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This thesis deals with the characterisation and engineering of new thermophilic PET hydrolases as potential candidates for an eco-friendly biocatalytic recycling approach for the upcycling or downcycling of polyethylene terephthalate (PET) on industrial scale. Furthermore, high-throughput screening methods are described that detect the products of PET hydrolysis. The high demand of PET in the packaging and textile industries with a global production of 82 million metric tons per year has significantly contributed to the global solid waste stream and environmental plastic pollution after its end-of-life. Although PET hydrolases have been identified in various microorganisms, only a handful of benchmark enzymes have been engineered for industrial applications. Therefore, the identification of new PET hydrolases from metagenomes or via protein engineering approaches, especially thermophilic PET hydrolases with optimal operating temperatures (i.e., increased thermostability and activity) near the glass transition temperature of the polymer PET, is a crucial step towards a bio-based circular plastic economy. Article I demonstrates that metagenome-derived thermophilic PET hydrolases can be significantly improved using different engineering approaches to achieve a similar activity level as the well-established leaf-branch-compost cutinase (LCC) F243I/D238C/S283C/Y127G variant (LCC ICCG). In Article II, thermostable variants of a mesophilic enzyme (PETase from Ideonella sakaiensis) were identified from a mutant library and characterised against PET substrates in various forms. Articles III and IV describe the application of high-throughput methods for the identification of novel PET hydrolases by directly assaying terephthalic acid (TPA), one of the monomeric building blocks of PET. Furthermore, Article IV describes the possibility of a one-pot conversion of the TPA-based aldehydes produced to their diamines as example for an open-loop upcycling method.
Promiscuous acyltransferases enable transesterification reactions in bulk water by preferentially catalyzing acyl transfer over hydrolysis. Until recently, only a small number of promiscuous acyltransferases have been described in the literature, exhibiting several limitations in terms of acyltransferase efficiency and applicability. This work focuses on the discovery of novel promiscuous acyltransferases and the engineering of promiscuous acyltransferases via rational design. Several promiscuous acyltransferases in the bacterial hormone-sensitive lipase family and family VIII carboxylesterases have been identified, demonstrating that promiscuous acyltransferase activity is not a rare phenomenon. Moreover, the efficiency and applicability of the enzymes could be improved via protein engineering in terms of acyltransferase activity, enantioselectivity, and substrate scope.
In their idealized forms, enzymes can facilitate complex reactions with extreme specificity and selectivity. Additionally, in this imaginative form, they only require mild reaction conditions, resulting in low energy consumption, and they are biodegradable, efficient, reusable, and sustainable. Unfortunately, this idealized form often deviates significantly from reality, where enzymes are more likely to be associated with marginal stability and low reaction rates, leaving them less than desirable for many industrial applications. As such, if we could master the process of engineering the configuration of a protein towards a given task, the implications could be staggering.
This thesis aims to contribute to the process of protein engineering, mainly how computational tools can be used to make the protein engineering process more efficient and accessible.
Article I explores the current state of the art in machine learning-guided directed evolution and serves as a foundation for Article II, which is a concrete application of these techniques to an engineering campaign. Despite successfully improving overall activity and selectivity, we also observe limitations and constraints within the methodology. Article III then delves into these drawbacks and attempts to lay the foundation for a more generalizable and, more importantly, efficient engineering workflow, balancing the strengths and weaknesses of computational techniques with advances in gene synthesis. We then validated this novel pipeline in Article IV, where we show the potential of this methodology. Article V describes a more standard protein engineering campaign on squalene-hopene cyclases for potentially interesting products in the flavor and fragrance industry. Lastly, Article VI outlines a PyMol plugin for molecular docking.
Die Hälfte der globalen Primarproduktion wird in den Ozeanen realisiert und dabei wird ein großer Anteil des fixierten CO2 genutzt, um Algenpolysaccharide zu synthetisieren. Diese Kohlenhydrate dienen als wichtige Kohlenstoff- und Energiequelle für marine Nahrungsnetze, wobei sie von kohlenhydrataktiven Enzymen zu monomeren Zuckern umgesetzt werden. Da bisher wenig über den enzymatischen Abbau von Algenpolysacchariden in den Ozeanen bekannt ist, war es das Ziel dieser Arbeit, zu einem tieferen Verständnis dieser Prozesse beizutragen.
O-Methylierungen stellen stabile Modifikationen an Zuckern in marinen und terrestrischen Polysacchariden dar. Es wurde in Artikel I gezeigt, dass Cytochrom P450 Monooxygenasen eine wichtige Funktion in enzymatischen Abbausystemen aus marinen Bakterien für Agar haben, wobei diese Enzyme die oxidative Demethylierung von 6-O-Methyl-D-galaktose, einem Monomer aus Rotalgenpolysacchariden, katalysieren. Diese Ergebnisse zeigen, dass es sich bei der P450-Subfamilie CYP236A um die zweite beschriebene Gruppe von kohlenhydrataktiven Monooxygenasen handelt. Die charakterisierten P450s sind hochspezifisch für 6-O-Methyl-D-galaktose und akzeptieren keine typischen P450-Substrate. Um die molekularen Faktoren für den spezifischen Umsatz dieses polaren Substrates aufzuklären, wurde Proteinkristallografie genutzt (Artikel II). Die Kristallstruktur der P450 Monooxygenase aus Z. galactanivorans mit gebundenem Substratmolekül zeigt, dass sowohl Wasserstoffbrückenbindungen als auch hydrophobe Interaktionen an der Substraterkennung beteiligt sind, was zusätzlich durch ITC sowie Mutationsstudien bestätigt wurde.
Schnellwachsende Grünalgen der Gattung Ulva führen weltweit zu gefährlichen Algenblüten. Ein Hauptbestandteil der gebildeten Biomasse stellt das anionische Polysaccharid Ulvan dar. Bisher war der enzymatische Ulvanabbau kaum verstanden, was die sinnvolle Nutzung von Ulva-Biomasse erschwerte. Die detaillierte biochemische Charakterisierung einer Ulvanlyase auf F. agariphila wird in Artikel III gezeigt. Dieses Enzym katalysiert den ersten Schritt im Ulvanabbau und die biochemischen Parameter stimmen mit den Umweltbedingungen in Küstenbereichen des gemäßigten Ozeans überein, dem Habitat, aus dem dieses Bakterium isoliert wurde. Alle nachfolgenden Schritte im kompletten enzymatischen Ulvanabbau wurden aufgeklärt und sind in Artikel IV zum ersten Mal beschrieben. Insgesamt 13 Enzyme aus den Klassen der Polysaccharidlyasen, Glykosidhydrolasen sowie Sulfatasen agieren in einer komplexen Kaskade zusammen, um schlussendlich monomere Zucker aus Ulvan bereitzustellen.
Die gezeigten Identifizierungen und Charakterisierungen von neuen kohlenhydrataktiven Enzymen tragen nicht nur zu einem besseren Verständnis der Vorgänge im marinen Kohlenstoffkreislauf bei, sondern bilden zudem die Grundlage für zukünftige biotechnologische Prozesse. Eine effiziente enzymatische Depolymerisation der Algenpolysaccharide ist nötig, um Bioraffineriekonzepte basierend auf Algenkohlenhydraten zu realisieren. Dabei können über mikrobielle Fermentation Biokraftstoffe der zweiten Generation oder andere nützliche Produkte hergestellt werden.
The aims of this thesis were the identification and development of whole-cell biocatalysts for the regio- and stereoselective hydroxylation of steroids, including hormones and bile acids by P450 monooxygenases. Steroids and their derivatives are applied as therapeutic agents. The chemical synthesis of such compounds depends on multi-step procedures, in a stereo- and regiospecific manner involving the protection and deprotection of functional groups and toxic reagents and intermediates. In this thesis, different P450 monooxygenases were investigated as ‘bio-based’ alternatives to chemical catalysts for the late-stage functionalization of steroids and bile acids and engineered by directed evolution procedures towards desired transformation activities. In Article I, the 16α-hydroxylation activity of the bovine CYP17A1 was enhanced by protein engineering to improve the transformation of progesterone into 16α-hydroxyprogesterone in Saccharomyces cerevisiae. Article II follows the same line of research and targets the selective synthesis of bile acid derivatives in Escherichia coli (E. coli) whole-cells. The P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus (S. antibioticus) was identified, which selectively hydroxylates bile acids like lithocholic acid (LCA) and deoxycholic acid (DCA) at the 6β-position, yielding murideoxycholic acid (MDCA), a gallstone solubilizing agent, and 3α-,6β-,12α-trihydroxy-5β-cholan-24-oic acid, respectively. The utilization of OleP as catalyst resulted in shorter synthesis routes for both compounds and additional in a higher yield for MDCA. Building on the results of Article II and the protein engineering approach from Article I, Article III deals with the switch of regioselectivity of the identified CYP107D1 from 6β- to 7β-hydroxylation to form the therapeutic agent ursodeoxycholic acid (UDCA) from LCA by direct hydroxylation. Following a rational protein engineering strategy, a variant with nearly perfect selectivity for UDCA formation was found. Until today, UDCA is either isolated from bile of catheterised farmed bears or produced semisynthetically through low-yielding multistep reactions starting from cholic acid (CA). Article III presents the first reported enzyme for the direct 7β-hydroxylation of LCA to UDCA.
S-adenosyl-L-methionine- (SAM) dependent methyltransferases (MTs) catalyse methylation of halide ions and the C, O, N, S, Se, and As atoms of biomolecules ranging from biopolymers to small molecules. They display different chemo-, regio- and stereoselectivity according to their specific functions. This thesis focuses on the engineering of O-methyltransferases (OMTs) and halide methyltransferases (HMTs) through rational design and directed evolution to study their structure-function relationship and to explore their catalytic promiscuity. The influence of substrate binding residues on the substrate scope and regioselectivity of a plant OMT against various phenolic substrates (Article I) and flavonoids (Article II) has been investigated. Article III describes the directed evolution of an HMT for the biocatalytic synthesis of diverse SAM analogues. With the evolved HMT, regioselective alkylation of phenolic compounds and flavonoids, as well as the SAM analogue regeneration, were achieved through an HMT-MT cascade reaction.
Article I Specific residues expand the substrate scope and enhance the regioselectivity of a plant O-methyltransferase.
It was reported in literature that an isoeugenol 4-OMT (IeOMT) can be engineered to a caffeic acid 3-OMT (CaOMT) by replacing three consecutive residues. In this article, we investigated the effect of these residues on substrate preference and regioselectivity of IeOMT. The triple mutant T133M/A134N/T135Q and the respective single mutants were constructed and tested against a series of phenolic compounds. The variant T133M had a universal effect to improve enzymatic activities against all tested substrates while the mutant A134N had enhanced regioselectivity. The triple mutant T133M/A134N/T135Q benefits from these two mutations, which not only expanded the substrate scope, but also enhanced the regioselectivity of IeOMT. On the basis of this work, regiospecific methylated phenolics can be produced in high purity by different IeOMT variants.
Article II Influence of substrate binding residues on the substrate scope and regioselectivity of a plant O-methyltransferase against flavonoids
Flavonoid OMTs (FOMTs), isoflavonoid OMTs (IOMTs) and phenylpropanoid OMTs (POMTs) display different substrate preferences. Sequence comparison showed that the substrate binding residues at positions 322 and 326 are different between these OMT groups and might be critical for the substrate discrimination. Residues at positions 322 and 326 in IeOMT (a POMT) were mutated to the commonly presented residues in FOMT and IOMT. The introduced mutants, in cooperation with the variant T133M, have improved or brought novel activities and regioselectivity against the tested flavonoids eriodictyol, naringenin, luteolin, quercetin, and also the isoflavonoid genistein compared to the wild-type IeOMT. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
Article III Directed evolution of a halide methyltransferase enables biocatalytic synthesis of diverse SAM analogs
Biocatalytic alkylations to obtain chemo‐, regio‐ and stereoselectively alkylated compounds can be achieved by MTs with the supply of SAM analogues. It was recently discovered that SAM can be directly synthesized from S adenosyl-L homocysteine (SAH) and methyl iodide, catalysed by an HMT. To explore the promiscuity of HMT in the synthesis of SAM analogues, we performed directed evolution of the Arabidopsis thaliana HMT based on a sensitive, colorimetric iodide assay. The identified variant V140T displayed activities against ethyl‐, propyl‐, and allyl iodides to produce the corresponding SAM analogues. With this HMT variant, regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde, as well as the SAM analogue regeneration, were achieved through this HMT-MT one-pot cascade reaction.
Unter promiskuitiver Acyltransferase-Aktivität versteht man die Eigenschaft bestimmter Hydrolasen, in wässriger Lösung bevorzugt Acyltransfer statt Hydrolyse zu katalysieren. Bis vor Kurzem waren nur wenige promiskuitive Acyltransferasen literaturbekannt. Dies führte zu der allgemeinen Annahme, dass diese Aktivität ein seltenes Phänomen in Hydrolasen ist. Diese Arbeit zeigt jedoch, dass promiskuitive Acyltransferase-Aktivität in der Familie der bakteriellen hormonsensitiven Lipasen und Carboxylesterasen der Familie VIII weit verbreitet ist. Detaillierte Struktur-Funktions-Analysen ermöglichen die sequenzbasierte Vorhersage und Optimierung der Acyltransferase-Aktivität in beiden Enzymfamilien. Insbesondere die Carboxylesterasen der Familie VIII überschreiten die Grenzen des bisher für möglich Gehaltenen, indem sie gute Enantioselektivität bei der kinetischen Racematspaltung sekundärer Alkohole zeigen und darüber hinaus die irreversible Bildung von Amiden und Carbamaten in Wasser katalysieren können. Die biokatalytische Acylierung von Zuckern in Wasser galt lange Zeit als unerreichtes Ziel der Biokatalyse. In dieser Arbeit wurde jedoch gezeigt, dass natürlich vorkommende und modifizierte Carboxylesterasen der Familie VIII die regioselektive Acetylierung von Glucose, Maltose und Maltotriose in Wasser mit hoher Effizienz katalysieren können.
In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols.
By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines.
The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.
In this thesis, new catalysts as well as unprecedented approaches for the
valorization of sustainable carbon sources were investigated. The first part deals with the design of catalysts for photocatalytic CO2 reduction (Articles I&II). The promiscuous activity of phenolic acid decarboxylase from Bacillus subtilis (BsPAD) was found to catalyze CO2 reduction (Article I). This cofactor-free enzyme could facilitate the replacement of (noble) metal catalysts regularly employed in CO2 reduction. Based on these findings, additional enzyme catalysts were identified for photocatalytic CO2 reduction. The second part (Articles III-VII) focuses on the valorization of resources obtained from biomass, such as olive mill waste water or lignin, by the promising acyltransferases/hydrolase PestE from Pyrobaculum calidifontis VA1 (Articles IV-VII). The potential of PestE for the valorization of sustainable sources has been demonstrated by enzyme engineering and use in (chemo)enzymatic cascade reactions leading to value-added products.
This work investigated the enzymatic degradation of polyethylene terephthalate (PET) (ArticlesI and II) and polyvinyl alcohol (PVA) (Article III). Physical or chemical degradation of plastic polymers is often performed under extreme conditions like high temperatures or pressure. In comparison to that, recycling of plastics with enzymes can be carried out at ambient temperatures and neutral pH. Enzymes themselves are non- toxic, environmentally friendly, and have been used successfully in a variety of industrial processes.
Enzymatic degradation of polyesters is well studied. Their heteroatomic backbone, which is connecting monomers via ester bonds offers a target for an enzymatic attack. Especially PET, one of the most common polyesters, has been in the focus of research. The first enzyme capable of degrading the polymer was found in 2005. Since then, researchers discovered several enzymes with similar functions and subjected them to enzyme engineering. Improving the enzyme's substrate affinity, activity, and stability aims at making PET recycling more efficient. Article I provides an overview of limitations that enzymatic PET recycling is still facing and the research carried out to overcome them. More precisely, enzyme−substrate interactions, thermostability, catalytic efficiency, and inhibition caused by oligomeric degradation intermediates are summarized and discussed in detail.
Article II further addresses one of the above-mentioned limitations, namely product inhibition of PET hydrolyzing enzymes. We elucidated the crystal structure of TfCa, a carboxylesterase from Thermobifida fusca (T. fusca), and applied semi-rational enzyme engineering. The article discusses the structure-function relationship of TfCa based on the apo-structure as well as ligand-soaked structures. Furthermore, it compares the structures of TfCa and MHETase, another PET hydrolase helper enzyme. Lastly, we determined the substrate profile of the carboxylesterase based on terephthalate-based oligo-esters of various lengths and one ortho-phthalate ester. In a dual enzyme system, TfCa degraded intermediate products derived from the PET hydrolysis of a variant of PETase hydrolase from Ideonella sakaiensis (I. sakaiensis). The dual enzyme system utilized PET more efficiently in comparison to solely PETase due to relieved product inhibition. Since TfCa successfully degraded oligomeric intermediates, the reaction not only released terephthalic acid as the sole product but also increased the overall product yield.
While PET contains an ester bond that can be attacked and hydrolyzed by esterases or lipases, PVA consists of a homoatomic C-C-backbone with repeating 1,3-diol units. The polymer is water soluble with remarkable physical properties such as thermostability and viscosity. PVA is often described as biodegradable, but microbial degradation is slow and frequently involves cost-intensive cofactors. In this study, we present an improved PVA polymer with derivatized side chains and an enzyme cascade that can degrade not only modified but also unmodified PVA in a one-pot reaction. The enzyme cascade consists of a lipase, an alcohol dehydrogenase (ADH), and a Baeyer-Villiger monooxygenase (BVMO). In comparison to the scarcely published research on PVA degradation with free enzyme, this cascade is not only independent from the frequently required cofactor pyrroloquinoline quinone (PQQ) but, in principle, contains an in vitro cofactor recycling mechanism.