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Buried Kex2 Sites in Glargine Precursor Aggregates Prevent Its Intracellular Processing in Pichia pastoris Muts Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters


Glargine is a long-acting insulin analog with less hypoglycemia risk. Like human insulin, glargine is a globular protein composed of two polypeptide chains linked by two disulfide bonds. Pichia pastoris KM71 Muts strains were engineered to produce and secrete insulin glargine through the cleavage of two Kex2 sites. Nevertheless, the recombinant product was the single-chain insulin glargine (glargine precursor) instead of the expected double-chain glargine. Molecular model analysis of the dimeric and hexameric forms of the single-chain glargine showed buried Kex2 sites that prevent intracellular glargine precursor processing. The effect of the methanol-feeding strategy (methanol limited fed-batch vs. methanol non-limited fed-batch) and the induction temperature (28 °C vs. 24 °C) on the cell growth and production parameters in bioreactor cultures was also evaluated. Exponential growth at a constant specific growth rate was observed in all the cultures. The volumetric productivities and specific substrate consumption rates were directly proportional to the specific growth rate. The lower temperature led to increased metabolic activity of the yeast cells, which increased the specific growth rate. The methanol non-limited fed-batch culture at 24 °C showed the highest values for the process parameters. After 75 h of induction, 0.122 g/L of glargine precursor was obtained from the culture medium.

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The authors thank Glen D. Wheeler for his stylistic suggestions in the preparation of the manuscript. A.C.-P. thanks Consejo Nacional de Ciencia y Tecnología (CONACYT) for his fellowship.


This work was supported by Universidad Autónoma de Nuevo León, Mexico (Grant number CN926-19, PAICYT).

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A.C.-P-: conceptualization, methodology, formal analysis, investigation, data curation, writing—original draft, writing—review and editing, visualization. J.M.V.-S.: conceptualization, methodology, formal analysis, data curation, writing—original draft, writing—review and editing, visualization, supervision, project administration. A.L.H.-E.: investigation, writing—original draft. J.A.F.-G.: investigation, data curation. M.G.-O.: conceptualization, methodology, resources, writing—review and editing, supervision, project administration, funding acquisition. All authors read and approved the final manuscript.

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Correspondence to Martha Guerrero-Olazarán.

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Caballero-Pérez, A., Viader-Salvadó, J.M., Herrera-Estala, A.L. et al. Buried Kex2 Sites in Glargine Precursor Aggregates Prevent Its Intracellular Processing in Pichia pastoris Muts Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters. Appl Biochem Biotechnol 193, 2806–2829 (2021).

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  • Single-chain insulin glargine
  • Pichia pastoris
  • Kex2 protease
  • Muts phenotype
  • Methanol limited fed-batch culture
  • Methanol non-limited fed-batch culture