The display of self-sufficient CYPs on the E. coli surface

Surface display was initiated by constructing the vector, containing the InaKtrBM3-encoding sequence for the surface display of CYP102A1 (from here on now denoted as BM3).

Subsequently, the heme domain of BM3 was substituted by the gene encoding the truncated CYP52A13 as described in previous chapter, without the artificial N-terminal sequence. As the CYPtrBMR construct from previous chapter was not active, it was decided to make an alternative chimer, in analogy with the molecular lego approach described by Gilardi et al [273].

Instead of using the natural BM3 linker sequence, CYPtr was directly attached to BMR using a flexible linker of five Gly residues [274], [275]. We chose the BMR domain in the production host E. coli over the available CPRtr construct as the corresponding gene was predicted to be better suited for this recombinant host, based on the GenScript Rare Codon Analysis Tool and the online tool ATGme. The obtained plasmids were transformed in E. coli strain JM109, a strain often used for recombinant production of CYPs [229], [230]. After overnight expression at 28 °C, the cells were collected and lysate, inner membrane (IM) and outer membrane (OM) samples were isolated for western blot analysis (Figure 4.6). First, it is clear that both enzymes are produced, with InaKtrBM3 (152 kDa) present to a much higher level compared to InaKtrCYPtrBMR (154 kDa) in all fractions. Both constructs were present in the OM fraction, indicating that the surface display was successful for both enzymes. It must be noted that the used protocol for OM isolation rather leads to OM enrichment and might not result in completely pure OM so additional experiments are required to confirm surface display. In all lanes, multiple bands are seen for both enzymes. Higher MW bands indicate aggregation, which might be the result of extensive heating during the sample preparation (boiling of sample in Laemmli in preparation of SDS-PAGE) or due to contamination with formed inclusion bodies, although these should have been removed by the intermediate centrifugation step performed after sonication to remove cell debris. Multiple bands at a lower MW are seen as well, which shows that degradation occurs. Based on the MW of the lower bands, it was hypothesized that proteolysis of the linker sequences occurs. Proteolysis of the linker sequence in between InaKtr and BM3 or CYPtrBMR leads to a signal at 118 kDa or 120 kDa, respectively. Release of the BMR sequence by proteolysis of the linker in between the heme and reductase domain theoretically gives a signal around 65 kDa, as determined by the Expasy translate and protparam tools.

Figure 4.6: Western blot analysis of the collected cell fractions, that is the lysate, the inner membrane (IM) and the outer membrane (OM), after either InaKtrBM3 (152 kDa) or InaKtrCYPtrBMR (154 kDa) production in JM109.

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Further characterization of the surface displayed enzymes was done by performing a whole-cell bioconversion assay. In case of InaKtrBM3, palmitic acid was used as the substrate. Following the bioconversion assay, the products were extracted and silylated for GC-MS analysis. The chromatogram obtained after a 20 min palmitic acid conversion by surface displayed InaKtrBM3 is shown in Figure 4.7. Two small peaks appeared in this chromatogram, i.e. at RT 7.971 min and 8.105 min. The MS spectra of both peaks are shown in Figure 4.8. Both spectra coincide with hydroxylated palmitic acid. The molecular ion of m/z 416 is not found, and it indeed is reported that the molecular ion of silylated OHFAs is only present in low abundance [366]. Instead the ion [M-15]⁺, missing one methyl group, and the ions [M-31]⁺ and [M-105]⁺, resulting from interactions between the two functional groups, are seen. In the MS spectrum of Figure 4.8A, the ion with m/z 131 is highly abundant, indicating that palmitic acid is hydroxylated at the ω-2 position. The spectrum of Figure 4.8B shows a highly abundant ion with m/z 117, indicating hydroxylation at the ω-1 position. InaKtrBM3 thus actively converts palmitic acid mainly to 14-, and 15-hydroxypalmitic acid.

Figure 4.7: A: GC chromatogram of silylated extracts after a 20 min whole-cell palmitic acid bioconversion assay using JM109 cells, surface displaying InaKtrBM3. Palmitic acid was hydroxylated at its ω-1 and ω-2 position.

B: GC chromatogram of silylated extracts of a blank, i.e. no palmitic acid substrate was added during bioconversion. The identified palmitic acid and stearic acid are contaminants, always observed by GC-MS analysis. Only peaks identified as silylated compounds, based on the presence in the MS spectrum of the characteristic peaks at m/z 73 and 75, are annotated.

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Figure 4.8: MS spectra of hydroxylated palmitic acid. A: MS spectrum of peak at RT 7.971 min (Figure 4.7), that is 14-hydroxypalmitic acid. B: MS spectrum of peak at RT 8.105 min (Figure 4.7), that is 15-hydroxypalmitic acid.

In case of InaKtrCYPtrBMR, oleic acid was used as the substrate for a whole-cell bioconversion assay. In one condition, a NADPH regeneration system was added. In the second condition, NADPH was discarded, serving as a blank. NADPH cannot migrate through the membrane so conversion linked to NADPH addition was hypothesized to be specific for the surface displayed construct. Unfortunately, no peaks coinciding with either OHFA or DCA were observed leading to the conclusion that no conversion occurred (data not shown).

CYPtr designed as described in previous chapter did not show any activity, neither in P. pastoris, nor in E. coli. Therefore, alternative truncations were performed, as described in Material and methods (see 2.3). In the same way as described for CYPtr, the heme domain of InaKtrBM3 was replaced by either CYPΔ32, CYPΔ25 or CYPnt. An expression test was performed and instead of isolating the lysate, IM and OM, surface display was assessed by means of a proteinase K accessibility assay. Only if the protein is present on the surface of E. coli, the protein will be degraded by proteinase K, as this protease cannot migrate through the membrane. Subsequent to proteinase K incubation, the OM was collected and analysed by SDS-PAGE (Figure 4.9A). A marked difference is seen between the OM samples isolated after proteinase K addition compared to a negative control (i.e. no addition of proteinase K). After

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proteinase K incubation, all protein in the higher MW region seems to be absent. On the bottom of the Coomassie-stained gel, an intense band is observed. These bands were cut out from all eight lanes and analysed by MALDI-TOF MS. In all eight bands, the outer membrane porin (Omp) C was identified and in case of the untreated samples (i.e. no proteinase K was added), a mixture of both OmpC and OmpF was found, based on the peptide mass fingerprint. MS/MS analysis of the most abundant peptides resulted in the identification of OmpC alone in all cases.

This confirms that indeed we have isolated the OM. The bands around 150 kDa were cut out as well for MALDI-TOF MS. Unfortunately, these could not be identified. Therefore, a western blot was executed (Figure 4.9B). Indeed all four constructs (InaKtrCYPtrBMR, InaKtrCYPΔ32BMR, InaKtrCYPΔ25BMR and InaKtrCYPntBMR) are present in the OM and were degraded upon proteinase K addition. All four chimers were thus successfully surface displayed. Of note, an alternative OM isolation protocol to the one used for OM isolation in Figure 4.6 was applied. This alternative protocol seems to lead to a much higher purity, as no contaminating signal is observed from the lysate or IM in the proteinase K-treated samples.

Figure 4.9: OM fraction, isolated after proteinase K accessibility assay. All four InaKtrCYPBMR, where CYP is either CYPtr, CYPΔ32, CYPΔ25 or CYPnt, were assayed. - : untreated. +: proteinase K-treated. A: SDS-PAGE analysis. The intense gel bands at the bottom of the Coomassie-stained gel were cut out and analysed by MALDI-TOF MS. Either OmpC or a mixture of OmpF and OmpC was identified. B: Western blot analysis.

A next step was to assay whether the surface displayed chimeric constructs were able to convert oleic acid to 18-hydroxy oleic acid and/or 1,18-octadecenedioic acid. As a blank, the NADPH regeneration system was left out of the reaction mixture. The resulting GC chromatograms from the CYPΔ32 construct are shown in Figure 4.10. The chromatograms from the other constructs were similar. Unfortunately, no OHFA nor DCA was observed so the alternative truncations did not lead to an active surface displayed chimer. Of note, oleic acid appeared to be consumed in absence of the NADPH regeneration system (Figure 4.10B). When only NADPH was discarded from the reaction mixture, thus still including G6P and G6PDH, oleic acid did not seem to be consumed in the blank (Figure 4.11). Oleic acid was thus degraded by β-oxidation in absence of the alternative energy source G6P. This highlights the disadvantage of working with whole-cells for the conversion of FAs, i.e. degradation of substrate (and formed products) by β-oxidation.

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Figure 4.10: GC chromatogram of silylated extracts after whole-cell oleic acid bioconversion assay using JM109 cells, surface displaying InaKtrCYPΔ32BMR. A:

Bioconversion assay including NADPH regeneration system. B: Bioconversion assay excluding NADPH regeneration system. No hydroxylated product of oleic acid was observed. Oleic acid appeared to be consumed in absence of an NADPH regeneration system. Only peaks identified as silylated compounds, based on the presence in the MS spectrum of the characteristic peaks at m/z 73 and 75, are annotated.

Figure 4.11: GC chromatogram of silylated extracts after whole-cell oleic acid bioconversion assay using JM109 cells, surface displaying InaKtrCYPΔ32BMR. A:

Bioconversion assay including NADPH. B: Bioconversion assay excluding NADPH but including the regeneration components G6P and G6PDH. No hydroxylated product of oleic acid was observed. Only peaks identified as silylated compounds, based on the presence in the MS spectrum of the characteristic peaks at m/z 73 and 75, are annotated.

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