Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture

Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological temperature of 37°. However, recent research has shown that the use of low-temperature conditions (30-33°) as a platform for cell-culture results in changes in cell characteristics, such as increased specific productivity and extended periods of cell viability, that can potentially improve the production of recombinant proteins. Furthermore, many recent reports have focused on investigating low-temperature mammalian cell culture of Chinese hamster ovary (CHO) cells, one of the principal cell-lines used in industrial production of recombinant proteins. Exposure to low ambient temperatures exerts an external stress on all living cells, and elicits a cellular response. This cold-stress response has been observed in bacteria, plants and mammals, and is regulated at the gene level. The exact genes and molecular mechanisms involved in the cold-stress response in prokaryotes and plants have been well studied. There are also various reports that detail the modification of cold-stress genes to improve the characteristics of bacteria or plant cells at low temperatures. However, there is very limited information on mammalian cold-stress genes or the related pathways governing the mammalian cold-stress response. This project seeks to investigate and characterise cold-stress genes that are differentially expressed during low-temperature culture of CHO cells, and to relate them to the various changes in cell characteristics observed in low-temperature culture of CHO cells. The gene information can then be used to modify CHO cell-lines for improved performance in the production of recombinant proteins.

Inverse Metabolic Engineering of Synechocystis PCC 6803 for Improved Growth Rate and Poly-3-hydroxybutyrate Production

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems.