Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production

Adenosine triphosphate (ATP), as a universal energy currency, takes a central role in many biochemical reactions with potential for the synthesis of numerous high-value products. However, the high cost of ATP limits industrial ATP-dependent enzyme-catalyzed reactions. Here, we investigated the effect of cell-surface display of phosphotransferase on ATP regeneration in recombinant Escherichia coli. By N-terminal fusion of the super-folder green fluorescent protein (sfGFP), we successfully displayed the phosphotransferase of Pseudomonas brassicacearum (PAP-Pb) on the surface of E. coli cells. The catalytic activity of sfGFP-PAP-Pb intact cells was 2.12 and 1.47 times higher than that of PAP-Pb intact cells, when the substrate was AMP and ADP, respectively. The conversion of ATP from AMP or ADP were up to 97.5% and 80.1% respectively when catalyzed by the surface-displayed enzyme at 37 °C for only 20 min. The whole-cell catalyst was very stable, and the enzyme activity of the whole cell was maintained above 40% after 40 rounds of recovery. Under this condition, 49.01 mg/mL (96.66 mM) ATP was accumulated for multi-rounds reaction. This ATP regeneration system has the characteristics of low cost, long lifetime, flexible compatibility, and great robustness.

A whole-cell recording database of neuromodulatory action in the adult neocortex

Background: The recent release of two large intracellular electrophysiological databases now allows high-dimensional systematic analysis of mechanisms of information processing in the neocortex. Here, to complement these efforts, we introduce a freely and publicly available database that provides a comparative insight into the role of various neuromodulatory transmitters in controlling neural information processing. Findings: A database of in vitro whole-cell patch-clamp recordings from primary somatosensory and motor cortices (layers 2/3) of the adult mice (2-15 months old) from both sexes is introduced. A total of 464 current-clamp experiments from identified excitatory and inhibitory neurons are provided. Experiments include recordings with (i) Step-and-Hold protocol during which the current was transiently held at 10 steps, gradually increasing in amplitude, (ii) 'Frozen Noise' injections that model the amplitude and time-varying nature of synaptic inputs to a neuron in biological networks. All experiments follow a within neuron across drug design which includes a vehicle control and a modulation of one of the following targets in the same neuron: dopamine and its receptors D1R, D2R, serotonin 5HT1f receptor, norepinephrine Alpha1, and acetylcholine M1 receptors. Conclusions: This dataset is the first to provide a systematic and comparative insight into the role of the selected neuromodulators in controlling cellular excitability. The data will help to mechanistically address how bottom-up information processing can be modulated, providing a reference for studying neural coding characteristics and revealing the contribution of neuromodulation to information processing.

Low error estimation of half-cell thermodynamic parameters from whole-cell Li-ion battery experiments: Physics-based model formulation, experimental demonstration, and an open software tool

Low C-rate charge and discharge experiments, plus complementary differential voltage or differential capacity analysis, are among the most common battery characterization methods. Here, we adapt the multi-species, multi-reaction (MSMR) half-cell thermodynamic model to low C-rate cycling of whole-cell Li-ion batteries. MSMR models for the anode and cathode are coupled through whole-cell charge balances and cell-cycling voltage constraint equations, forming the basis for model-based estimation of MSMR half-cell parameters from whole-cell experimental data. Emergent properties of the whole-cell, like slippage of the anode and cathode lithiation windows, are also computed as cells cycle and degrade. A sequential least-square optimization scheme is used for parameter estimation from low-C cycling data of Samsung 18650 NMC|C cells. Low-error fits of the open-circuit cell voltage (e.g., under 5 mV mean absolute error for charge or discharge curves) and differential voltage curves for fresh and aged cells are achieved. We explore the features (and limitations) of using literature reference values for the MSMR half-cell thermodynamic parameters (reducing our whole-cell formulation to a 1-degree-of-freedom fit) and demonstrate the benefits of expanding the degrees of freedom by letting the MSMR parameters be tailored to the cell under test, within a constrained neighborhood of the half-cell reference values. Bootstrap analysis is performed on each dataset to show the robustness of our fitting to experimental noise and data sampling over the course of 600 cell cycles. The results show which specific MSMR insertion reactions are most responsible for capacity loss in each half-cell and the collective interactions that lead to whole-cell slippage and changes in useable capacity. Open-source software is made available to easily extend this model-based analysis to other labs and battery chemistries.

Metabolomics-Guided Analysis of the Biocatalytic Conversion of Sclareol to Ambradiol by Hyphozyma roseoniger

The biocatalytic conversion of sclareol to ambradiol, a valuable component in the fragrance industry, using whole-cell biotransformation by the dimorphic yeast Hyphozyma roseoniger, was investigated using metabolomics tools. An integrated approach was used to identify and quantify the participating intermediates in this bioconversion using both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to mass spectrometry (LC–MS). This study entailed growth stage-dependent analysis of H. roseoniger suspensions grown in batch culture over a 14-day period, beginning with a three-day induction period using 20 mg/200 mL sclareol, followed by a further 1 g/200 mL sclareol dose to enable ambradiol production. The progress of the bioconversion and the resulting dynamic changes to the metabolome were monitored using NMR analysis and semi-targeted LC–MS metabolomics. This outlined the molecular conversions occurring within the matrix and no novel intermediates participating in the sclareol to ambradiol conversion could be identified. This study presents new findings about the transformative capabilities of H. roseoniger as a whole cell biocatalyst, highlighting its potential utility in similar applications.

Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.