Providing the redox partner .1 Addition of a separate redox partner

After initial production is established, other parameters could be optimized in order to obtain high yielding CYP. Frequently co-expression of the chaperones GroES/EL is performed, resulting in higher protein yields and 5-ALA is almost always added for increased production [229], [230]. Another factor is the choice of vector and host strain. The vectors pCWOri, containing a tac promotor, and the commercial pET vector with a the T7 promotor/lac operator are often used. In fact, the pCWOri vector appears to be the better choice for CYP production, although high yields have been reported with the pET vector as well [229], [230]. Several E. coli strains are commercially available and have been used for recombinant CYP production, JM109 often appearing as the strain of choice. However, as this strain does not carry a copy of phage T7 RNA polymerase, this cannot be used in combination with the pET vector [229], [230]. Of interest, high yields were reported as well when using the so-called Walker strain C41(DE3), a BL21(DE3) derivative for membrane protein overexpression [229]. Lastly, culture parameters could further alter the CYP yield, e.g. temperature, pH, rpm, addition of trace elements, point of induction, IPTG concentration, etc. [229], [230].

2.6.4 Providing the redox partner 2.6.4.1 Addition of a separate redox partner

CYPs from class II require one redox partner, i.e. CPR. In some cases an additional redox partner is involved, namely cytochrome b5. Eukaryotic expression hosts contain endogenous CPR and can sustain heterologous CYP activity. However, endogenous CPR might not be sufficient to sustain the overexpressed CYP and therefore, co-expression of the redox partner is often applied [229]. In yeast, this has been reported to lead to increased CYP stability [237].

In E. coli, no endogenous CPR is present and CPR co-expression is necessary for activity.

However, it was shown that the endogenous flavodoxin and reductase of E. coli are able to reduce class II CYP enzymes [263]. Using whole cells for bioconversion, it has been observed that finetuning this co-expression is necessary, both in yeast and E. coli. In natural circumstances, CYP and CPR are not present in a 1:1 ratio and CPR sustains multiple CYPs, as well as other enzymes. In the production of artemisin in S. cerevisiae (see 2.1), it proved to be advantageous to express cpr from a weaker promotor [132]. Also the production of taxadien-5α-ol in E. coli (see 2.1) improved upon increasing the CYP:CPR ratio [129].

The first choice to select a redox partner for co-expression would be the native CPR redox partner. On the other hand, creating a single strain overexpressing one general CPR for the production of CYPs from different origins is often done, e.g. in screening systems. For example, a S. cerevisiae strain overexpressing its endogenous CPR, was used for a functional screening of a plethora of fungal CYPs [236]. Also in the creation of chimeric constructs, many examples

45 can be found where a reductase domain of a self-sufficient CYP was chosen in order to mimic the natural self-sufficient system (see further). However, care should be taken when selecting a redox partner. Using heterologous redox partners, it was seen that these could majorly influence the activity. For example, a selection of human CYPs were co-expressed in S. pombe with human CPR, CPR from the plant Ammi majus and the endogenous CPR from S. pombe. While human CPR proved to be the better partner for the CYP3 family, this did not necessarily hold true for others. For two human CYPs, they saw that not human CPR but one of the other CPRs resulted in the highest activity [264]. Fungal CYP53A19 of F. oxysporum performed best with its endogenous redox partner, converting the substrates benzoic acid, 3-methoxybenzoic acid and 3-hydroxybenzoic acid. Both benzoic acid and 3-methoxybenzoic acid, but not 3-hydroxybenzoic acid, were converted when using S. cerevisiae CPR and only benzoic acid was hydroxylated in combination with C. albicans CPR [265]. In another example, Chang et al reported that fusion of the fungal CYP57B3 from Aspergillus oryzae to the reductase domain of CYP102A1 mainly resulted in 6-hydroxydaidzein. When using the native redox partner, production of 8-hydroxydaidzein was reported and with the CPR from S. cerevisiae, a 3’-hydroxyl derivative was produced [266]. A remarkable example, be it not from a class II-type CYP, is given by Zhang et al, who observed that separately adding the reductase domain of the class VII CYP116B2 to the bacterial CYP MycG resulted in a completely different reactivity [267].

In CYP characterization studies, CPR enzymes can be added separately, either isolated from their natural host or recombinantly produced and purified. Similar to CYP, CPR is a membrane protein, integrated in the microsomes with an N-terminal anchor. However, the hydrophobicity of the enzyme is lower than CYP and deleting this anchor appears sufficient to obtain a soluble enzyme. There is still some controversy about the function of this anchor, as discussed before.

2.6.4.2 Chimeric constructs

Inspired by the high catalytic activity of the self-sufficient CYP102A1 from B. megaterium, together with the high coupling efficiency, many chimeric enzymes have been constructed, fusing a microsomal CYP with a microsomal CPR (Figure 1.21A). In this way, the heme, FMN and FAD domain are found in the same polypeptide chain, eliminating the need for two separate enzyme partners. Only NADPH and the substrate need to be added in this case. The very first example of such a construct was produced in 1987 by Murakami et al [268], where rat CYP1A1 was fused to rat CPR using a short (three amino acids) linker. The CPR was N-terminally truncated in order to remove the membrane anchor, 56 amino acids were removed in accordance to the trypsin digested construct for crystallization (see 2.5). CYP was not N-terminally modified and expression was performed in S. cerevisiae. Both the isolated microsomes and the purified chimer showed to be active. The same group extended its research by making fusion constructs between bovine CYP17A1 and S. cerevisiae CPR, where they tested alternative CPR truncations with or without a three amino acid linker. The CPR where only part of the N-terminus, cleaved of by papain digest, was removed, proved to be the most active construct [269]. The group of Estabrook used the same approach to construct chimers between microsomal CYPs and truncated microsomal CPRs, using a Ser-Thr linker [270]. In this case,

46

also the N-terminus of CYP was modified in order to enable E. coli expression. This group and others continued to make other constructs using similar approaches, which have been reviewed comprehensively in [271]. Varying success has been achieved with this approach. Some chimers indeed showed increased activity upon purification together with good coupling efficiencies. However, not all chimers were equally successful. Lipid and detergent addition was required for activity and cytochrome b5 was necessary for increased coupling efficiency in several cases. Due to the necessity of cytochrome b5 in some instances, three-protein-fusions have been created as well [272]. Next to the mammalian chimers, also other fusion proteins using class II CYPs have been constructed, e.g. between Thlaspi arvensae CYP71B1 and Catharanthus roseus CPR and between C. roseus CYP71D12 and its CPR [271]. The recent example of CYP52M1 from S. bombicola fused to CPR from A. thaliana in an engineered S. cerevisiae strain for OHFA production further illustrates how creating a chimeric construct can improve catalytic properties. Of note, the heterologous redox partner CPR from A. thaliana resulted in higher conversion yields over the homologous redox partner [58].

Figure 1.21: Different approaches for the construction of a self-sufficient CYP. A: fusion of the class II CYP to a class II redox partner CPR. B: fusion of the reductase domain of a natural self-sufficient CYP to a class II CYP.

C: Protein engineering of a natural self-sufficient CYP.

Another approach was proposed by Gilardi et al, where a so-called molecular lego method was applied [273]. Instead of fusing microsomal CYPs to a microsomal redox partner, the reductase domain of CYP102A1 was introduced C-terminally of a truncated CYP (Figure 1.21B). This technique showed to be successful in creating a self-sufficient system for several mammalian microsomal CYPs and no additional proteins were needed. On top, solubility was increased and the enzyme was active in the absence of detergents and lipids [271]. Further studies showed the importance of the linker regarding coupling efficiency, catalytic activity and substrate binding [274], [275]. It was seen that using a longer five glycine-residue linker resulted in improved properties. The reductase domain of CYP116B2 of class VII has emerged as a possible fusion partner as well (Figure 1.21B). This was already exemplified in the ω-hydroxylation of FAs by the bacterial CYP153 and showed promise for bacterial CYPs [276]. This reductase domain showed to support the activity of eukaryotic CYPs upon fusion as well [277].

On the one hand, artificial chimers can be constructed. On the other hand, natural chimers could be used in biotechnological applications, as self-sufficient CYPs have been identified throughout the years. These have been reviewed more recently in [137]. Current research thus also focusses on engineering these natural chimers (Figure 1.21C), either by rational design or directed evolution in order to obtain foreseen activity and selectivity. This has been exemplified by engineering CYP102A1 to enable selective ω-oxidation of FAs (see 1.2.2).

A B C

47 2.6.4.3 Other electron delivery systems

In both approaches described above, obtaining high coupling efficiencies remains challenging.

Additionally, NADPH is required in stoichiometric amounts, unless cofactor regeneration is in place. Cofactor regeneration is established metabolically in whole-cell biocatalysts and some enzymes have been put forward for in vitro cofactor regeneration (see further). Other ways of circumventing the need for NADPH and/or a redox partner in CYP catalysis have been investigated.

A first example has already been mentioned before (see 2.4), i.e. making use of the peroxide shunt pathway. In this case, instead of using oxygen together with electron delivery from NAD(P)H, hydrogen peroxide is used by CYP. The high spin species formed upon substrate binding is then immediately converted to the ferric hydroperoxo intermediate Compound 0.

Also other so-called oxygen surrogates were applied in this context, such as iodosylbenzene, sodium chlorite, cumene hydroperoxide and m-chloroperbenzoic acid [176], [278]. Remember, m-chloroperbenzoic acid was used in order to spectroscopically characterize Compound I (see 2.4). Several examples are found in which mammalian CYPs could use one or more of these oxygen surrogates [279]–[282]. However, stability issues and heme inactivation arises when using hydrogen peroxide. Additionally, most CYPs need to be mutated in order to use oxygen surrogates in an efficient way. The group of Frances Arnold, for example, created a CYP102A1 mutant by directed evolution, able to use hydrogen peroxide for the hydroxylation of p-nitrophenoxydodecanoic acid, FAs and styrene [283]. Notably, some CYPs evolved to be actual peroxygenases and use hydrogen peroxide as the oxidant [278].

An electrochemical approach where CYPs are immobilized on electrodes, serves as a potential alternative. Electrodes can deliver electrons directly to the heme. In order to do this efficiently, the electrode surface is best engineered. Different electrode materials have been used, such as graphite, glassy carbon, gold and other metals. Many surface modifications have been applied for enzyme immobilization. The surface has been coated with surfactants, polymers, gold nanoparticles and self-assembled monolayers. Stable vesicular phospholipid dispersions have been used in order to create a lipid film and microsomes have been immobilized on the electrodes. The immobilization strategy, involving also enzyme orientation, the use of spacers, etc, greatly affects the stability and selectivity of the enzyme. Also protein engineering has been proposed and the chimeric constructs, as described above, have been applied [284], [285]. This electrochemical approach is of particular interest in the context of biosensors [286]. However, heme reduction by the electrode was stated to be insufficient in context of industrial biocatalysis and issues concerning CYP stability upon electrode fixation need to be solved [122].

More recently, light-driven electron delivery has been put forward as a promising strategy. The idea is that a photosensitizer transfers light energy to the chemical reaction for electron supply.

Different photosensitizers have been under investigation in CYP catalysis; quantum dots, deazaflavins, isoalloxazine flavins riboflavin, FAD and FMN, eosin Y, Ru(II)-diimine complexes and photosystem I (remember redirecting the metabolic pathway to the chloroplast of plants and the use of cyanobacteria). Not all photosensitizers deliver the electrons in the same

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way. Firstly, some photosensitizers transfer electrons to oxygen, resulting in peroxide which than continues through the peroxide shunt pathway. Secondly, photosensitizers deliver electrons to the heme via a redox partner. Thirdly, no redox partner is involved an the photosensitizer directly reduces the heme group. These light-driven reactions have recently been reviewed in [287]. Such systems still require a lot of optimization regarding the stability and activity of the CYP enzymes and in case of the direct heme reduction, coupling efficiency needs to be optimized as well.