Understanding and Overcoming the Limitations of Bacillus badius and Caldalkalibacillus thermarum Amine Dehydrogenases for Biocatalytic Reductive Amination

The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a growing field of research in both chemocatalysis and biocatalysis. Recent research has focused on the enzyme engineering of amino acid dehydrogenases (to obtain amine dehydrogenases), and this technology promises to be a potentially exploitable route for chiral amine synthesis. However, the use of these enzymes in industrial biocatalysis has not yet been demonstrated with substrate loadings above 80 mM, because of the enzymes’ generally low turnover numbers (<i>k</i>_cat < 0.1 s^–1) and variable stability under reaction conditions. In this work, a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase (PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase (AmDH) from Bacillus badius for both kinetic and thermostability parameters, with the former exhibiting an increased thermostability (melting temperature, <i>T</i>_m) of 83.5 °C, compared to 56.5 °C for the latter. The recruited enzyme was further used in the reductive amination of up to 400 mM of phenoxy-2-propanone (<i>c</i> = 96%, ee (<i>R</i>) < 99%) in a biphasic reaction system utilizing a lyophilized whole-cell preparation. Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs, compared to their PheDH counterparts

Effects of various concentrations of HAI-1 and AI-2 on LuxN- and LuxQ- mediated phosphorylation of LuxU.

<p>LuxN- and LuxQ-bearing membrane vesicles, together with purified LuxP and LuxU, were incubated with 100 μM [γ-³²P] ATP, and the effects of AI-2 and HAI-1 on the initial rate of LuxU phosphorylation were tested. AI-2 and HAI-1 were added at physiological concentrations (see Fig. 2), indicated in the lower part of the graph (HAI-1 in black, AI-2 in gray). Phosphorylated LuxU was quantitatively analyzed as described in Fig. 7. The degree of inhibition is expressed as the percentage reduction in the initial rate of LuxU phosphorylation measured in the presence of the indicated concentrations/blends of autoinducers relative to that seen in the absence of autoinducers. All experiments were performed in triplicate, and error bars indicate standard deviations of the mean.</p

Dose-dependent effects of HAI-1 and AI-2 on bioluminescence and exoproteolytic activity of <i>V. harveyi.</i>

<p>The autoinducer synthase negative mutant <i>V. harveyi</i> MM77 (<i>luxM</i>::Tn<i>5 luxS</i>::Cm^r) was used to analyze the dose-dependent effects of HAI-1 and AI-2. Strain MM77 was cultivated in the presence of varying concentrations (0, 0.1, 0.3, 0.5, 2.5, 5, 25 and 50 μM) of HAI-1 and/or AI-2, and levels of bioluminescence (A) and exoproteolytic activity (B) in the culture fluids were determined. Light levels and exoproteolytic activities were expressed relative to the optical density of the culture, and values are displayed in a 3D mesh. All experiments were performed in triplicate, and mean values are shown. The standard deviations were below 5%.</p

Alterations in CAI-1 activity during growth of <i>V. harveyi</i>.

<p>(A) CAI-1 activity was determined in cell-free culture fluids (the same samples as described in Fig. 2) using a bioassay with <i>V. cholerae</i> MM920 as reporter strain. Levels of CAI-1 mediated bioluminescence are indicated by light gray dots. A curve is presented to guide the eye. The optical density (OD₆₀₀) is plotted as crosses. All experiments were performed at least in triplicate, and error bars indicate standard deviations of the mean. (B, C) Unbiased GC-TOF-MS profiling was used to identify signaling molecules that accumulated in the medium after 7 h of growth. (B) Single-ion responses with defined retention indices (RI) close to that expected for Ea-C8-CAI-1 were tested for significant increases between 7 h and 19 h of cultivation, p<1.0 10⁻⁴. The data were presented as x-fold accumulation in comparison to the 7 h time point. The replicate mass spectrum and respective retention index may be retrieved from the Golm Metabolome Database (<a href="http://gmd.mpimp-golm.mpg.de/" target="_blank">http://gmd.mpimp-golm.mpg.de/</a>) using the identifier code A158016 (m/z 356_RI 1586.20). (C) Representative mass spectrum of candidate signaling molecules possibly representing Ea-C8-CAI-1. The mass of compound A158016 corresponds to Ea-C8-CAI-1 (arrow), which was modified with trimethylsilylated methoxyamine. Its chemical structure is shown. All experiments were performed at least in triplicate. Error bars indicate standard deviations of the mean. Time courses were interpolated by smoothed lines using MS-EXCEL software.</p

Transcriptional analysis of AI-regulated genes.

<p>Cells of the wild type (BB120) and the autoinducer-negative mutant JMH634 were grown as described in Fig. 2. Total RNA was isolated at four different time points (marked by the arrows in Fig. 2A), which are characterized by different concentrations/blends of the AIs: 1– early exponential growth phase  =  low concentration of AI-2; 2– mid-exponential growth phase  =  high concentration of AI-2; 3– late exponential growth phase  =  blend of AI-2 and HAI-1; 4– stationary phase  =  blend of AI-2, HAI-1 and CAI-1. Levels of <i>luxR</i> (A), <i>luxA</i> (B), <i>vhpA</i> (C), <i>vopN</i> (D), <i>vscP</i> (D) and <i>recA</i> (as reference) transcripts were determined by qRT-PCR for each time point. Changes in transcript levels (expressed relative to <i>recA</i>) were calculated using the C_T method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048310#pone.0048310-Schmittgen1" target="_blank">[49]</a>. Since transcript levels of the corresponding genes in mutant JMH634 did not change significantly over time, only one time point (3) is shown. All experiments were performed in triplicate, and error bars indicate standard deviations of the mean.</p

Time course of HAI-1 and AI-2 production (A, C), bioluminescence and exoproteolytic activity (B, D) during growth of <i>V. harveyi</i>.

<p>Cells of an overnight culture of <i>V. harveyi</i> BB120 were diluted 5,000-fold in fresh AB medium and cultivated aerobically at 30°C. Samples were taken at the times indicated and autoinducer concentrations in the medium, bioluminescence levels and exoproteolytic activity were determined. (A, B) Extracellular HAI-1 concentrations were determined by UPLC (black squares). AI-2 was captured with the binding protein LuxP, and quantified by bioassay (gray triangles). In parallel, the CFU and the optical density (OD₆₀₀, black crosses) were determined. Closed symbols (A) indicate the extracellular concentrations of the autoinducers. Open symbols (B) indicate autoinducer concentrations normalized relative to the OD₆₀₀ value. The arrows (A) mark the time points chosen for transcriptional analysis (see Fig. 6). (C, D) The same samples were analyzed for bioluminescence level (light units, LU) and exoproteolytic activity (AU). Closed symbols (C) indicate bioluminescence levels (black diamonds) and exoproteolytic activity (gray circles) as absolute values; open symbols (D) are normalized to the corresponding optical density. All experiments were performed in triplicate and error bars indicate standard deviations of the mean.</p

The quorum sensing circuit in <i>Vibrio harveyi</i>.

<p>In <i>V. harveyi</i> the three autoinducers HAI-1, AI-2 and CAI-1 are synthesized by the synthases LuxM, LuxS and CqsA. The cognate hybrid sensor kinases LuxN, LuxQ together with LuxP, and CqsS detect each autoinducer and effectively measure their concentrations: the higher the autoinducer concentration, the lower is the autophosphorylation activity of the hybrid kinases. The phosphoryl groups are transferred via phosphorelay including the histidine phosphotransfer protein LuxU to the σ⁵⁴-dependent transcriptional activator LuxO. Phosphorylated LuxO in turn activates transcription of five regulatory sRNAs, four of which (Qrr1-4) are active. Together with the RNA chaperone Hfq, these sRNAs destabilize the transcript that codes for the master regulator LuxR. The LuxR content is further regulated by additional feedback regulation (see text for details). Autoinducers activate genes required for bioluminescence, biofilm formation and proteolysis and repress genes involved in type III secretion and siderophore production. Dashed lines indicate phosphotransfer reactions. <i>H</i> (histidine) and <i>D</i> (aspartate) denote the phosphorylation sites. <i>CM</i>, cytoplasmic membrane; <i>CP</i>, cytoplasm; <i>PP</i>, periplasm.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Exoproteolytic activity of <i>V. harveyi</i> mutants.

<p>(A) Exoproteolytic activity was analyzed in cell-free culture fluids of the wild type BB120 (blue) in comparison to the autoinducer-independent, constitutively active mutant JAF78 (Δ<i>luxO</i>) (green), and the quorum sensing negative mutant JAF548 (<i>luxO-</i>D47E) (red). Furthermore, the exoproteolytic activity produced by the autoinducer synthase mutant MM77 (<i>luxM</i>::Tn<i>5 luxS</i>::Cm^r) in the absence (black) or in the presence of AI-2 (gray) or HAI-1 and AI-2 (each 10 μM) (light gray) was determined. Culture fluids were obtained from cells grown to the stationary phase. All experiments were performed in triplicate, and error bars indicate standard deviations of the mean. To classify the type of exoprotease detected, the metalloprotease inhibitor ethylenediaminetetraacetic acid (EDTA, 5 mM) (white, striped to the right) or the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 1 mM) (white) was added to the activity assay. (B) Time courses of the exoproteolytic activity of growing cells of strains BB120 (wild type, blue circles), JAF78 (autoinducer-independent, constitutively active mutant, green squares), and BB120 in the presence of synthetic HAI-1 (10 μM), which was added at time point 0 (dark gray triangles). All experiments were performed in triplicate, and error bars indicate standard deviations of the mean.</p

LAI-1 reverses Icm/Dot-dependent inhibition of migration by <i>L</i>. <i>pneumophila</i>.

<p>(A) <i>D</i>. <i>discoideum</i> Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) <i>D</i>. <i>discoideum</i> Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005307#ppat.1005307.s007" target="_blank">S7 Fig</a>)) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001).</p