AI Article Synopsis

  • Phenoxy radical coupling reactions, guided by dirigent proteins, are essential for lignan biosynthesis, creating specific compounds like (+) or (-)-pinoresinol from coniferyl alcohol.
  • A new high-yield production process using Pichia pastoris has significantly increased the amount of the dirigent protein AtDIR6 to over 47 mg L⁻¹, improving upon previous methods by more than 250 times.
  • Biochemical analysis confirmed that AtDIR6 produced in P. pastoris closely matches that from plant cultures in terms of structure and function, highlighting the importance of glycan structures for the protein's activity and stability.

Article Abstract

Phenoxy radical coupling reactions are involved in the biosynthesis of lignans in planta. Interestingly, the reaction can be guided by dirigent proteins, which mediate the stereoselective formation of either (+) or (-)-pinoresinol from coniferyl alcohol. So far, the mechanism is poorly understood, and for detailed mechanistic studies, a heterologous expression platform which allows the cost-effective, fast, and robust expression in high yields is needed. We established a reliable, high-yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg L⁻¹ of the dirigent protein AtDIR6, which represents a more than 250-fold increase compared to previous studies. Biochemical characterization of AtDIR6 produced with P. pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum. CD spectroscopy verified the β-barrel structure proposed by earlier studies and bioconversion experiments revealed similar activities to plant-derived protein, validating P. pastoris as a suitable expression system for dirigent proteins. Compared to the complex glycan structures of most plant cells, proteins produced with P. pastoris have the advantage that they can be enzymatically deglycosylated under non-denaturating conditions. With this study, we demonstrate that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein as deglycosylation induced conformational changes leading to the complete loss in dirigent activity and subsequent protein aggregation.

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Source
http://dx.doi.org/10.1007/s00253-012-4579-xDOI Listing

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