Ω-Transaminases As Promising Biocatalysts for the Chiral Synthesis of Β-Amino Acids
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ω-Transaminases as Promising Biocatalysts for the Chiral Synthesis of β-Amino Acids Zur Erlangung des akademischen Grades eines DOKTORS DER INGENIEURWISSENSCHAFTEN (Dr.-Ing.) der Fakultät für Chemieingenieurwesen und Verfahrenstechnik des Karlsruher Instituts für Technologie (KIT) vorgelegte Genehmigte DISSERTATION von M. Sc. Oliver Buß aus Weinheim a. d. Bergstraße Referent: Prof. Dr. rer. nat. Christoph Syldatk Korreferent: Prof. Dr. rer. nat. Jürgen Pleiss Tag der mündlichen Prüfung: 18.10.2018 I Preamble Preamble Parts of this dissertation are based on submitted manuscripts for publication or on peer-review research articles. The publications are based on work carried out between January 2015 and July 2018 for this dissertation. Sections based on publications are marked at the beginning of each section. The text of these sections may be largely identical to the publications, but has been supplemented in parts by further information as well as illustrations. I Preamble You may never know what results come of your actions, but if you do nothing, there will be no results. (Mahatma Gandhi) II Abstract Abstract The enzyme family of ω-transaminases (ω-TA) catalyzes a stereoselective transfer of an amino group from an amino donor to an acceptor molecule (with ketone/aldehyde function). ω-TA are of great interest for many pharmaceutical processes and synthesis strategies, as they enable the production of enantiomerically pure amino-drugs. This thesis, which is part of the Molecular Interaction Engineering (MIE) project funded by the German Federal Ministry of Education and Research, deals with enzyme production and modification using the synthesis of β-amino acids and their degradation by microorganisms as examples. Therefore, the focus of this work was set on the transamination of β-keto acids/esters, the necessary enzyme engineering, as well as the characterization and cataloguing of the ω-transaminase family. The primary goal was to demonstrate the synthesis of the chiral cancer drug component paclitaxel (Taxol), β-phenylalanine(ester), mediated by ω-transaminases. In summary, the following points could be achieved in this thesis: o The β-phenylalanine converting Variovorax paradoxus ω-TA gene was codon optimized for expression in E. coli BL21, purified by fast protein liquid chromatography (FPLC) using Ni-NTA columns and thermostabilized by in silico guided site directed mutagenesis. o The ω-transaminase engineering database (oTAED) was developed as a helpful starting point for protein engineering and discovery of ω-TA. o Functional amino acid positions in the V. paradoxus ω-TA were mutated and the effects on activity were investigated. o The protein stability of ω-TA from V. paradoxus was improved by targeted mutagenesis (single mutation) while maintaining the enzymatic activity. o The difficulties in the synthesis of β-phenylalanine (β-PA) by a lipase-ω-TA reaction cascade were demonstrated. Alternative synthesis methods were proposed and at least one established. o Therefore two ω-TA for the synthesis of (R)- and (S)-β-phenylalanine ethyl ester were identified by screening an ω-TA library. o The ω-TA 3FCR_4M showed potential for up-scaling by a factor of 200 for the synthesis of (S)-β-phenylalanine ethyl ester (200 mL - 30 mM product concentration). III Abstract o The degradation of β-phenylalanine by two bacteria was analyzed in detail under controlled conditions. Additionally for the first time the simultaneous degradation of both enantiomers was shown for one bacterium. IV Abstract Chapter 1 introduces the importance of α- and β-amino acids and presents the various synthesis processes (chemical and enzymatic). The mode of action of the enzymatic catalyst is also described in detail. In addition, it is discussed how enzymes can be specifically modified by simulation methods in order to modify important properties such as substrate selectivity or protein stability. This also presupposes that the enzyme family(s) are systematically catalogued and characterized in order to predict and underline properties such as enantioselectivity, substrate selectivity and to adapt reaction conditions. Therefore, a publicly available transaminase database was created which is described in Chapter 3. In this chapter, the evolutionary conserved amino acid positions within the two ω-TA families (fold type I and type IV) are shown and their functions are analyzed. It could also be shown that by standardizing the amino acid positions (standard numbering), the properties of certain amino acid positions can be compared and transferred between different ω-TA. As an example, a mutation study conducted at a particular ω-TA can be transferred to a second poorly characterized ω-TA (within the same family). In order to demonstrate the standardization of amino acid positions, data from the literature were compared with each other, with the result that different mutation studies (at different ω-TA) had actually mutated and investigated the same standard amino acid positions. This illustrates the need for standard numbering of amino acid positions within an enzyme family, in particular with regard to ω-TA engineering, since the large and extensive search for engineering sites within the long peptide chain of enzymes is no longer necessary. Within the ω-TA family of (S)-selective enzymes, a relatively large group of β-PA converting transaminases was determined within the database. This group also includes ω-TA from V. paradoxus which show high activity towards this substrate. Therefore, chapter 4 deals with the characterization, enzyme production and the targeted improvement of the protein stability of ω-TA from V. paradoxus. This particular ω-TA allows the conversion of β-PA under mild reaction conditions in a buffered aqueous reaction system. This enzyme should be modified for further processing by specific mutations within the protein sequence in such a manner that the protein stability and long-term activity of the enzyme will be increased. This was achieved using the FoldX protein stabilization algorithm and potentially energy-stabilizing mutations were identified and tested. The amino acid changes predicted on this basis were introduced into the gene of ω-TA by mutagenesis PCR. The activities of the resulting ω-TA variants were investigated and the resulting stabilization was analyzed using the protein melting point. The starting point of protein melting was shifted by approx. 4°C for the best variant. This increase V Abstract in thermostability allows maintaining the enzymatic activity over a longer period of time. Furthermore, this improvement can be used as a basis for further enzyme mutation experiments, as mutations often lead to a reduction in protein stability. Based on the systematic analysis of mainly the (S) and (R)-selective ω-TA family, Chapter 5 discusses the enzymatic synthesis of β-phenylalanine ethyl ester from the substrate ethyl 3-oxo phenylpropanoate (β-keto ester) using mutant and natural ω-TA. It was therefore shown for the first time that ω-TA are able to convert this category of aromatic substrate. For this reason, mutation experiments were carried out for the ω-TA from V. paradoxus, as it converts the product, β-phenylalanine, with a high turnover rate. The amino acid positions of the ω-TA, which should have an influence on substrate selectivity, were determined and mutated by methods developed in Chapter 3. At all, the wild type enzyme showed no activity towards the β-keto ester substrate. Mainly amino acid residues within the enzyme were altered which should have an influence on the substrate binding, namely: R41, Y76, Y159 and R398. R41, for example, is an important arginine residue that binds the negatively charged carboxyl group of β-phenylalanine via its positive charge. This residue was replaced because the substrate of interest does not have a free carboxyl group, but an ester functionality. However, no activity against ethyl 3-oxo-phenylpropanoate could be detected for most variants. Only the variant R41K-R398K could be regarded as active in qualitative terms, but the activity was too low to allow a quantitative statement about the activity. Since no clearly active variant with a quantifiable turnover was found, an ω-TA library of the Bornscheuer working group (University of Greifswald) was screened. For this purpose, a chromophoric screening test was used which allows a quick selection between non-active and active transaminases. At least two transaminases with activity were detected in this screening, one (R)-selective, the other (S)- selective. These two transaminases are no longer wild type enzymes, but contain several mutations. The (R)-selective enzyme was determined as the transaminase ATA117, which was created for the synthesis of sitagpliptin by Savile et al.. The (S)-selective ω-TA is an enzyme which was engineered by the Bornscheuer research group for the conversion of large aromatic ketones. The (S)-selective reaction was used in a preparative approach with an (S)-selective enzyme to demonstrate that the detected ω-TA also enables up-scaling. In this context, a preparative purification method using automated column chromatography was also established. In contrast to the enzyme conversion, however, little is generally known about the degradation of β-amino acids by microorganisms. Therefore, chapter 6 deals with the characterization of β-PA degradation by β-Proteobacteria Paraburkholderia PsJN and BS115. BS115 is a strain isolated from potting soil enriched with soy-peptone. Type strain PsJN, on the other hand, has VI Abstract originally been isolated from plants and is closely involved with the nitrogen cycle in soil. The aim was to discover new, as yet uncharacterized ω-TA and maybe additional enzymes and to investigate the microbial resolution of the racemate as an alternative to an enzymatic resolution. This degradation process was investigated under controlled conditions in a 2.5L bioreactor and the temporal degradation and biomass formation was investigated. It could be shown that racemic β-PA is degraded stereoselectively by PsJN. During this process, (S)-β-PA was completely degraded while the (R)-enantiomer was completely retained in the fermentation medium.