The Mut+ strain of Komagataella phaffii (Pichia pastoris) expresses PAOX1 5 and 10 times faster than Muts and Mut− strains: evidence that formaldehyde or/and formate are true inducers of PAOX1

Abstract

The Mut + strain of Komagataella phaffii (Pichia pastoris) expresses PAOX1 5 and 10 times faster than Mut s and Mutstrains: Evidence that formaldehyde or/and formate are true inducers of AOX.

PAOX1expression in mixed-substrate continuous cultures ofKomagataella phaffii(Pichia pastoris) is completely determined by methanol consumption regardless of the secondary carbon source

“…When methanol + glycerol and methanol + sorbitol were fed to a continuous culture, the variation of the substrate concentrations with D was consistent with the characteristic pattern described above. In the dual-limited regime, both substrates were completely consumed — up to D = 0.08 h −1 ≈ 0.11 h −1 = μ m | methanol (Singh and Narang, 2020) in Fig. 2a and D = 0.03 h −1 = μ m | sorbitol h −1 in Fig.…”

“…2.1 Microorganism and growth medium A K. phaffii Mut + strain, GS115 (his4) was procured from J. M. Cregg, Keck Graduate Institute, Claremont, CA, USA and was genetically modified to express a recombinant β-galactosidase protein. Details of the strain construction have been presented elsewhere (Singh and Narang, 2020). The resulting strain was called Mut + (pSAOH5-T1) and was used for this study.…”

“…We are interested in measuring the steady state concentrations of biomass X , primary carbon source S 1 , and secondary carbon source S 2 as well as the specific activity of enzyme E 1 . These quantities are denoted x , s 1 , s 2 , and e 1 , respectively, and satisfy the mass balances: where s f ,1 , s f ,2 denote the respective feed concentrations of S 1 , S 2 ; and μ , r s ,1 , r s ,2 , r e ,1 denote the respective specific rates of growth, consumption of substrate, and expression of a stable intracellular protein (Pfeffer et al., 2011; Singh and Narang, 2020). It follows from Eqs.…”

“…denote the respective specific rates of growth, consumption of substrate, and expression of a stable intracellular protein (Pfeffer et al, 2011;Singh and Narang, 2020). It follows from Eqs.…”

“…The foregoing strategies have led to reduced methanol consumption, but they can also result in decreased recombinant protein expression. Recently, we found that host strain engineering decreases recombinant protein expression substantially -the specific productivities of the engineered Mut s and Mutstrains are respectively 5-and 10-fold lower than that of the Mut + strain (Singh and Narang, 2020). Since these three strains differ only with respect to their capacity for methanol uptake, it is clear that the methanol uptake rate is an important determinant of the PAOX1 expression rate.…”