This application aims to test a new Scarab E. coli strain, MDS42pdu, for commercial fermentation to produce biopharmaceuticals, amino acids and biofuels. It is designed for a very low rate of point mutations and Insertion Sequence transposition, especially in the stress conditions of recombinant protein production. We propose here to study the impact on a fundamental problem of large scale fermentation: Darwinian evolution of bacteria in the fermenter toward loss of productivity. Random mutations occurring in culture can produce cells freed from the burden of product formation and these have a selective advantage. Soon these overtake the culture and reduce or eliminate productivity. In short, cultures deteriorate. Selection of mutations can also undermine product purity. We would like to slow or eliminate this degradation of performance to improve stability, quality and efficiency of current fed batch methods and possibly, to support a more continuous fermentation protocol in the future. The proposed studies use periodic total genomic sequencing of extended cultures to compare ordinary production strains with the new low mutation strain to see if the period of culture productivity before degradation is extended, and whether we can identify the types of mutations involved in performance degradation. The two aims propose periodic whole genome sequencing of serially transferred cultures in shake flasks to quantify inexpensively the performance of the low mutation strain in a wide variety of cases, to be followed by more detailed study using continuous flow fermentation. We anticipate that control cultures using ordinary E. coli strains will become non productive considerably more quickly than MDS42pdu. If this anticipated result is found, we will determine the maximum number of generations before problems emerge in the new production sytem. If a greatly extended productive lifetime is achieved we will consider it a major milestone toward commercialization of our product for fed batch fermentation. Sequencing will also help determine whether additional mutational mechanisms may be active that could be beneficially inactivated in future work. An extremely long productive culture life will indicate the feasibility of a continuous fermentation process usig this strain.
Public Health Relevance Statement: Public Health Relevance: This proposal further develops Scarab Genomics' Clean Genome(R) E. coli to engineer a genetically stable strain for use in fermentation processes for biomanufacturing. Improved stability will provide higher quality bio- products and extended culturing will lead to higher yields from more efficient and cheaper production.
Project Terms: Agreement; Amino Acids; Animal Feed; Antibodies; Bacteria; Biological Assay; Biological Products; Biomanufacturing; Businesses; Categories; Cell Line; Cells; Chemical Industry; Cloning; commercialization; Computers; Consult; Cytolysis; Data; design; Development; DNA; DNA Insertion Elements; Engineering; Escherichia coli; Evaluation; Evolution; experience; Fee-for-Service Plans; feeding; Fermentation; Flagella; flasks; Future; Gel; Generations; Genetic; Genome; genome sequencing; Genomic Segment; Genomics; Growth; Hormones; Immunoglobulin Fragments; improved; Infant formula; IS Elements; Lead; Legal patent; Length; Licensing; Life; Literature; Lymphokines; Mails; Marketing; Measures; meetings; Methods; Molecular Weight; Mutation; Organism; payment; Peptide Hydrolases; Peptides; Performance; Pharmacologic Substance; Phase; plasmid DNA; Plasmids; Point Mutation; Process; Production; Productivity; productivity loss; Prophages; Proteins; Protocols documentation; public health relevance; Publications; Reader; Recombinant Proteins; Recombinants; Research; Route; Sales; Sampling; Solutions; Staging; Stress; Swimming; Testing; Threonine; Time; web site; Work
