive fermentor produces the biological product under inducer-containing culture medium, and culture medium from the first fermentor inoculates the second for at least two weeks with recovery from the second.
Reduced genome Escherichia coli lacking transposable genetic elements
The reduced genome Escherichia coli are genetically engineered to lack transposable genetic elements for the continuous fermentation producing the biological product.
Induced production in a second successive continuous fermentor inoculated from the first
The second successive continuous fermentor produces the biological product using culture medium comprising an inducer and an amount of culture medium from the first fermentor sufficient to inoculate the second fermentor, with culture under suitable conditions to produce the biological product for at least two weeks.
MDS69 meta ΔrecA reduced-genome cells
The process is carried out using reduced genome bacteria cells that are MDS69 meta ΔrecA cells.
Dual feed approach separating phosphate in glucose minimal salts medium
Each fermentor is fed a glucose minimal salts medium via a dual feed approach that separates phosphate delivery from other media components.
Gravimetric feeding of culture medium to the fermentors
The culture medium in the fermentors is fed gravimetrically.
Multigravimetric system using multiple pumps and multiple balances
Gravimetric feed is performed using a multigravimetric system with a plurality of pumps and a plurality of balances to control the flow through the fermentors.
Culture stabilization by an OD600 threshold
E. coli cultures in each successive fermentor are stabilized at an optical density at 600 nm (OD600) of at least about 200.
Overall, the claims cover a continuous multi-stage chemostat process using reduced-genome E. coli lacking transposable genetic elements, with an uninduced seed fermentor and an inducer-driven production fermentor inoculated by transfer from the first. Dependent claims further specify particular reduced-genome cells (MDS69 meta ΔrecA), a dual-feed phosphate-separated glucose minimal salts medium, gravimetric feeding with a multigravimetric control architecture, and an OD600-based stabilization threshold (≥ about 200).
Stated Advantages
Maintains high productivity for weeks without culture collapse compared to conventional strains.
Documented Applications
Production of biological products including CRM197, rEPA, and human gelsolin using the described continuous multi-stage fermentation approach with reduced-genome Escherichia coli lacking transposable genetic elements and long-duration operation.
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