Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter

Protein Expr Purif. 2013 Dec;92(2):235-44. doi: 10.1016/j.pep.2013.08.018. Epub 2013 Oct 16.


The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.

Keywords: Bovine chymosin; Fed-batch fermentation process; Methanol-inducible AOX1 promoter; Optimization; Pichia (Komagataella) pastoris; Purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde Oxidase / genetics*
  • Animals
  • Ascorbic Acid / metabolism
  • Bioreactors
  • Cattle
  • Chymosin / analysis
  • Chymosin / chemistry
  • Chymosin / genetics
  • Chymosin / metabolism*
  • Culture Media
  • Fermentation
  • Hydrogen-Ion Concentration
  • Pichia / genetics
  • Pichia / metabolism*
  • Promoter Regions, Genetic / genetics*
  • Recombinant Proteins / analysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Sorbitol / metabolism
  • Temperature


  • Culture Media
  • Recombinant Proteins
  • Sorbitol
  • Aldehyde Oxidase
  • Chymosin
  • Ascorbic Acid