Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode

Abstract

The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

“…Then, this culture was inoculated in 3 L BSM medium held in a 5 L fermenter with initial OD 600 of 0.6. Using the standard method reported previously (Liu and Zhu, 2015;Wang et al, 2017), the fermentation operation was divided into three phases: P1, a glycerol batch phase; P2, a glycerol fed-batch phase; and P3, the methanol fed-batch phase. Glycerol fed-batch cultivation was applied to increase the cell density for improved FBG1 production.…”

Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

“…The expression levels of the heterologous enzymes in S. cerevisiae significantly affect the production of the target products. The transcriptional activator HAC1 is a major regulator of the unfolded protein response in many eukaryotes, which can up-regulate the expression of the heterologous enzymes 34 . We previously showed that the titers of the unnatural ginsenosides 3 β - O -Glc-DM and 20 S - O -Glc-DM in the engineered yeasts were increased by 1.6- and 1.5-fold, respectively, by overexpressing HAC1 24 .…”

Astragalus–Scorpion Drug Pair Inhibits the Development of Prostate Cancer by Regulating GDPD4-2/PI3K/AKT/mTOR Pathway and Autophagy

“…The study found that BmK_AGAP was also presented in the scorpion. On the one hand, BmK_AngM1 and BmK_AGAP were reported to have significant analgesic effects ( Wang QH. et al, 2017 ; Kampo et al, 2019 ).…”