Providing the redox partner for the nontruncated CYP52A13 .1 Chimeric construct

CYP needs a redox partner for the delivery of electrons and for initial enzymatic assays, it was hypothesized that the endogenous CPR of P. pastoris might serve to this purpose. As no active system was obtained for designs based on this assumption, recombinant production of CPRtr was explored for the external addition in enzymatic assays. As described above, only low activity samples could be obtained and addition to either CYPtr or CYPnt, present in the microsome, did not lead to oleic acid-converting microsomes. Therefore, an artificial chimer was created between the CYPnt and BMR in the same way as described for CYPtr. Expression tests were performed in parallel with CYPnt and expression was difficult to prove for the chimeric construct as well. It was hypothesized that also in this case, only a very low yield was obtained. This was evidenced by a cytochrome c reductase assay, shown in Figure 3.32. A 100 ml expression test was executed after which the microsomes were isolated, taking along CYPnt. The reductase activity of 0.1 mg microsomal protein after CYPntBMR production was compared to the reductase activity of 0.1 mg microsomal protein after CYPnt production and showed only a minor increase. Referring to Figure 3.27, presence of the BMR domain results in a marked reductase activity increase in the CYPtrBMR chimeric construct. A similar increase in reductase activity is not observed here, thereby confirming the low enzyme yield. Due to this low yield, no further efforts were done to construct CYPntCPRtr chimers.

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Figure 3.32: Cytochrome c reductase assay of microsome samples. A: CYPnt. B: CYPntBMR.

3.4.2.2 Co-expression of the nontruncated CPR-a

No active enzyme was yet obtained and several reasons could be ascribed to that. No suitable redox partner was present, that is neither the endogenous CPR from P. pastoris nor CPRtr or the BMR domain were sufficient. The truncations might lead to loss of activity, which could not be verified using the nontruncated form due to its low production yield. An attempt was done to prove the activity of CYP52A13 in P. pastoris by creating a positive control where CYPnt was co-expressed with a nontruncated form of CPR-a from C. viswanathii (CPRnt) in the ER of P. pastoris. It was hypothesized that co-expression of the natural redox partner CPRnt might be beneficial for CYPnt production as it has been reported that CYP production and stability are dependent on the co-expressed CPR [237], [327]. Interestingly, Gudiminchi et al optimized a high-throughput screening method for CYP production in P. pastoris by measuring the CO-bound difference spectrum of whole cells. They applied their protocol to investigate in which case CYP52A13 yields were the highest and saw that co-expressing CPR increased the CYP yield [327]. To this end, a co-expression plasmid was created where the cprnt expression cassette was cloned into the cypnt vector using a Golden Gate-inspired molecular cloning approach. To be able to linearize the obtained plasmid with the PmeI restriction enzyme for efficient integration into the genome of P. pastoris, the PmeI restriction site was removed from the cprnt vector by site directed mutagenesis before cloning the cprnt expression cassette into the cypnt vector. For rapid detection, constructs including a C-terminal Histag were used. A 100 ml expression test was performed, taking along the CYPntHis construct. Microsomes were isolated and it was already evident from the collected pellet that CPRnt was present as the colour was markedly different from the microsomes containing CYPntHis alone (Figure 3.33A). The pellet appears to be more green-brown, the greenish colour coinciding with the presence of a blue semiquinone flavin and a yellow fully oxidized flavin [183]. Western blot analysis (Figure 3.33B) shows a very intense signal, not only at the expected MW of 78 kDa (CPRnt) and 60 kDa (CYPnt), but a smear is seen. Therefore, it could not be concluded whether one of the two or both enzymes were produced and what the ratio between the two enzymes was.

A B

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Figure 3.33: A: Microsomal pellet isolated after (left) CYPntHis production and (right) CYPntHis-CPRntHis production. B: Western blot analysis of these isolated microsomes. No signal is seen for CYPntHis (60 kDa). After co-expression, a smear is observed, instead of two distinct signals coinciding with CYPntHis (60 kDa) and CPRntHis (78 kDa).

The next step was to use these microsomes in an in vitro enzymatic assay. Not one but five different enzymatic assays were set up, selected from different sources, all assaying ω-hydroxylation (see 2.13.1). Subsequent to each assay, the extracted products were analysed by GC-MS upon silylation. Unfortunately, not one chromatogram showed the presence of 18-hydroxyoleic acid or 1,18-octadecenedioic acid (data not shown). Here again it could be questioned whether this was actually due to an inactive CYPnt. The western blot in Figure 3.33 shows an intense signal together with a lot of degradation whereas CYPntHis shows no signal at all. Even though CPRntHis co-expression could lead to an increased yield of CYPntHis, it was considered that most of the signal came from CPRntHis, based on other co-expression experiments (see below). This would mean that instead of a CYPntHis:CPRntHis ratio higher than one, as present in natural systems, the ratio is lower than one and an excess of CPRntHis is present. Indeed, Paddon et al reported it to be advantageous to express cpr from a weak promotor [132].

A B

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