Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

; ; ; ; ; (). Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter. Applied Microbiology and Biotechnology 1-14. Peer reviewed.

As Pichia pastoris (syn. Komagataella sp.) yeast
can secrete pure recombinant proteins at high rates, it is a
desirable production system. The function of a novel synthetic
variant of the AOX1 promoter was characterised comprehensively
using a strain secreting Candida antarctica lipase B
(CALB) as a model. A new time-saving approach was introduced
to determine, in only one experiment, the hitherto unknown
relationship between specific product formation rate
(qp) and specific growth rate (μ). Tight control of recombinant
protein formation was possible in the absence of methanol,
while using glycerol as a sole carbon/energy source. CALB
was not synthesised during batch cultivation in excess glycerol
(>10 g l-1) and at a growth rate close to μmax (0.15 h−1).
Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch
cultures, basal levels of qp > 0.4 mg g−1 h−1 CALB were
reached, independent of the μ at which the culture grew. At
μ > 0.04 h−1, an elevated qp occurred temporarily during the
first 20 h after changing to fedbatch mode and decreased
thereafter to basal. In order to accelerate the determination of
the qp(μ) relationship (kinetics of product formation), the entire
μ range was covered in a single fedbatch experiment. By
linearly increasing and decreasing glycerol addition rates, μ
values were repeatedly shifted from 0.004 to 0.074 h−1 and
vice versa. Changes in qp were related to changes in μ. A
rough estimation of μ range suitable for production was
possible in a single fedbatch, thus significantly reducing the
experimental input over previous approaches comprising several
experiments.