A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris

Abstract

BackgroundPichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets.ResultsHere, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate qs, specific productivity qp and the adaption time (Δtimeadapt) of the culture to methanol. Based on qs, an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for qs were increased stepwise until a qs max of 2.0 mmol·g-1·h-1 resulted in the highest specific productivity of 11 U·g-1·h-1.ConclusionsOur strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts.

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…However, production characteristics such as titres, yields and specific productivities obtained for this set of operational parameters used to determine µ max for Mut + and Mut S strains/clones (Dietzsch et al, 2011a, Krainer et al, 2012 do not reflect the highest possible productivity (Table 3). The best producing strain/clone could not therefore, be selected based on q p -data obtained in such batch experiments.…”

“…5). Unlike pulsed strategies (Dietzsch et al, 2011a), the specific growth rate is controlled by the rate of substrate addition at a defined value lower than or equal to its maximum (µ max ) (Zhang et al, 2007). The added substrate is immediately utilised and therefore cells can only grow as fast as the rate of substrate supply.…”

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastorisMut+ phenotype in continuous culture

“…Even, q Meth has been reported as basic criteria to design induction operation of high productive protein production process. 38 It has recently been reported that the implementation of methanol feeding based on constant q Meth in fed-batch operation achieves a more stable production profile of galactose oxidase, with similar volumetric productivity to that obtained at pre-programmed feeding. 39 In the present kinetic study, the composition of the mixture feeding considered as a process variable, together with continuous cultivation, allowed for assessing the effect of feeding rate without changing cell growth rate.…”

High Expression of Human Cathepsin S by Recombinant <b><i>Pichia pastoris</i></b> with Cod Skin as an Organic Co-Nitrogen Source

“…As is well known, heterologous protein expression by methylotrophic P. pastoris controlled by the AOX promotor takes place in the presence of methanol as the sole carbon source. In some studies, carbon sources, such as sorbitol, xylose, ribose, and glucose as co-substrates, were reported for P. pastoris growth [Dietzsch et al, 2011;Wang et al, 2016]. When they had been exhausted, the step of methanol induction just began [Iglesias-Figueroa et al, 2016].…”