Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations

Abstract

Recombinant protein synthesis in Pichia pastoris is generally controlled by the strong methanol inducible AOX1 promoter which is repressed by glucose and glycerol. In shake flasks, commonly one or two methanol pulses are added per day for induction. Such pulse feeding procedure leads to carbon starvation phases, which may enhance proteolytic activities and, therefore, cause product losses. Starvation between the methanol pulses could be avoided with a continuous enzymatic feed of glucose from a glucose-based polymer. The amount of glucose was low enough to prevent AOX1 repression by glucose. Energy and carbon were continuously supplied for cell maintenance resulting in significantly increased cell densities and product activities, as shown here at the example of a fungal lipase expressed in P. pastoris. A threefold improvement in measured product activity was obtained by applying enzymatic glucose feed and a further improvement was achieved by applying a defined mixture of ammonium compounds. The strategy described here simplifies the general procedure in shaken cultures by allowing the direct continuation of the cultivation from glucose to the methanol-based production phase without a medium change. It is easily applicable to multiwell plates and thus beneficial for high throughput applications.

Comparison of Yeasts as Hosts for Recombinant Protein Production

“…For example, to avoid the transitional period prior to methanol induction, a feed strategy was established with slow enzymatic glucose feed to maximize the expression of Rhizopus oryzae lipase (ROL). This feed strategy maintains a low glucose concentration while avoiding cellular starvation and inactivation of pAOX1, which resulted in 3- and 6-fold higher cell density protein activity, respectively, when compared to [ 144 ].…”

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli

“…Ruth et al [ 82 ] successfully applied the Feed Bead ® Technology to study transcription factor of P. pastoris and Ashoor et al [ 83 ] to produce human immunoglobulin receptors with EnPresso in P. pastoris . In the approach by Panula-Perälä et al [ 84 ] the slow glucose release of the EnBase technology was used as a continuous background energy and carbon supply bridging the gaps between methanol pulses needed for protein expression under the AOX1 promotor. Such a background feeding provided a significant increase in cell densities and product activities.…”

Improved microscale cultivation of Pichia pastoris for clonal screening

“…This problem has been circumvented by providing substrate via diffusion [ 61 , 62 ]. Another possibility is the enzymatic substrate release from oligo- or polymeric inert compounds like starch [ 31 , 60 , 63 ] or sucrose [ 43 ] at a constant rate. Contrary to exponential substrate feeding, which can be applied during lab-scale bioreactor cultivation to adjust a constant specific growth rate, a constant substrate release rate will lead to a decreasing specific growth rate over time.…”