Studies of the toluene dioxygenase from Pseudomonas putida F1: influence of active-site positions on hydroxylations of mono- and bicyclic aromatics

Abstract

Till today, the selective dearomatizing cis-dihydroxylation of aromatics cannot be performed chemically. Hence, it is truly exceptional how nature evolved Rieske non-heme iron dioxygenases (ROs), capable of performing such challenging reactions. These enzymatic multicomponent systems are not only able to catalyze cis-dihydroxylation reactions with outstanding activity, but also with an excellent enantioselectivity (>99% ee) for a broad range of aromatic substrates. In their natural host, ROs are involved in the initial catabolism of aromatic compounds into intermediates of the tricarboxylic acids cycle. In biocatalysis, the oxyfunctionalizing capabilities of ROs can be utilized to synthetize cis-dihydrodiols from mono- and polycyclic arenes. These hydroxylated compounds are valuable synthons, frequently employed in the synthesis of natural products, pharmaceuticals, and fine chemicals. One of the best experimentally characterized ROs reported in literature is toluene dioxygenase (TDO), from Pseudomonas putida F1, which substrate scope comprises over 100 different compounds. Nevertheless, a restraint hampering TDO application as a biocatalyst for the targeted cis-dihydroxylation of aromatics, is the observed decrease in conversion as substrate size increases, resulting in modest product yields. Therefore, fundamental studies revealing the influence of TDO active-site positions, enabling a better conversion of mono- and especially bulky bicyclic aromatics are compulsory. Such need prompted the present thesis, which focused on TDO as model biocatalysts, since it displays remarkable substrate promiscuity and advantageous features for preparative biotransformations. The central task of this work was fostering the biocatalytic capabilities of TDO toward the cis-dihydroxylation of four aromatic model substrates. The first step was the development of an enhanced recombinant TDO system in Escherichia coli (E. coli), for an efficient dioxygenase overexpression. Therefore, the new platform E. coli BW25113 pBAD18-TDO was established, which exhibited outstanding product formation for the bicyclic substrate naphthalene. Nevertheless, in the attempt to apply such platform for the conversion of small monocyclic aromatics, an unforeseen downstream reaction catalyzed by E. coli, dehydrogenating the generated cis-dihydrocatechols to the corresponding catechols, was discovered. By performing a systematic screening of dehydrogenase deficient single knock-out strains from the KEIO collection, the enzyme glycerol dehydrogenase (GldA) from E. coli was identified as the main responsible for such degradation. These findings drove the development of the enhanced platform E. coli BW25113 ΔgldA pBAD18-TDO, allowing the abolishment of the unwanted secondary reaction. Thus, a semi-preparative biotransformation of benzene was performed utilizing the customized TDO platform, resulting in the isolation of 141 mg (31%) of cis-dihydrocatechol as sole product. The system was also tested for the semi-preparative biotransformation of the bicyclic substrate naphthalene, yielding 287 mg (89%) of enantiopure (1R,2S)-1,2-dihydro-1,2-naphthalenediol. Once the TDO platform was established and successfully applied, the next task was to explore the influence of TDO active-site position F366 in naphthalene conversion by generating and investigating a set of nine TDO variants at this position. Strikingly, the single point variant TDOF366V revealed that the enantioselectivity could be switched completely. Furthermore, semi-preparative naphthalene biotransformations with TDOF366V enabled the synthesis of 101 mg (31%) enantioenriched (1S,2R)-1,2-dihydro-1,2-naphthalenediol (90% ee). It is worth to mention that before this study, this enantiomer was never directly generated either chemically or biocatalytically. The next aim was to expand the TDO-based biocatalyst portfolio by generating a semi-rational designed TDO single- and double mutant library. Thus, the library consisting out of 176 variants was tested for the conversion of the bicyclic substrates naphthalene, 1,2,3,4-tetrahydroquinoline, and 2-phenylpyridine in the pursue to enhance product formation and/or chemo-, regio- and enantioselectivity. These studies highlighted that introduced mutations at the active site hot-spot positions M220, A223 and F366, strongly influences chemo-, regio- and enantioselectivity. Moreover, mutations at positions M220 and A223 also exerted substantial effects on product formation during the conversion of bicyclic (hetero)aromatics. In addition, since the TDO mutant library addressed all 14 non-conserved active site amino acids, it was noticeable that the active site is highly tolerant to the introduction of mutations. Additionally, it could be assessed that the combination of the outperforming mutations into the double variant TDOF114H_A223T entirely abolished the formation of the side product quinoline in 1,2,3,4-tetrahydroquinoline biotransformations. This approach enabled in a semi-preparative biotransformation the selective production of 106 mg (71%) of enantioenriched (R)-1,2,3,4-tetrahydroquinoline-4-ol (94% ee). Further, double variant TDOM220A_V309G exhibited an astonishing 15.1-fold higher conversion of the substrate 2-phenyl-pyridine, in comparison to TDO wild type. This enhancement enabled for the first time, the TDO-catalyzed production of 114 mg (60%) enantiopure (1S,2R)-3-(pyridin-2-yl)cyclohexa-3,5-diene-1,2-diol, along with reduced amounts of 6 mg (4%) 2-phenylpyridin-3-ol, as side product. These compelling findings meet the scope of this thesis, in terms of fostering TDO product formation as well as chemo-, regio- and enantioselectivity for bulky bicyclic (hetero) aromatics. Hence, this dissertation highlights the importance of both, the improvement of the recombinant E. coli BW25113 platform, as well as the enhancement of the biocatalyst TDO via enzyme engineering for the generation of valuable cis-dihydroxylated compounds.