Table 1.

Overview of the fed-batch cultivations.

Recombinant protein	None		EcPGA		CaLB			TlXynA
Strain	Pp1 (control)	Pp1 (control)	Pp4	Pp5 (control)	Pp10	Pp10	Pp10	Pp14
sfGFP expression	PKAR2	PKAR2	PKAR2	No promoter (x)	PKAR2	PKAR2	PKAR2	PKAR2
μmethanol (h−1) set	0.016	0.032	0.016	0.016	0.016	0.032	0.008	0.016
μmethanol (h−1) reached	0.021 ± 0.001	0.039 ± 0.001	0.019 ± 0.002	0.021 ± 0.002	0.018 ± 0.001	0.036 ± 0.001	0.007 ± 0.001	0.019 ± 0.001

P. pastoris strains producing three different recombinant proteins and/or containing a reporter of UPR up-regulation were constructed. The recombinant proteins were EcPGA, CaLB, and TlXynA. The reporter of the up-regulation of UPR was a genome-integrated cassette consisting of the KAR2 promoter and sfGFP gene. Three strains contained the UPR reporter and produced a recombinant protein—EcPGA (Pp4), CaLB (Pp10), or TlXynA (Pp14). A control strain to verify the suitability of our UPR reporter produced EcPGA and contained a genome-integrated cassette carrying the sfGFP gene, but no promoter in front of this gene (Pp5). A control strain to examine the UPR up-regulation by cultivation conditions contained the UPR reporter, but produced none of the three recombinant proteins of interest (Pp1). In all processes, temperature was maintained at 30°C and pH at 5.5. All processes consisted of a batch phase with glycerol (30 g l⁻¹), a short (2.5 h) growth fed-batch with an exponential feeding of glycerol to maintain the specific growth rate at 0.2 h⁻¹, and a production fed-batch with an exponential feeding of methanol to maintain the specific growth rate at 0.008, 0.016, or 0.032 h⁻¹.