Recombinant production of a hard‐to‐express membrane‐bound cytochrome P450 in different yeasts—Comparison of physiology and productivity

Abstract

Cytochrome P450s comprise one of the largest protein superfamilies. They occur in every kingdom of life and catalyse a variety of essential reactions. Their production is of utmost interest regarding biotransformation and structure‐function elucidation. However, they have proven hard to express due to their membrane anchor, their complex co‐factor requirements and their need for a redox‐partner. In our study, we investigated and compared different yeast strains for the production of the plant cytochrome P450 chalcone 3‐hydroxylase. To our knowledge, this is the first study evaluating different yeasts for the expression of this abundant and highly significant protein superfamily. Saccharomyces cerevisiae and three different strains of Pichia pastoris expressing chalcone 3‐hydroxylase were cultivated in controlled bioreactor runs and evaluated regarding physiological parameters and expression levels of the cytochrome P450. Production differed significantly between the different strains and was found highest in the investigated P. pastoris MutS strain KM71H where 8 mg P450 per gram dry cell weight were detected. We believe that this host could be suitable for the expression of many eukaryotic, especially plant‐derived, cytochrome P450s as it combines high specific product yields together with straightforward cultivation techniques for achieving high biomass concentrations. Both factors greatly facilitate subsequent establishment of purification procedures for the cytochrome P450 and make the yeast strain an ideal platform for biotransformation as well.

Functional expression of eukaryotic cytochrome P450s in yeast

“…Kolar et al (2007) expressed active human P450 (CYP17) to produce 17‐hydroxyprogesterone and 16‐hydroxyprogesterone from progesterone, which was the first report for heterologous expression of a functional human steroidogenic P450 in P. pastoris . In addition, three P. pastoris strains KM71H, SMD1168H, and GS115 as well as S. cerevisiae strain INVSc1 were used to express a plant P450 (chalcone 3‐hydroxylase), and KM71H was found to have the highest production of the P450 enzyme, reaching 8 mg/g dry cell weight (Hausjell et al, 2020). Similarly, Geier et al (2012) compared the expression level of CYP2D6 (one of the most important drug‐metabolizing P450s in human) in four commonly used expression systems and P. pastoris was determined to be the optimal host.…”

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

“…A purification procedure for the membrane-bound Dv CH3H, recombinantly cultivated in controlled bioreactor runs in P. pastoris KM71H (for more details see 40 ), was established by systematic investigation of each purification step. An overview is shown in Fig.…”

“…Extraction of mRNA and cDNA synthesis were performed with the µMACS mRNA isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and are described in detail in the SI and Supplementary Table S2 . The cloning into the appropriate vectors, the transformation into Pichia pastoris as well as the recombinant expression were all performed as described previously 40 . The cultivated cells were harvested by centrifugation at 7000× g for 20 min at 4 °C, the supernatant was discarded and the cell biomass was frozen at − 20 °C until further use.…”

Heterologous expression of a novel linoleic acid isomerase BBI, and effect of fusion tags on its performance

“…As a methylotrophic yeast, P. pastoris combines the eukaryotic expression system for protein processing, folding, and post-translational modification with special traits, such as ease of operation, relatively fast expression times, low cost, and high expression levels, like those of the prokaryotic expression systems ( Karbalaei et al, 2020 ). For example, P. pastoris has been shown an optimal host for the expression of the membrane-bound protein cytochrome P450, and can support the needs of this class of membrane proteins during expression, including specific membrane-anchored conformations, complex co-factors, and redox chaperones ( Byrne, 2015 ; Hausjell et al, 2020 ). In addition, water channel AQP family members, ion channel protein K2P4.1 ( Brohawn et al, 2012 ), P-glycoprotein transporters ( Bai et al, 2011 ), and G protein-coupled receptors ( Hino et al, 2012 ; Singh et al, 2012 ) with a seven-fold transmembrane structure are expressed in high amounts in P. pastoris ; the system has been further used in the elucidation of their enzyme activities and crystal structures.…”