Reduced methanol input induces increased protein output byAOX1promoter in atrans-acting elements engineeredPichia pastoris

Abstract

High oxygen consumption and heat release caused by methanol catabolism usually bring difficulties to industrial scale-up and cost for protein expression driven by methanol-induced AOX1 promoter in Pichia pastoris. Here, reduced methanol feeding levels were investigated for expression of insulin precursor in a trans-acting elements engineered P. pastoris strain MF1-IP. Insulin precursor expression level reached 6.69 g/(L supernatant) at the methanol feeding rate of 6.67 mL/(h·L broth), which was 59% higher than that in the wild-type strain WT-IP at the methanol feeding rate of 12 mL/(h·L broth). Correspondingly, the insulin precursor expression level in fermentation broth and maximum specific insulin precursor production rate was 137 and 77% higher than the WT-IP, respectively. However, oxygen consumption and heat evolution were reduced, and the highest oxygen consumption rate and heat evolution rate of the MF1-IP were 18.0 and 37.7% lower than the WT-IP, respectively.

Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology

“…When methanol is the only carbon source, P. pastoris intracellular alcohol oxidase 1 (Aox1, EC 1.1.3.13) efficiently catalyzes the oxidation of methanol to formaldehyde (Rodriguez et al, ; Xuan et al, ). The AOX1 promoter (P AOX1 ) is very efficient and is one of the most widely used promoters in recombinant protein expression systems (Higgins & Cregg, ; Y. Li et al, ; J. Wang et al, ). However, the presence of other carbon sources, such as glycerol, can inhibit the metabolism of methanol.…”

“…The AOX1 promoter (P AOX1 ) is very efficient and is one of the most widely used promoters in recombinant protein expression systems (Higgins & Cregg, 1998;Y. Li et al, 2003;J. Wang et al, 2018).…”