Reduced methanol input induces increased protein output by AOX1 promoter in a trans-acting elements engineered Pichia pastoris
- 447 Downloads
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.
KeywordsPichia pastoris Insulin precursor Methanol induction AOX1 promoter Protein expression
This work was supported by the Shanghai Science and Technology Innovation Action Plan (17JC1402400), Fundamental Research Funds for the Central Universities (22A201514040), National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204) and Talent Program of School of Biotechnology in East China University of Science and Technology.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 2.Brierley R, Siegel R, Bussineau C, Craig W, Holtz G, Davis G (1990) Mixed feed recombinant yeast fermentation. US Patent WO9003431Google Scholar
- 7.Gong X, Ding C, Liu L, Wu J (2013) Enhancement of human insulin precursor production by increasing the copy number in Pichia pastoris. Acta Microbiol Sin 53:545–552Google Scholar
- 8.Gurramkonda C, Polez S, Skoko N, Adnan A, Gäbel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas N (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Fact 9:31CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313:1441–1443CrossRefPubMedGoogle Scholar
- 18.Pais-Chanfrau JM, García Y, Besada V, Castellanos-Serra L, Cabello CI, Hernández L, Mansur M, Plana L, Hidalgo A, Támbara Y, Abrahantes-Pérez MC, del Toro Y, Valdés J, Martínez E (2004) Improving the expression of mini-proinsulin in Pichia pastoris. Biotechnol Lett 26:1269–1272CrossRefPubMedGoogle Scholar
- 22.Tadayuku I, Shuichi A (1976) A convenient method to estimate the rate of heat evolution in fermentation. Appl Chem Biotechnol 26:559–567Google Scholar