Fundamental Investigations On Overproduction Of Metabolic Intermediate And Heterologous Proteins From Yeast Systems

Yeast is one of the widest distributed in the nature, the closest related with human being and the safest microorganisms. Yeast cell has played an important role in traditional fermentation industry as well as modern biotechnology. In this work, the overproduction of intermediate metabolite fructose- 1,6-diphosphate (FDP) by yeast cells was investigated in depth, the high level expressions of mouse a-amylase and monellin by genetic engineering yeast cell were evaluated and optimized. The results offer abundant experimental data and theoretical guidance for the further research and development of above processes.1. Overproduction of FDP by metabolic regulation of yeast cellsThe mechanism and technology of the bioconversion via yeast cells for FDP overproduction were studied. The methods to increase cell wall permeability were evaluated for the purposes of changing metabolic pathway and accumulating FDP in large quantity. The effects of environmental conditions, such as temperature, pH, carbon sources, inorganic phosphate, metal ions as well as dissolved oxygen etc, on the FDP overproduction were studied. The operation conditions were optimized for the FDP production by using waste brewer's yeast cells. Then, the fed-batch operation strategy was formulated for FDP production, and the operation time was optimized with the production cost as a main concern. The highest FDP concentration and glucose conversion ratio reached 135.65 g/1 and 28.2, respectively.The metabolic pathways and flux were analyzed for the FDP overproduction via permeabilized yeast cell. The mechanism for FDP accumulation was discussed. The concentrations of substrates, intermediates and product during the bioconversion process were measured and the metabolic flux model was proposed to describe the glucose metabolization and FDP overproduction by using permeabilized yeast cells. The model was applied to analyze the kinetic characteristics during FDP accumulation processes. The results indicated that the intracellular ATPase deactivation, BMP pathway and ethanol as well as glycerol sub-pathways were the main concerns in FDP accumulation. The phosphofructokinase (PFK) was the controlling point in the metabolic pathway. The rate limiting was not caused by the low concentration offructose-6-phosphate, but the insufficient supply of another substrate ATP. The pyruvate kinase (PK) is the rigid point in regulating the metabolic flux distribution. The limitation in PK flux will lead metabolic flux to the glycerol sub-pathway, which is the major reason in insufficient ATP supply and low recovery of FDP.A new process for FDP separation and purification was proposed. The residue inorganic phosphate was precipitated by zinc salt and FDP was recovered. After ion exchange dissociation and organic solvent precipitation, the purity and recovery yield were reached 86.17% and 52.24%, respectively.2. Overproduction of heterogeneous protein by the culture of recombinant Saccharomyces cerevislaeThe secretive expression of heterogeneous protein mouse a-amylase by using recombinant S. cerevisiae was studied as a model system. The promoter SUC2 is regulated by glucose concentration, and the selective pressure for plasmid stabilization is auxotrophy (TRP). The basic principles for high expression of mouse a-amylase were explored in this study.The favorable cultivation conditions for secretive expression of mouse a-amylase were evaluated experimentally. It is necessary to eliminate the glucose inhibition to the promoter SUC2, and to supply sufficient natural nitrogen, such as yeast extract and corn steep. A proper carbon/nitrogen ratio in cell growth stage can make the recombinant yeast cell with high activity in expression stage and lower the plasmid loss. The favorable carbon/nitrogen ratio is C/N = 5:1-4:1. The dissolved oxygen (DO) concentration is also an important factor for protein synthesis, and the DO value should higher than 1.5 mg/1.As mentioned before, the high expression of heterogeneous protein of recombinant yeast...