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53 This PhD was started as part of the project Enzymes for Added Sustainability and Efficiency (EnzymASE). The project was funded by The Flemish Agency for Innovation and Entrepreneurship (VLAIO) and supported by Catalisti. EnzymASE is a cooperation between different industrial partners and knowledge centres, including Ghent University. Its aim is to develop enzymes and corresponding biocatalytic processes for a variety of applications; novel enzyme tools, feed additives, coatings and adhesives. One of the industrial partners was interested in saturated long-chain α,ω-DCAs for the use in repellent materials. Developing a sustainable production process for these compounds was known to be challenging and was therefore presented as a PhD project. Long-chain α,ω-DCA production is not only of interest in this specific application. In a broader context, these compounds are of value as a monomer in polyamides and polyesters and as precursors for pharmaceuticals, perfumes, lubricants, adhesives, plasticizers, powder coatings and corrosion inhibitors. Using long-chain α,ω-DCAs gives attractive properties to the polymers compared to currently used short-to-medium-chain DCAs, such as decreased water absorbance, which affects stability and mechanical performance, and more polyethylene-like properties are obtained, i.e. higher melting temperatures and a higher tendency to crystallize. Moreover, these long-chain DCAs can lead to entirely new polymers.
Two different processes are in place in industry for the production of long-chain α,ω-DCAs.
Chemical conversions are industrialized, of which only the metathesis of monounsaturated FAs delivers long-chain α,ω-DCAs. This chemical conversion process is accompanied with a number of disadvantages. Byproduct formation occurs by cross-metathesis reactions when the substrate is a mixture of monounsaturated FAs, and by isomerization. This leads to an extensive purification process, elevating the costs. Only half of the FA substrate is included in the final product and expensive rare catalysts are used for the chemical conversion, which cannot be recovered and contaminate the final product. Lastly, saturated DCAs are of specific interest in this project so this chemical conversion would require an extra hydrogenation step. Many of these disadvantages are overcome in the second production process in industry, that is the fermentation process using Candida viswanathii. Unfortunately, this organism is classified as pathogenic in Europe (risk group class two) and the necessary safety precautions that need to be taken, leads to increased prices. Additionally, both approaches are protected by patents [308], [309]. A sustainable production process was pursued, which is not only lower in costs, but is also more flexible and yields products with higher purity and consistency. An in vitro approach was of specific interest. This circumvented issues regarding substrate uptake limitations of long-chain FAs and unwanted side reactions in the cellular environment (e.g.
degradation of FAs and DCAs via β-oxidation and the inclusion of FAs in lipid bodies), encompassed in fermentative approaches. Furthermore, often higher productivities are obtained in an in vitro process and products do not need to be recovered from the complex fermentation broth, enabling higher purities. Additionally, integration of a biocatalytic process in established chemical companies is more straightforward, as they have little know-how on fermentative production processes and huge investments regarding installations would be required for fermentation.
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Two strategies were considered in this dissertation for the development of an in vitro cell-free production process (Figure 2.1A and B, respectively). In a first strategy, the recombinant production of a soluble variant of CYP52A13 from the industrial DCA producer C. viswanathii was investigated. A soluble enzyme allows for a facilitated implementation in the enzyme reactor and provides a flexible system. In a second strategy, in vivo immobilization of the same biocatalyst on the surface of Escherichia coli-derived outer membrane vesicles (OMVs) was explored. It was hypothesized that immobilization on these OMVs would enhance enzyme stability. Moreover, OMVs might be recovered after biocatalysis for recycling, important for reducing the costs of the overall production process. In both strategies, the production of a self-sufficient CYP was pursued. Creating a chimeric construct where both the CYP and the redox partner CPR are encoded in the same polypeptide chain, eliminates the necessity of producing two individual enzymes. Additionally, it has been reported that fusing CYP and CPR can significantly enhance the coupling efficiency. Furthermore, cofactor regeneration using FDH was addressed. As NADPH is an expensive cofactor, large-scale application of this system would not be feasible if the NADPH has to be added in stoichiometric amounts. FDH was chosen as it is irreversible, requires an inexpensive co-substrate and the formed CO2 is inert and can be easily removed.
Figure 2.1: Proposed in vitro systems. A: soluble self-sufficient CYP, produced in P. pastoris. B: self-sufficient CYP, produced in E. coli and displayed on E. coli-derived outer membrane vesicles (OMV). Cofactor regeneration is pursued by recombinantly producing the FDH from Burkholderia stabilis.
The first experimental chapter (chapter 3) describes the recombinant production of a soluble self-sufficient CYP using the heterologous host Pichia pastoris. On the one hand, both CYP52A13 and CPR-a were produced separately in a soluble and secreted form, after which a chimeric construct was made. On the other hand, the nontruncated CYP52A13 was produced, intended for optimization of the chimeric construct in the native ER environment, before continuing to further solubilization efforts. The second experimental chapter (chapter 4) reports the search for an in vivo immobilization strategy using OMVs. The natural self-sufficient CYP102A1 from Bacillus megaterium was used as a model self-sufficient CYP in order to deliver a proof-of-concept. In a next step, the self-sufficient CYP52A13 was first surface displayed and subsequently displayed on OMVs. The third chapter (chapter 5) then focusses on the recombinant production of FDH for cofactor regeneration. The NADP+-dependent FDH from Burkholderia stabilis was selected for recombinant production in two different hosts, i.e.
P. pastoris and E. coli.
A B