Computer Modeling Explains the Structural Reasons for the Difference in Reactivity of Amine Transaminases Regarding Prochiral Methylketones

Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the kcat/Km of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with &gt;99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.

Synthesis of Sitagliptin Intermediate by a Multi-Enzymatic Cascade System Using Lipase and Transaminase With Benzylamine as an Amino Donor

Herein, we report the development of a multi-enzyme cascade using transaminase (TA), esterase, aldehyde reductase (AHR), and formate dehydrogenase (FDH), using benzylamine as an amino donor to synthesize the industrially important compound sitagliptin intermediate. A panel of 16 TAs was screened using ethyl 3-oxo-4-(2,4,5-trifluorophenyl) butanoate as a substrate (1). Amongst these enzymes, TA from Roseomonas deserti (TARO) was found to be the most suitable, showing the highest activity towards benzylamine (∼70%). The inhibitory effect of benzaldehyde was resolved by using AHR from Synechocystis sp. and FDH from Pseudomonas sp., which catalyzed the conversion of benzaldehyde to benzyl alcohol at the expense of NAD(P)H. Reaction parameters, such as pH, buffer system, and concentration of amino donor, were optimized. A single whole-cell system was developed for co-expressing TARO and esterase, and the promoter engineering strategy was adopted to control the expression level of each biocatalyst. The whole-cell reactions were performed with varying substrate concentrations (10–100 mM), resulting in excellent conversions (ranging from 72 to 91%) into the desired product. Finally, the applicability of this cascade was highlighted on Gram scale, indicating production of 70% of the sitagliptin intermediate with 61% isolated yield. The protocol reported herein may be considered an alternative to existing methods with respect to the use of cheaper amine donors as well as improved synthesis of (R) and (S) enantiomers with the use of non-chiral amino donors.

Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathway of gentamicin has been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are converted to C1a and C2a by concerted actions of two PLP (pyridoxal 5'-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then re-aminated by GenB3 using an amino donor. Crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes' functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed new light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

Mechanistic insights into dideoxygenation in gentamicin biosynthesis

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathway of gentamicin has been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are converted to C1a and C2a by concerted actions of two PLP (pyridoxal 5'-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then re-aminated by GenB3 using an amino donor. Crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes' functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed new light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

Efficient Amino Donor Recycling in Amination Reactions: Development of a New Alanine Dehydrogenase in Continuous Flow and Dialysis Membrane Reactors

Transaminases have arisen as one of the main biocatalysts for amine production but despite their many advantages, their stability is still a concern for widespread application. One of the reasons for their instability is the need to use an excess of the amino donor when trying to synthesise amines with unfavourable equilibria. To circumvent this, recycling systems for the amino donor, such as amino acid dehydrogenases or aldolases, have proved useful to push the equilibria while avoiding high amino donor concentrations. In this work, we report the use of a new alanine dehydrogenase from the halotolerant bacteria Halomonas elongata which exhibits excellent stability to different cosolvents, combined with the well characterised CbFDH as a recycling system of L-alanine for the amination of three model substrates with unfavourable equilibria. In a step forward, the amino donor recycling system has been co-immobilised and used in flow with success as well as re-used as a dialysis enclosed system for the amination of an aromatic aldehyde.

Identification and biochemical characterisation of tyrosine aminotransferase from Anthoceros agrestis unveils the conceivable entry point into rosmarinic acid biosynthesis in hornworts

Main conclusion Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for l-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. Abstract The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of l-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying l-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC–MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

Abstractω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

Metabolic Engineering of Escherichia Coli for Ectoine Production With the Fermentation Strategy of Supplementing Amino Donor

Background: Ectoine, a compatible solute, has broad application prospects in food biotechnology, agriculture, medicine, and cosmetics because of its protective action on biological compounds. Industrially, ectoine is produced by halophilic bacteria in a complex process. Recently, various works focus on improving ectoine production by using engineered strains, but there are still problems of low yield and low ectoine production efficiency.Results: To overcome the drawback, a final metabolic engineered strain E. coli ET08 was constructed by eliminating lysine synthesis branch and by-product metabolic pathways, and ectoine production reached 10.2 g/L through culture medium optimization. Compared with nitrate, addition of ammonium salt contributed more to the ectoine synthesis. Furthermore, the ammonium sulphate boosted more ectoine titers than ammonium chloride and sodium glutamate. The analysis of transcriptional levels revealed that the ammonium sulfate enhanced ectoine biosynthesis by enhancing metabolic flux toward ectoine biosynthesis and providing affluent synthetic precursors. Ultimately, the ectoine production and yield of the E. coli ET08 reached 36.5 g/L and 0.3 g/g glucose with supplementing amino donor in a 7.5 L bioreactor.Conclusions: a novel potential metabolic engineered Escherichia coli for ectoine production was constructed. optimizing amino donor and analyzing the transcription levels conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis. It is the first report about the effect of exogenous amino donor on ectoine fermentation by metabolic engineered strain. The maximum ectoine production and yield from glucose synthesized by E. coli were obtained by two-stage feeding fermentation with supplementing amino donor. It provides a novel strategy for the synthesis of ectoine by engineered strain in industry. This research provides the basis for an effective process for ectoine production, together with the further applications of ectoine in food and cosmetics, and could also be used to produce other high value amino acid derivative.

Metabolic Engineering of Escherichia Coli for Ectoine Production with the Fermentation Strategy of Supplementing Amino Donor

Background: Ectoine, a compatible solute, has broad application prospects in food biotechnology, agriculture, medicine, and cosmetics because of its protective action on biological compounds. Industrially, ectoine is produced by halophilic bacteria in a complex process. Recently, various works focus on improving ectoine production by using engineered strains, but there are still problems of low yield and low ectoine production efficiency.Results: To overcome the drawback, a final metabolic engineered strain E. coli ET08 was constructed by eliminating lysine synthesis branch and by-product metabolic pathways, and ectoine production reached 10.2 g/L through culture medium optimization. Compared with nitrate, addition of ammonium salt contributed more to the ectoine synthesis. Furthermore, the ammonium sulphate boosted more ectoine titers than ammonium chloride and sodium glutamate. The analysis of transcriptional levels revealed that the ammonium sulfate enhanced ectoine biosynthesis by enhancing metabolic flux toward ectoine biosynthesis and providing affluent synthetic precursors. Ultimately, the ectoine production and yield of the E. coli ET08 reached 36.5 g/L and 0.3 g/g glucose with supplementing amino donor in a 7.5 L bioreactor.Conclusions: a novel potential metabolic engineered Escherichia coli for ectoine production was constructed. optimizing amino donor and analyzing the transcription levels conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis. It is the first report about the effect of exogenous amino donor on ectoine fermentation by metabolic engineered strain. The maximum ectoine production and yield from glucose synthesized by E. coli were obtained by two-stage feeding fermentation with supplementing amino donor. It provides a novel strategy for the synthesis of ectoine by engineered strain in industry. This research provides the basis for an effective process for ectoine production, together with the further applications of ectoine in food and cosmetics, and could also be used to produce other high value amino acid derivative.