Doctoral Thesis
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Amine transaminases are versatile biocatalysts for the production of pharmaceutically and agrochemically relevant chiral amines. They represent an environmentally benign alternative to waste intensive transition metal catalysed synthesis strategies, especially because of their high stereoselectivity and robustness. Therefore, they have been frequently used in the (chemo)enzymatic synthesis of amines and/or became attractive targets for enzyme engineering especially in the last decade, mainly in order to enlarge their substrate scope. Certainly, one of the most notable examples of amine transaminase engineering is the
manufacturing of the anti-diabetic drug Sitagliptin in large scale after several rounds of protein engineering. Thereby, the target amine was produced in asymmetric synthesis mode which is the most convenient and favored route to a target chiral amine, starting from the corresponding ketone. The choice of the amine donor is highly relevant for reaction design in terms of economical and thermodynamic considerations. For instance, the use of alanine as the natural amine donor is one of the most common strategies for the amination of target ketones but needs the involvement of auxiliary enzymes to shift the reaction equilibrium towards product formation. In fact, isopropylamine is probably one of the most favored donor molecules since it is cheap and achiral but it is supposed to be accepted only by a limited number of amine transaminases.
This thesis focusses on the optimization and application of amine transaminases for asymmetric synthesis reactions en route to novel target chiral amines using isopropylamine as the preferred amine donor.
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.
The focus of this thesis is the engineering and analysis of the enantioselectivity of esterases using 3-phenylbutyric acid (3-PBA) as model substrate. An ultra high throughput assay for identification of enantioselective esterases has been developed, based on the combination of in vivo selection and flow cytometry. The in vivo selection medium consists of a couple of pseudo-enantiomers of 3-PBA; one enantiomer is coupled to glycerol (GE), and hydrolysis of this substrate will enable cell survival. The other enantiomer is coupled to the toxin 2,3-dibromopropanol (BE), the hydrolysis of this substrate will cause cell death. Thus, cell survival is a function of the enantioselectivity of the enzyme expressed. The pseudo-enantiomeric substrates are structurally similar to allow selection for enantioselectivity instead of selection for enzyme substrate affinity. Next, esterase BS2 was chosen as negative control to establish the selection system since it hydrolyses both pseudo-enantiomers with low enantioselectivity (E~3 and 1, respectively). High enantioselective esterases towards 3-PBA: esterases PestE and CL1 (E > 100, both (R)-selective) were identified in a screening and used as positive controls. Further, the hyperthermophilic esterase PestE was crystallized. After elucidation of the enzyme structure, the high enantioselectivity of the enzyme towards 3-PBA could be explained by molecular modelling. The optimal concentration of the pseudo-enantiomeric substrates was set to be 5 mM for GE (higher concentrations were toxic) and 20 mM for BE (lower concentrations did not completely inhibit bacterial growth). The in vivo selection system was established together with the identification of a flow cytometric method to differentiate bacterial physiological status. The combination of Syto9 and PI was chosen as staining technique, because it allowed differentiation of the viable and the dead cell populations, and of these from the background. After viability detection by flow cytometry was established, esterases PestE and BS2 were cultivated in selection ((R)-GE and (S)-BE) and anti-selection medium ((S)-GE and (R)-BE). Clear differences in the culture viability depending on the enantioselectivity of the enzyme expressed appeared: cells expressing the (R)-enantioselective PestE could proliferate in selection medium, but could not proliferate in anti-selection medium. Cells expressing the non-selective BS2 did not grow in any media. Further, cultures containing mixtures of BS2/PestE or BS2/CL1 expressing cells were incubated in selection and anti-selection medium, and the viable clones were detected by flow cytometry analysis, sorted out and plated on agar. When the mixtures were incubated in selection medium, enrichment of the (R)-selective enzyme (PestE or CL1) over the non-selective enzyme (BS2) was observed. When the enzyme mixtures were incubated in anti-selection medium, very few colonies grew on agar, indicating that cell survival was a function of enzyme enantioselectivity. The successfully developed assay was used to identify variants with increased enantioselectivity in a mutant library of esterase PFEI (E ~ 3, (R)-selective) created by saturation mutagenesis. After library expression, 108 clones were in vivo selected and analyzed by flow cytometry. The viable cells were sorted out and plated on agar. The 28 resulting colonies were transferred to one microtiterplate and their activity and enantioselectivity (Eapp) was investigated using p-nitrophenyl derivatives. Four interesting mutants were identified: Table 1. Enantioselectivity of the in vivo selected mutants. Mutant Eapp[a]Etrue[b]Etrue[c]Etrue[d]Etrue[e] Mutations C4 80 4 4 3 1 V121I, F198G, V225A E7 >100 2 n.d. 3 n.d. V121S E8 2 25 16 50 >100 V121S, F198G, V225A F5 5 13 15 18 80 F121I, F198C [a] with separate (R)- or (S)-enantiomers of p-nitrophenyl-3-phenylbutanoate. [b] towards GE with cell lysate or [c] pure enzyme. [d] towards Et-3-PB with cell lysate or [e] pure enzyme. n.d. not determined. The mutants were purified and activity and enantioselectivity were determined in kinetic resolutions towards Et-3-PB and GE (Table 1). Mutants identified as highly enantioselective in the Eapp-assay (C4 and E7) were low selective in kinetic resolutions. On the contrary, mutants E8 and F5, which showed low enantioselectivity towards p-nitrophenyl-3-phenylbutanoate, hydrolyzed the 3-phenylbutyric esters with good to excellent enantioselectivities. This confirms that Eapp values can differ much from Etrue values as “you get what you screen for”, and supports that the here described method is very suitable for identification of enantioselective esterases. In this PhD thesis a novel strategy for identification of enantioselective esterases has been developed. This method allows a very high throughput (≥ 108 mutants/day) and opens the bottleneck of variant analysis, which exists in protein engineering technology.
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.