Suchergebnisse

Abstract. 4-Hydroxy-2-ketoacid derivatives are versatile building blocks for the synthesis of amino acids, hydroxy carboxylic acids and chiral aldehydes. Pyruvate aldolases are privileged catalysts for a straightforward access to this class of ketoacid compounds. In this work, a Class II pyruvate aldolase from Escherichia coli K-12, 2-keto-3-deoxy-L-rhamnonate aldolase (YfaU), was evaluated for the synthesis of amino acid derivatives of proline, pipecolic acid, and pyrrolizidine-3-carboxylic acid. The aldol addition of pyruvate to N-protected amino aldehydes was the key enzymatic aldol addition step followed by catalytic intramolecular reductive amination. The corresponding N-Cbz-amino-4-hydroxy-2-ketoacid (Cbz=benzyloxycarbonyl) precursors were obtained in 51%-95% isolated yields and enantioselectivity ratios from 26:74 to 95:5, with chiral -substituted N-Cbz-amino aldehydes. (S)-N-Cbz-amino aldehydes gave aldol adducts with preferentially (R)-configuration at the newly formed stereocenter, whereas the contrary is true for (R)-N-Cbz-amino aldehydes. Addition reactions to achiral amino aldehydes rendered racemic aldol adducts. Molecular models of the pre-reaction ternary complexes YfaU-pyruvate enolate-acceptor aldehyde were constructed to explain the observed stereochemical outcome of the reactions. Catalytic reductive amination of the aldol adducts yielded 4-hydroxy-2-pipecolic acid, and unprecedented C5 substituted 4-hydroxy proline and pyrrolizidine-3-carboxylic acid derivatives. ; This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 635595 (CarbaZymes), the Ministerio de Economía y Competitividad (MINECO) and the European Regional Development Fund (FEDER) (grant CTQ2015-63563-R to P.C. and CTQ2015-64436-P to T.P.) and COST action CM1303 Systems Biocatalysis. ; Peer reviewed

Thiamine diphosphate (ThDP) dependent enzymes are useful catalysts for asymmetric C-C bond formation through benzoin-type condensation reactions that result in α-hydroxy ketones. A wide range of aldehydes and ketones can be used as acceptor substrates; however, the donor substrate range is mostly limited to achiral α-keto acids and simple aldehydes. By using a unifying retro-biosynthetic approach towards acyl-branched sugars, we identified a subclass of (myco)bacterial ThDP-dependent enzymes with a greatly extended donor substrate range, namely functionalized chiral α-keto acids with a chain length from C4 to C8 . Highly enantioenriched acyloin products were obtained in good to high yields and several reactions were performed on a preparative scale. The newly introduced functionalized α-keto acids, accessible by known aldolase-catalyzed transformations, substantially broaden the donor substrate range of ThDP-dependent enzymes, thus enabling a more general use of these already valuable catalysts. ; We thank the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant RTI2018-094637-B-I00), and Programación Conjunta Internacional (PCI2018-092937), through the initiative ERA CoBioTech (Tralaminol), and the DFG (FOR 1296) and the European Union's Horizon 2020 Research and Innovation Programm (Grant Agreement no. 635595) for financial support. Many thanks to Dr. Stephen Seah, University of Gulph, for providing the plasmid of hpaI. We would also like to thank Dr. Philippe Bisel and Sascha Ferlaino, University of Freiburg, for NMR measurements and constructive suggestions for the manuscript, as well as Dr. Kay Greenfield for her careful proofreading. Open Access funding enabled and organized by Projekt DEAL. ; Peer reviewed

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S- or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee values of >99%. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 635595 (CarbaZymes), the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to P.C.), and COST action CM1303 Systems Biocatalysis. ; We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

Aldolase and transaminase coexpressed in Escherichia coli cells and lyophilized (i.e., lyophilized whole-cell biocatalyst (LWCB)) were used as biocatalysts for the one-pot cascade synthesis of l-homoserine with substrate cycling. The kinetic analysis of enzymes within lyophilized cells was performed to evaluate the behavior of the system. The best result among the performed fed-batch reactor experiments achieved was 640.8 mM (76.3 g L–1) of l-homoserine with a volume productivity of 2.6 g L–1 h–1. This is comparable with the results of the same cascade synthesis using cell-free extracts (CFEs) and significantly better than the reports in the literature applying fermentation technology. The approach applied here can serve as guidance for the design of microbial cells with an optimal ratio of expressed enzymes that act as biocatalysts in the cascade, resulting in lower biocatalyst cost, no need for the addition of expensive coenzymes, and enhanced enzyme stability as compared with cell-free extracts. ; This project has received funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement no. 635595 (CarbaZymes). This communication reflects only the beneficiary's view, and the European Commission is not responsible for any use that may be made of the information it contains. The authors gratefully acknowledge Prozomix Ltd. (United Kingdom) for providing the lyophilized whole cells. The authors also acknowledge the Ministerio de Ciencia e Innovación (MICIN), the Fondo Europeo de Desarrollo Regional (FEDER) (grant RTI2018-094637-B-I00), and Programación Conjunta Internacional (PCI2018-092937), through the initiative ERA CoBioTech (Tralaminol) ; Peer reviewed