Physiological Characterization And Kinetics Of Porcine Insulin Precursor Production In Multi-copy Pichia Pastoris

Insulin is a potent drug which is currently used in the treatment of diabetes. Recombinant Pichia pastoris Mut+ strains harboring different porcine insulin precursor (PIP) gene copy number (from 1,6,12 to 18) were genetically constructed in our lab eariler. In this thesis, the effect of PIP gene dosage on physiological characterization of recombinant Pichia pastoris cells was investigated and kinetics of PIP production was established. A mixed feeding strategy for PIP production by multi-copy strains was developed as well.(1) The effects of PIP gene copy number on the growth, cell viability, genetic stability and oxygen uptaken rate (OUR) of multi-copy P. pastoris cells were studied in 5L fermenter fed-batch culture. The results showed that there were no significant changes in cell growth of multi-copy P. pastoris from the glycerol phase to the early methanol induction phase. However, after 24h of methanol induction, the specific growth rates of both recombinant strain A2 harboring 12-copy PIP gene and strain A3 harboring 18-copy PIP gene remarkably decreased as compared with the low multi-copy strains G1 (single PIP copy) and G6 (six PIP gene copies). Also, the cell viability of 18-copy strain A3 was only 78% and its PIP gene loss rate was up to 19.4% in genetic stability experiments. The maximum PIP production of 702mg/L was achieved by the 12-copy P. pastoris strain A2 grown in fed-batch culture among 4 multiple PIP copies P. pastoris strains tested here.(2) Kinetic models of cell growth, PIP formation and total methanol consumption amount in methanol fed-batch culture were proposed and kinetic model parameters ofμm, Xm,α,β,κ1 andκ2 were determined. The experimental data were basically consistent with the model predicted values, indicting that the kinetic models established here, theoretically, provided a reasonable description for multi-copy P. pastoris cells growth in methanol fed-batch culture. With the copy number increasing in recombinant P. pastoris cells, the coefficient of PIP production a was closely associated with the cells growth, indicating that to enhance the specific growth rates of multi-copy strain cells is an effective way to improve PIP production.(3) To improve the growth rate of high-pip-copy strains, a mixed feeding strategy with sorbitol and methanol was developed. Compared to methanol as the sole carbon source and energy source, PIP production level has been improved with an increase of 1.9% for strain G1, 42.6% for strain G6,34.7% for strain A2 and 80.9% for strain A3, respectively, using co-feeds of methanol and sorbitol during the induction phase. It is mainly due to increases in cell viability and in the specific growth rates of high-copy strains grown by co-substrates sorbitol and methanol.