Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii

Abstract

Background: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.

Results: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.

Conclusions: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Keywords: Komagataella phaffii; Bi-directionalized promoter; Derepressed feeding; ERAD; Methanol-free; Recombinant protein production; UPR; Unspecific peroxygenase.

Fig. 1

Representative plasmid map of bi-directionalized expression constructs pBSY5Z_AnoUPO-PDI_promoterPDI. Vectors contain the coding sequence for the AnoUPO (KAB8223135.1) with its native signal sequence and the P_DF as promoter controlling its expression, the PDI with different promoter sequences controlling its expression, parts necessary for bacterial propagation (pUC ori), selection in bacteria and yeast (Zeocin resistance cassette: P_ILV5-P_EM72-ZeoR-AOD_TT) and a unique restriction site for linearization of the vector prior to transformation (SmiI)

Fig. 2

The mechanistic dependence of q_p and q_{s glycerol} is shown. Controlled fed-batch cultivations at different specific glycerol uptake rates q_{s glycerol} were performed; the specific productivity for AnoUPO (q_p) was investigated as a function of q_{s glycerol} for both recombinant yeast strains (A and B, respectively)

Fig. 3

Cultivations performed at conditions resulting in highest q_p at respective conditions: “Mixed feed cultivation” with 30% of q_{s, glycerol, max} = adjusted q_{s, glycerol} of 75 mg/g/h with q_{s, methanol} at 20 mg/g/h and “Derepressive cultivation” with q_{s, glycerol, max} at 20% = adjusted q_{s, glycerol} of 50 mg/g/h A) Biomass concentration for the AnoUPO and AnoUPO-PDI strains are shown for derepressed and mixed feed cultivation over time of induction; B) indicating the biomass-specific expression of the enzyme AnoUPO for the respective cultivation of the two strains; C) The total protein concentrations of the respective supernatants for the AnoUPO and AnoUPO-PDI strains in the respective cultivation conditions; D) the protein-specific expression (i.e., purity) of the enzyme AnoUPO for the respective cultivation of the two strains

Fig. 4

The two strains, AnoUPO and AnoUPO-PDI, were cultivated in a non-dynamic fed-batch mode and induced via mixed-feed or methanol-free derepression. Samples were taken at indicated induction time points: one sample prior to induction, a sample representing the switch proposed by the induction conditions (either switch in feeding rate or methanol pulse), and two further samples were taken in 20 h intervals to monitor the time effect of the induction period; the total RNA was isolated, and the relative transcript levels of the indicated genes were determined by an RT-qPCR assay normalized to the reference sample (indicated by an asterisk) using the reference genes RSC1 and TAF10 for normalization. Gene expression for these timed-dependent cultivations is shown for HAC1 spliced (A), wtPDI (B), CDC48 (C), PNG1 (D), SEC61 (E), and HEM13 (F)