Materials and methods for extended continuous flow fermentation of reduced genome bacteria
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Publication Number
US-10604736-B2
Publication Date
2020-03-31
Expiration Date
Abstract
A two-vessel continuous flow system in conjunction with low mutation reduced genome bacterial strains provides a platform for long term extended fermentations. Such systems require modification of standard fermentation devices such as probes, pumps and monitoring systems as well as improved procedures for feed delivery, culture monitoring and product harvesting methods. An optimized two-vessel system for producing large quantities of fermentation products from small volume, long duration continuous fermentations represents a significant improvement over existing fermentation strategies. 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 that produces a biological product by culturing reduced genome Escherichia coli bacteria cells comprising an inducible expression vector encoding the biological product. The process uses at least two successive fermentors, each configured as an independent continuous flow chemostat, to support long-term production. In the first fermentor, the cells are cultured under uninduced conditions, and in the second fermentor the cells are cultured under suitable conditions to produce the biological product for at least two weeks, and the biological product is recovered from the second fermentor.
A key aspect is sequential inducer-separated operation, where the first fermentor provides culture medium for inoculating the second fermentor. The second fermentor is operated with inducer and receives an amount of culture medium from the first fermentor sufficient to inoculate the second fermentor. The described scheme is aligned with long-term extended fermentation in reduced genome bacteria and is presented for biological-product recovery after prolonged operation.
The reduced genome E. coli are based on E. coli K-12 strain MG1655 and have deleted therefrom a defined set of genes. The summary further describes an optimized two-vessel continuous flow C-flow chemostat system with modifications for gravimetrically controlled dual or multi-feed delivery, inducer-separated seed and production operation, enhanced sparger and gas handling, and high-OD flow-cell monitoring. The document also describes optional reduced-genome strain engineering to improve stability and presents comparative results showing reduced-genome E. coli sustaining high production and avoiding culture collapse versus commonly used strains.
Claims Coverage
The independent claim is directed to a continuous chemostat fermentation scheme with inducible expression vector production in at least two successive fermentors, including uninduced seed conditions in the first fermentor and induced production in the second fermentor for at least two weeks. The claim coverage centers on two primary inventive features, supported by dependent claims that narrow the produced biological product, constrain genome reduction and fermentation performance, and define additional feed and strain specifics.
Inducer-separated at-least-two successive continuous flow chemostats with uninduced first fermentor and induced second fermentor
A continuous fermentation process for producing a biological product by culturing reduced genome E. coli comprising an inducible expression vector in at least two successive fermentors, each configured as an independent continuous flow chemostat, where the first fermentor culture medium is used to inoculate a second successive continuous fermentor, the first fermentor operates under uninduced conditions and the second fermentor operates with inducer to produce the biological product for a period of at least two weeks, and the biological product is recovered from the second fermentor.
Reduced genome E. coli based on MG1655 with defined gene deletions
The process uses reduced genome E. coli whose native parent strain is E. coli K-12 strain MG1655 and where the reduced genome E. coli have deleted therefrom at least the listed genes.
Overall, the claim coverage centers on inducer-separated operation across at least two successive continuous flow chemostats to enable long-term biological-product production in reduced genome E. coli, combined with reduced-genome definition based on MG1655 with specified gene deletions.
Stated Advantages
Enables production for a period of at least two weeks.
Supports long-term extended fermentation and allows recovery of biological product from the second fermentor.
Comparative results are described as showing reduced-genome E. coli sustaining high production and avoiding culture collapse versus commonly used strains.
Documented Applications
Continuous production of biological products using reduced-genome E. coli in an inducible expression system, with comparative results reported for CRM197, rEPA, and human gelsolin in C-flow versus fed-batch.
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