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.

Keywords: Pichia pastoris; Saccharomyces cerevisiae; chalcone 3-hydroxylase; physiology; recombinant protein production; yeast.

Figure 1

Carbon dioxide evolution rate (CER) over time during the cultivation of Mut+ strain GS115 (A) and MutS strain KM71H (B). The CER during the methanol pulses is zoomed out (right). The adaption time was calculated from addition of the pulse until the maximum of the CER was reached, indicated by the star. The different phases during the cultivation are numbered chronologically: The glycerol batch phase (1) was followed by a glycerol fed‐batch (2). Thereafter, the adaption pulse of 0.5 % methanol was added (3) and a second methanol pulse with 1 % was applied after complete metabolization of the first (4). After that, a methanol fed‐batch was started (5) which lasted for 72 h

Figure 2

Comparison of the different yeast strains regarding physiology and productivity. Yields during the batch phase (A), fed‐batch phase (B) and induced fed‐batch phase (C) are shown as well as the obtained dry cell weight concentrations (D), product titres (E) and product yields (F) at the end of the cultivations.”I” indicates S. cerevisiae strain INVSc, “G” P. pastoris strain GS115, “S” P. pastoris strain SMD1168H and “K” P.pastoris strain KM71H. “+” is written for strains expressing CH3H, “‐“for empty strains. For (A), (B) and (C) only the errors on the C‐balances (calculated by error propagation) are shown for better readability. Errors on the respective yields can be found in Tables 2 and 3. For (D), (E), and (F) errors were calculated from at least three measurements. Differences in productivity were determined as significant at a 95 % confidence level

Figure A1

Additional file 1: Western Blot