P22: Feed-In-Time Fed-batch Media for Yeast: Approval with Lipase expressing Pichia pastoris strain in Clone Screening and High Cell Density Fermentation at Microscale

The new Feed-In-Time (FIT) fed-batch media are introduced with an exemplary study of Lipase expressing Pichia pastoris clones. The FIT fed-batch-media possess the capability to release glucose from polysaccharides by enzymatic hydrolysis directly in the medium and therefore, are ideally suited for small scale applications. The poster presents the impact of batch versus fed-batch fermentation mode on product formation with the pAOX1 driven secretory expression of Lipase (RoL) in Pichia pastoris. In the initial clone screening using Glucose-fed-batch-medium, significant differences in clone ranking were observed compared to classical batch screening. Thus, in early screening the fermentation profile is of great influence for selecting productive clones. Furthermore, the FIT media was improved to conduct high cell density fermentation without the need of pH re-adjustment. In consequence, the application of Glucose/Methanol mixed-feed resulted in high cell densities (OD₆₀₀ ≈ 300) and high enzyme expression levels (≈ 25 kU/L). The results demonstrated that Glucose-fed-batch in combination with Methanol induction of P. pastoris is an attractive and economically interesting alternative to conventional Glycerol-fed-batch processes, especially for the production of industrial enzymes. In summary, a consequent study of the typical biotechnological workflow was conducted, beginning with evaluation of clone screening strategy, the clone screening itself and the optimization of HCDC fed-batch fermentation strategy. The combination of high-throughput experimentation in the BioLector and the FIT fed-batch-media now provide an interesting new possibility to study clones and culture media with different growth rates in fed-batch mode at small scale and high-throughput, thus, enabling faster development times.