Improving phytase production in Pichia pastoris fermentations through
de-repression and methanol induction optimisation
Abstract
Pichia pastoris ( Komagataella phaffii) is a fast-growing
methylotrophic yeast with the ability to assimilate several carbon
sources such as methanol, glucose, or glycerol. It has been shown to
have outstanding secretion capability with a variety of heterologous
proteins. In previous studies, we engineered P. pastoris to
co-express E. coli AppA phytase and the HAC1 transcriptional
activator using a bidirectional promoter. Phytase production was
characterised in shake flasks and did not reflect industrial conditions.
In the present study, phytase expression was explored and optimised
using instrumented fermenters in continuous and fed-batch modes. First,
the production of phytase was investigated under glucose de-repression
in continuous culture at three dilution factors, 0.5 d
-1, 1 d -1, and 1.5 d
-1. The fermenter parameters of these cultures were
used to inform a kinetic model in batch and fed-batch modes for growth
and phytase production. The kinetic model developed aided to design the
glucose feeding profile of a fed-batch culture. Kinetic model
simulations under glucose de-repression and fed-batch conditions
identified an optimal phytase productivity at the specific growth rate
of 0.041 h -1. Validation of the model simulation with
experimental data confirmed the feasibility of the model to predict
phytase production in our newly engineered strain. Methanol was used
only to induce the expression of phytase at high cell densities. Our
results showed that high phytase production required two stages, the
first stage used glucose under de-repression conditions to generate
biomass while expressing phytase, and stage two used methanol to induce
phytase expression. The production of phytase was improved 3.5-fold by
methanol induction compared to the expression with glucose alone under
de-repression conditions to a final phytase activity of 12.65 MU/L. This
final volumetric phytase production represented an approximate 36-fold
change compared to the flask fermentations. This two-phase strategy
allowed to optimise phytase productivity by producing phytase during
both growth and methanol induction phases. Finally, the phytase protein
produced was assayed to confirm its molecular weight, and pH and
temperature profiles. This study highlights the importance of optimising
protein production in P. pastoris when using novel promoters and
presents a general approach to performing bioprocess optimisation in
this important production host.