Improving the enzymatic synthesis of semi-synthetic beta-lactam antibiotics via reaction engineering and data-driven protein engineering

Semi-synthetic beta-lactam antibiotics are the most prescribed class of antibiotics in the world. Chemical coupling of a beta-lactam moiety with an acyl side chain has dominated the industrial production of semi-synthetic beta-lactam antibiotics since their discovery in the early 1960s. Enzymatic coupling of a beta-lactam moiety with an acyl side chain can be accomplished in a process that is much more environmentally benign but also results in a much lower yield. The lower yield in the enzymatic synthesis can be attributed to the fact that the enzymes that catalyze the reaction, penicillin G acylase (PGA) or alpha-amino ester hydrolase (AEH), have the ability to catalyze the undesired primary hydrolysis of the side chain acyl donor and the secondary hydrolysis of the antibiotic, in addition to the desired synthesis reaction. The goal of the research presented in this dissertation is to improve the enzymatic synthesis of beta-lactam antibiotics via reaction engineering, medium engineering and data-driven protein engineering.;Reaction engineering was employed to demonstrate that the hydrolysis of penicillin G to produce the beta-lactam nucleus 6-aminopenicillanic acid (6-APA), and the synthesis of ampicillin from 6-APA and (R)-phenylglycine methyl ester ((R)-PGME), can be combined in a cascade conversion. In this work, PGA was utilized to catalyze the hydrolysis step, and PGA and AEH were both studied to catalyze the synthesis step. Two different reaction configurations and various relative enzyme loadings were studied. In all reaction configurations the two-enzyme system that utilized PGA and AEH outperformed the one-enzyme system that utilized only PGA. The one-pot, two step system, in which the PGA catalyzed hydrolysis of penicillin G was allowed to proceed prior to addition of the acyl donor and AEH catalyzed synthesis, achieved 47%, yield and secondary hydrolysis was minimized by optimizing relative enzyme loadings. The one-pot, one-step system, a batch process in which all substrates and enzymes are present at the beginning of the reaction, achieved 39% yield, but could be advantageous due to its operational ease and faster cycle times. Both configurations present a promising alternative to the current two-pot set-up which requires intermittent isolation of the intermediate, 6-aminopenicillanic acid (6-APA).;Medium engineering is primarily of interest in beta-lactam antibiotic synthesis as a means to suppress the undesired primary and secondary hydrolysis reactions. The synthesis of ampicillin from 6-APA and (R)-PGME in the presence of ethylene glycol was chosen for study after a review of the literature. It was found that the yield enhancement observed in syntheses in 30% (v/v) ethylene glycol was dependent on the amount of (R)-PGME used for the reaction. Furthermore, it was discovered that the transesterification product of (R)-PGME and ethylene glycol, (R)-phenylglycine hydroxyethyl ester ((R)-PGHEE), is transiently formed during the synthesis reactions. This never reported side reaction has the ability to positively affect yield by re-directing a portion of the consumption of (R)-PGME to an intermediate that could be used to synthesize ampicillin, rather than to an unusable hydrolysis product. (R)-PGHEE was synthesized and its ability to act as an acyl side chain donor for ampicillin synthesis was confirmed. Also, ampicillin synthesis was performed with (R)-phenylglycine amide, an acyl side chain donor that does not undergo transesterification by ethylene glycol, and the ampicillin yield enhancement in 30% (v/v) ethylene glycol was found to be minimal. Based on these results, there is evidence that transesterification by a co-solvent can positively affect yield by participating in an in-situ mixed donor process, where both (R)- PGME and (R)-PGHEE contribute to the synthesis of ampicillin.;Protein engineering was utilized to alter the selectivity of wild-type PGA with respect to the alpha carbon of its substrates. This work represents the first time that the selectivity of PGA has been studied to synthesize a diastereomerically pure beta-lactam antibiotic from racemic substrates. Using existing crystal structures of PGA, residues were targeted for site-saturation based on their proximity to the alpha carbon of the inhibitor penicillin G sulfoxide. Four residues were identified that had altered selectivity toward the desired product, (R)-ampicillin. The most selective variant, betaPhe24Ala, improved the wild-type diastereomeric excess (d.e.) value from 37% to a d.e. value of 98%. Furthermore, our (R)-selective variants improved the yield from pure (R)-PGME up to 2-fold and significantly decreased the amount of secondary hydrolysis present in the reactions. Protein engineering techniques should be pursued to further improve the selectivity of these PGA variants to a d.e. value greater than 99.8% in order to eliminate the need to prepare enantiomerically pure substrates for semi-synthetic beta-lactam antibiotics.;Overall, we have expanded the applicability of PGA and AEH for the synthesis of semi-synthetic beta-lactam antibiotics. We have shown the two enzymes can be combined in a novel one-pot cascade, which has the potential to eliminate an isolation step in the current manufacturing process. Furthermore, we have shown that the previously reported ex-situ mixed donor synthesis of ampicillin for PGA can also occur in-situ in the presence of a suitable side chain acyl donor and co-solvent. Finally, we have made significant progress towards obtaining a selective PGA that is capable of synthesizing diastereomerically pure semi-synthetic beta-lactam antibiotics from racemic substrates.