Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

Abstract

Background Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. Results A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3′hydroxyibuprofen. Conclusions For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Structural and Biochemical Studies Enlighten the Unspecific Peroxygenase from Hypoxylon sp. EC38 as an Efficient Oxidative Biocatalyst

“…Strain Generation. Plasmids were obtained from bisy (bisy GmbH, Hofstaehen, Austria) and generated as described in ref 40. These vectors are optimized to drive the expression of the full-length protein fused with the Mating-Factor signal sequence [MKSLSFSLALGFGSTLVYS], allowing the secretion of the target polypeptide under the control of a bidirectional promoter P PDF .…”

Screening of yeast co‐culture using crude hydrolysate for co‐fermentation of pentose and hexose

“…This screening strategy allows for the evaluation of different culture conditions simultaneously with several repetitions, providing a replicable culture environment and saving experimental time and material. Small‐scale processes in bioreactors have shown reproducible results 22–24 . However, for accurate analysis of some parameters, such as aeration, specialized equipment, such as a stirred‐tank bioreactor, may be required to have more than a general idea of the aeration condition.…”

“…Small-scale processes in bioreactors have shown reproducible results. [22][23][24] However, for accurate analysis of some parameters, such as aeration, specialized equipment, such as a stirred-tank bioreactor, may be required to have more than a general idea of the aeration condition.…”