Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale

Neha Kaushik,Urpo Lamminmäki,Gaurav Batra,Navin Khanna

doi:10.1038/s41598-020-63995-5

Abstract

Cultivation of yeast Pichia pastoris in the microtiter plate, for optimisation of culture conditions, and expression screening of transformants has gained significance in recent years. However, in the microtiter plate, it has been challenging to attain cell densities similar to well-aerated shake-flask culture, due to the poor mixing resulting in oxygen limitation. To solve this problem, we investigated the influence of multiple cultivation parameters on P. pastoris cell growth, including the architecture of 96-deepwell plate (96-DWP), shaking throw diameter, shaking frequency, culture volume/well, and media composition. In the optimised conditions, a cell density of OD600 ~50 (dry cell weight ~13 g/L) with >99% cell viability was achieved in the casamino acids supplemented buffered-minimal-media in 300 to 1000 μl culture volume/well. We have devised a simplified method for coating of the culture supernatant on the polystyrene surface for immunoassay. Clones for secretory expression of envelope domain III of dengue virus serotype-1 under the control of inducible and constitutive promoter were screened using the developed method. Described microscale cultivation strategy can be used for rapid high-throughput screening of P. pastoris clones, media optimization, and high-throughput recombinant protein production. The knowledge gained through this work may also be applied, to other suspension cultures, with some modifications.

Highlights

Cultivation of yeast Pichia pastoris in the microtiter plate, for optimisation of culture conditions, and expression screening of transformants has gained significance in recent years
In the first series of experiments, we examined the impact of the geometry of 96-deepwell plate (96-deepwell plates (DWP)) and other conditions i.e., shaking throw diameter, shaking frequency and culture volume per well on P. pastoris cell growth
When shaking frequency of 25 mm throw diameter shaker was increased from 300 rpm to 400 rpm, further enhancement in cell growth was observed (OD600 19.5 to 55 in 1000 μl to 300 μl in V-bottom and OD at 600 nm (OD600) 17.5 to 56 in 1000 μl to 300 μl in U-bottom 96-DWP) (Fig. 1A,E)

Summary

Introduction

Cultivation of yeast Pichia pastoris in the microtiter plate, for optimisation of culture conditions, and expression screening of transformants has gained significance in recent years. In the microtiter plate, it has been challenging to attain cell densities similar to well-aerated shake-flask culture, due to the poor mixing resulting in oxygen limitation To solve this problem, we investigated the influence of multiple cultivation parameters on P. pastoris cell growth, including the architecture of 96-deepwell plate (96-DWP), shaking throw diameter, shaking frequency, culture volume/well, and media composition. High throughput parallel production of recombinant proteins in microwells has gained popularity, for combinatorial biology applications requiring an efficient microscale cultivation process for handling a large number of different variants[6]. 48-well plate was applied for the screening of engineered constitutive promoters using yeast-enhanced green fluorescent protein as a reporter in P. pastoris[9] In yet another studies, 96-DWP was utilized with 500 μl to 600 μl culture volumes for expression screening of enzyme variant and monoclonal antibodies[10,11]. The reported values indicate remarkably lower growth in DWPs in comparison to that achievable in well-aerated shake flask[3]

Methods

Results

Conclusion