Identification and engineering efflux transporters for improved L-homoserine production in Escherichia coli.

AIMS: This study aimed to functionally identify the potential L-homoserine transporters in Escherichia coli, and to generate the promising beneficial mutants by targeted directed evolution for improving the robustness and efficiency of microbial cell factories. METHODS AND RESULTS: By constructing a series of gene deletion and overexpression strains, L-homoserine tolerance assays revealed that RhtA was an efficient and major L-homoserine exporter in E. coli, whereas RhtB and RhtC exhibited relatively weak transport activities for L-homoserine. Real-time RT-PCR analysis suggested that the expression levels of these three target mRNAs were generally variably enhanced when cells were subjected to L-homoserine stress. Based on in vivo continuous directed evolution and growth-couple selections, three beneficial mutations of RhtA exporter (A22V, P119L, and T235I) with clearly increased tolerance against L-homoserine stress were quickly obtained after two rounds of mutagenesis-selection cycles. L-homoserine export assay revealed that the RhtA mutants exhibited different degrees of improvement in L-homoserine export capacity. Further studies suggested that a combination of these beneficial sites led to synergistic effects on conferring L-homoserine-resistance phenotypes. Moreover, the introduction of RhtA beneficial mutants into the L-homoserine-producing strains could facilitate increased amounts of L-homoserine in the shake-flask fermentation. CONCLUSIONS: In this study, we provided further evidence that RhtA serves as a major L-homoserine exporter in E. coli, and obtained several RhtA beneficial mutants, including A22V, P119L, and T235I that contributed to improving the L-homoserine resistance phenotypes and the production efficiency in microbial chassis.

Recent advances of pH homeostasis mechanisms in Corynebacterium glutamicum.

Corynebacterium glutamicum is generally regarded as a safe microorganism, and widely used in the large-scale production of various amino acids and organic acids, such as L-glutamate, L-lysine and succinic acid. During the process of industrial fermentation, C. glutamicum is usually exposed to varying environmental stresses, such as variations in pH, salinity, temperature, and osmolality. Among them, pH fluctuations are regarded as one of the most frequent environmental stresses in microbial fermentation. In this review, we summarize the current knowledge of pH homeostasis mechanisms adopted by C. glutamicum for coping with low acidic pH and high alkaline pH stresses. Facing with low pH environments, C. glutamicum develops a variety of strategies to maintain intracellular pH homeostasis, such as lowering intracellular reactive oxygen species, the improvement of potassium transport, the regulation of mycothiol-related pathways, as well as the repression of sulfur assimilation. While during alkaline pH stresses, the Mrp-type Na+/H+ antiporters are shown to play a dominant role in conferring C. glutamicum cells resistance to alkaline pH. Furthermore, we also discuss the general strategies and prospects on metabolic engineering of C. glutamicum to improve alkaline or acid resistance.

Asp141 and the Hydrogen-Bond Chain Asp141-Asn109-Asp33 Are Respectively Essential for GT80 Sialyltransferase Activity and Structural Stability.

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. In this study, the activity of a putative sialyltransferase (Pm0160) harboring an inherent mutation D141Y in the conserved DDG motif, which has been identified in GT52 and GT80 families, was restored by reverse mutation. More interestingly, a hydrogen-bond chain was found to form between three conserved residues (Asp141, Asn109, and Asp33) of GT80 sialyltransferases based on recently determined crystal structures. Our mutagenesis experiments demonstrated that the hydrogen-bond chain connecting the general base Asp141 with Nß4, Nß1, and Nα1 plays an essential role in maintaining protein structural stability other than keeping the general base Asp141 in a productive orientation for sialic acid transfer.

Triterpenoids and Flavonoids from the Leaves of Astragalus membranaceus and Their Inhibitory Effects on Nitric Oxide Production.

Four new cycloartane triterpenes, named huangqiyegenins V and VI and huangqiyenins K and L (1-4, resp.), together with nine known triterpenoids, 5-13, and eight flavonoids, 14-21, were isolated from a 70%-EtOH extract of Astragalus membranaceus leaves. The structures of the new compounds were elucidated by detailed spectroscopic analyses, and the compounds were identified as (9ß,11α,16ß,20R,24S)-11,16,25-trihydroxy-20,24-epoxy-9,19-cyclolanostane-3,6-dione (1), (9ß,16ß,24S)-16,24,25-trihydroxy-9,19-cyclolanostane-3,6-dione (2), (3ß,6α,9ß,16ß,20R,24R)-16,25-dihydroxy-3-(ß-D-xylopyranosyloxy)-20,24-epoxy-9,19-cyclolanostan-6-yl acetate (3), and (3ß,6α,9ß,16ß,24E)-26-(ß-D-glucopyranosyloxy)-16-hydroxy-3-(ß-D-xylopyranosyloxy)-9,19-cyclolanost-24-en-6-yl acetate (4). All isolated compounds were evaluated for their inhibitory activities against LPS-induced NO production in RAW264.7 macrophage cells. Compounds 1-3, 14, 15, and 18 exhibited strong inhibition on LPS-induced NO release by macrophages with IC50 values of 14.4-27.1 µM.

High-Efficiency Electromagnetic Interference Shielding from Highly Aligned MXene Porous Composites via Controlled Directional Freezing.

Lightweight porous composite materials (PCMs) with outstanding electromagnetic interference (EMI) shielding performances are ideal for aerospace, artificial intelligence, military, and other fields. Herein, a three-dimensional Ti3C2Tx MXene/sodium alginate (SA)/carbon nanotubes (CNTs) (MSC) PCMs was prepared by a controlled directional freezing process. This method constructs a directionally ordered porous structure, which can make the incident electromagnetic waves reflect and scattered several times in the PCMs. The introduction of CNTs into the MSC PCMs can form three-dimensional conductive networks with MXene, thus improving the conductivity and further improving the electromagnetic shielding performance. Furthermore, the SA with abundant hydrogen bonding can strengthen the interlayer interaction between MXene and CNTs. Profiting from the controlled directional freezing and highly aligned porous structure, the MSC PCMs with 75 wt % CNTs exhibit ultrahigh conductivity of 1630 S m-1, an ultrahigh EMI shielding effectiveness of 48.0 dB in X-band for electromagnetic waves incident perpendicular to the hole growth direction, and compressive strength of 72.3 kPa. The as-prepared MSC PCMs show excellent EMI shielding and mechanical properties and have significant applications in the preparation of an entirely novel type of EMI shielding materials with an absorption-based mechanism.

Directed evolution of an EamB transporter for improved L-cysteine tolerance and production in Escherichia coli.

Engineering transporter for improved efflux efficiency provides a powerful strategy to alleviate product-associated cytotoxicity and promote microbial production of desired compounds. However, the scarcity of efficient transporters limits its application in the construction of microbial cell factories. Here, we sought to improve the transport performance of the EamB transporter, a L-cysteine exporter from Escherichia coli. A total of four EamB variants (A31V, I83M, G156A, and N157M) were firstly obtained by random mutagenesis and screening, and two other improved mutant (G156S and N157S) were also identified by site-specific saturation mutagenesis. The transport assays revealed that the G156S and N157S mutants had increased L-cysteine export capacity relative to the native EamB transporter. A combinatorial mutagenesis approach was used to generate the best mutant G156S/N157S, which conferred cells optimal resistance to L-cysteine and highest yields of L-cysteine in shake flask fermentation. Taken together, our results offer several EamB mutants with improved efflux properties, highlighting the potential of these exporters in L-cysteine fermentative production.

Coupling sprinkler freshwater irrigation with vegetable species selection as a sustainable approach for agricultural production in farmlands with a history of 50-year wastewater irrigation.

Soil contamination and crop risks of heavy metal(loid)s are widely reported after the long-term irrigation of treated wastewater, causing an adverse influence on agricultural sustainability. Here, we collected soils after 50 years of wastewater irrigation to cultivate cabbage (Brassica pekinensis L.), rape (Brassica chinensis L.), carrots (Daucus carota L.), and potatoes (Solanum tuberosum L.), using surface and sprinkler irrigation with freshwater and wastewater. In general, we found the statistically insignificant influence of short-term freshwater irrigation on the soil and vegetable metal(loid) concentrations. Most of the vegetables had potential adverse health risks with the relatively lower risks in carrots and potatoes, and most of the risks were contributed by As and Cd. Nevertheless, we observed negligible health risks for all studied metal(loid)s in potatoes under the freshwater irrigations. Besides, compared to wastewater irrigations, freshwater irrigations produced lower Cd health risks in all four vegetable species. Sprinkler irrigation with freshwater was a favorable approach for reducing the uptake of metal(loid)s from soils and the metal(loid) concentrations in aboveground parts. Our study highlights the possibility of reducing vegetable metal(loid) risks in contaminated farmlands via a combined approach of coupling the short-term decrease in their levels in irrigation water with vegetable species selection.

[Expression, purification and characterization of catalase from Corynebacterium glutamicum].

Catalase catalyzes the decomposition of H2O2 to H2O and O2, and has a wide range of industrial applications. However, most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop alkaline catalase. In this study, a catalase from Corynebacterium glutamicum was expressed in Escherichia coli, and the expression conditions were optimized. The recombinant catalase was purified by Ni-chelating affinity chromatography, and the recombinant enzyme was characterized. The optimal conditions of producing the recombinant catalase were: an IPTG concentration of 0.2 mmol/L, a culturing temperature of 25 °C and a culturing time of 11 h. The purified catalase had a specific activity of 55 266 U/mg, and it had a high activity in the pH range of 4.0 to11.5, with the highest activity at pH 11.0. When treated in pH 11.0 for 3 h, the enzyme retained 93% of its activity, indicating that the enzyme was qualified with a favorable stability under high-alkaline condition. The recombinant catalase had maximal activity at 30 °C, and showed a satisfactory thermal stability at a range of 25 °C to 50 °C. The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/(minmg), respectively. Besides, different inhibitors, such as sodium dodecyl sulfate (SDS), urea, NaN2, ß-mercaptoethanol, and EDTA had different degrees of inhibition on enzyme activity. The catalase from C. glutamicum shows high catalytic efficiency and high alkaline stability, suggesting its potential utilization in industrial production.

Engineering of L-glutamate oxidase as the whole-cell biocatalyst for the improvement of α-ketoglutarate production.

L-glutamate oxidase (LGOX) catalyzes the oxidative deamination of l-glutamate to α-ketoglutarate (α-KG) with the formation of ammonia and hydrogen peroxide. Consequently, identifying a novel LGOX with high enzymatic activity is a prime target for industrial biotechnology. In this study, error-prone PCR mutagenesis of Streptomyces mobaraensis LGOX followed by high-throughput screening was performed to yield four single point mutants with improved enzymatic activity, termed F94L, S280T, I282M and H533R. Moreover, site-saturation mutagenesis at these four residues was employed, yielding two additionally improved mutants, termed I282L and H533L. Subsequently, we employed combinatorial mutagenesis of two, three and four point mutants, and the best mutant S280TH533L showed 90 % higher enzymatic activity than the wild-type control. The data also showed that the presence of these point mutations greatly enhanced enzymatic activity, but did not alter its optimum temperature and pH. Furthermore, the S280TH533L mutant had the maximal velocity (Vmax) of 231.3 µmol/mg/min and the Michaelis-Menten constant (KM) of 2.7 mM, which were the highest Vmax and lowest KM values of LGOX reported so far. Finally, we developed a whole-cell biocatalyst for α-KG production by co-expression of both S280TH533L mutant and KatE catalase. Randomized ribosome binding site (RBS) sequences were introduced to generate vectors with varying expression levels of S280TH533L and KatE, and two optimized co-expression strains were obtained after screening. The α-KG production reached a maximum titer of 181.9 g/L after 12 h conversation using the optimized whole-cell biocatalyst, with a molar conversion rate of substrate higher than 86.3 % in the absence of exogenous catalase, while the molar conversion rate of substrate using the wild-type biocatalyst was less than 30 %. Taken together, these data suggest that the engineering of LGOX has great potentials to enhance the industrial production of α-KG.

A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs.

Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70 °C and high thermostability over a broad range of temperatures (20-80 °C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30 °C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55 °C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs.

[Ln2(C2O4)2(pyzc)2(H2O)2]n[Ln = Pr (1), Er (2)]: novel two-dimensional lanthanide coordination polymers with 2-pyrazinecarboxylate and oxalate.

We report for the first time hydrothermal synthesis of lanthanide-pyzcH polymers, [Ln2(C2O4)2(pyzc)2 (H2O)2]n [Ln = Pr (1), Er (2)], in which pyzcH was decomposed into C2O42- and thus caused novel two-dimensional hexagonal lattice networks. The magnetic property of polymer 1 has been studied by an approximate treatment being enlightened by McPherson et al. (Inorg. Chim. Acta 1988, 148, 265), leading to Delta = -4.3 cm-1, zJ' = -11.73 cm-1, and g = 0.79. Complex 2 displays an intense room-temperature, liquid-state luminescent emission.