Journal of biotechnology最新文献

Nickel-NTA affinity chromatography is the current standard method for purifying His-tagged recombinant proteins. However, this process involves repetitive tasks, can be time-consuming, and reduces protein yield. Here, we present a simple, fast, and handy method for purifying His-tagged proteins using free Ni²⁺. This approach allows the fractional precipitation of His-tagged proteins directly from E. coli cell lysates. We successfully applied this Ni²⁺-based method to purify three His₆-tagged recombinant proteins overexpressed in E. coli. We found that Ni²⁺ at a final concentration of as low as 1 mM precipitates the His-tagged proteins with near-complete specificity as confirmed by SDS-PAGE analysis. The Ni²⁺-precipitated proteins were dissolved by adding 10 % acetic acid and further purified by reverse-phase HPLC. The final yields were between 3.5 and 8.0 mg per 200 mL culture, similar to or even higher than purification using conventional Ni-NTA chromatography. The purified proteins exhibited natively folded characteristics, as assessed by CD, SLS, and DLS, and binding activity, as assessed by ELISA and BLI, demonstrating the method's potential in both small and large-scale settings.

In our previous study, the whole cells containing an aldo-keto reductase (yhdN) and glucose dehydrogenase (GDH) were constructed and applied in a stereoselective carbonyl reduction reaction to prepare (S)-NEMCA-HEPE, being a key chiral intermediate of (S)-Rivastigmine which is widely prescribed for the treatment of Alzheimer's disease. Although the conversion and enantiomeric excess (e.e.) could reach to 78.2 % and 99 %, respectively, ionic liquid as an additive was required to improve the permeability of cell membrane. To further simplify the reaction, the molecular docking and saturation mutagenesis technology were used here to obtain an activity-improved yhdN variant such as G19A. And then, both excellent conversion and e.e. of 99 % for (S)-NEMCA-HEPE could be achieved within 40 min by using only G19A-GDH whole cell as a catalyst without any additive. However, the use of the whole cells still faces the issues of poor operation stability and adverse application prospect. Subsequently, a hydrophobic "cell-in-shell" complex of G19A-GDH@O-Silica was constructed by using a silica nanocoated technology. The obtained G19A-GDH@O-Silica exhibited an excellent conversion towards the asymmetric carbonyl reduction, and a good tolerance in changing thermal, pH, and storage environmental. Giving 76.3 % of reaction conversion even after the 11th cycle of reuse, indicated that G19A-GDH@O-Silica also possessed ideal recyclability. The aim of this study is to provide a rapid, and cost-effective nanocoated whole-cell biocatalyst for efficient preparation of (S)-NEMCA-HEPE. The simplicity and robustness of the immobilization approach may become a powerful tool to utilize whole-cell catalysts towards organic catalysis.

Chondroitin sulfate (CS) is a structurally complex anionic polysaccharide widely used in medical, cosmetic and food applications. Enzymatic catalysis is an important strategy for synthesizing CS with uniform chain lengths and well-defined structures. However, the industrial application of glycosyltransferases is hindered by limitations such as low expression yields, poor stability, and challenges in reuse. We developed a mild and rapid one-step synthetic method for the efficient immobilization of chondroitin synthase (KfoC). The resulting KfoC@ZIF-90 composite exhibits high catalytic activity, thermal stability, and pH adaptability. Notably, KfoC@ZIF-90 exhibited 5-fold enhanced thermal stability at 40°C and retained 86 % relative activity at pH 10, while also maintaining 90 % activity in organic solvents, surpassing the performance of free KfoC. Molecular docking analysis revealed that the binding capability of encapsulated KfoC with substrate was stronger than that of free KfoC, thereby improving catalytic performance. Furthermore, KfoC@ZIF-90 can be easily separated from the reaction solution by centrifugation, simplifying product isolation and purification while enabling enzyme reuse. These attributes significantly enhance operability and reduce processing costs, making enzymatic CS synthesis more feasible for industrial applications.

The search for new non-animal textile materials has increased yearly as environmental awareness and veganism continue to spread, driving the development of greener fabrics. Concurrently, β-keratin, a fibrous, resistant, and insoluble protein shows great potential for producing innovative biomaterials. However, β-keratin is naturally abundant in animal feathers. Therefore, the recombinant production of β-keratin from Gallus gallus feathers was proposed using a strategy of parallel expression in different vectors. Statistical tools of experimental design were employed to improve the production of soluble biosynthetic keratin. It was shown that β-keratins fused to His₆MBP had better performance regarding soluble expression. In addition, the optimized regions for the values of induction temperature, induction time, and induction absorbance were obtained. As a result, a yield of 185.3 ± 1.4 mg/L of soluble His₆MBP-Chr2.FK4 was achieved, representing the highest yield reported to date.

Melanin with antioxidant and antibacterial properties can be used in food, cosmetics, biotechnology, and other fields, but its insolubility become a main challenge hindering for its application. In this study, water-soluble melanin produced by the novel species Streptomyces vilmorinianum YP1 was characterized using scanning electron microscopy (SEM), UVvisible spectroscopy (with an absorption peak at 220 nm), and Fourier transform infrared (FTIR) spectroscopy. The glycosyltransferase gene ORF14 was knocked out, which improved the production of water-soluble melanin by inhibiting competitive pathway. In order to further enhance production of melanin, PlackettBurman and response surface methodology statistical design was employed to screen for key factors and determine the optimal combination. The maximum melanin production (4.00 g/L) was obtained under the conditions: amylodextrine concentration of 40 g/L, soya peptone concentration of 7 g/L, tryptone concentration of 5 g/L, NaCl concentration of 5.4 g/L, pH of 6.7 and temperature of 36 °C for 180 h. The physicochemical properties and bioactivity of melanin were further investigated, revealing that melanin had a good stability across a pH range of 4-12, antioxidant (with a survival rate of over 85 %), and resistance to reducing agents (with a survival rate of over 99 %). The results underscored that S. vilmorinianum YP1 is a promising candidate for water-soluble melanin production.

Efficient methods and universal DNA elements are eagerly required for the expression of proteins and the production of target chemicals in synthetic biology and metabolic engineering. This paper develops a customized-design approach by utilizing the host-independent T7 expression system (HITES), which facilitates the rational design and rapid construction of T7 expression systems. Firstly, the E_iL (Upper-limit value of initial enzyme activity) value is discovered to play a pivotal factor in the successful construction of the T7 expression system, different host strains exhibit varying E_iL values, and this study presents a method to measure the E_iL values. Secondly, E. coli DH5α is chosen as the host strain, and it demonstrates that various strategies to modulate T7 RNA polymerase activity can efficiently construct the HITES T7 expression system in E. coli DH5α under the guidance of E_iL. Lastly, the customized-design of HITES enables the efficient expression of sfGFP and D-MIase proteins across 13 host strains, guided by E_iL values. This customized-design method of HITES offers a streamlined pathway for T7 system construction across a broad range of hosts and serves as an enabling tool for synthetic biology, metabolic engineering, and enzyme engineering.

Efficient recombinant protein production requires mammalian stable cell lines or often relies on inefficient transfection processes. Baculoviral transduction of mammalian cells (BacMam) offers cost-effective and robust gene transfer and straightforward scalability. The advantages over conventional approaches are, no need of high biosafety level laboratories, efficient transduction of various cell types and transfer of large transgenes into host cells. In our study, we aim to develop a high expression cassette to increase yields of baculoviral transduction. The establishment follows a sequential approach by first identifying the strongest promoter, followed by intron and WPRE sequences as enhancer elements for transcription and translation. The resulting REMBAC-cassette was compared to conventional transfection in suspension and adherent cells. Irrespective of the cell line, transduction reached nearly 100 % efficiency and led to almost 10-fold increases of gene expression levels. We confirmed these results in larger scale with batch and fed-batch cultivations. Finally, expression of different soluble proteins with high degrees of complexity confirmed the versatility of our established cassette. Overall, the REMBAC-cassette incorporated into the BacMam platform is a manifold tool offering advantages over standard transfection, in the scalability, efficiency and gene expression, which results in higher yields, shorter cultivation times and consequently cost-effective production processes.

Aromatic amino acids and their derivatives are high value chemicals widely used in food, pharmaceutical and feed industries. Current preparation methods for aromatic amino acid products are fraught with limitations. In this study, the efficient biosynthesis of aromatic amino acid compound tyrosol was investigated by epigenetic modification-based regulation and optimization of the biosynthetic pathway of aromatic amino acids. The production of tyrosol was significantly improved by the overexpression of m⁶A modification writer Ime4 and reader Pho92, and the positive regulator Gcr2. Introduction of Bbxfpk and deletion of Gpp1 further improved tyrosol production. Then the feedback inhibition of the shikimate pathway was relieved by the mutants Aro4^K229L and Aro7^G141S. The final tyrosol producing engineered strain was constructed by the deletion of PHA2, replacement of the native promoter of ARO10 with the strong promoter PGK1p, and introduction of tyrosine decarboxylase PcAAS. In the background of m⁶A modification regulation, this strain ultimately produced 954.69 ± 43.72 mg/L of tyrosol, promoted by 61.7-fold in shake-flask fermentation.

We demonstrate the proof of concept of increasing the bioavailability of carbon substrates, derived from plastic waste, for their conversion to the biodegradable polymer polyhydroxyalkanoate [PHA] by bacteria and test various approaches to PHA accumulation through batch, fed batch and continuous culture. Styrene, ethylbenzene, and toluene are produced from the pyrolysis of mixed plastic waste (Kaminsky, 2021; Miandad et al., 2017), but they are volatile and poorly soluble in water making them difficult to work with in aqueous fermentation systems. By chemically converting these aromatic compounds to benzoic acid, and subsequently to its sodium salt, we increased the solubility and reduced the volatility of the substrate supplied to Pseudomonas putida CA-3 to accumulate polyhydroxyalkanoates. 1 L scale batch, fed batch, and continuous fermentations were carried out; the fed batch fermentation resulted in the maximum volumetric PHA productivity of 61.67 ± 7.34 mg L^-1 h^-1; while batch and continuous, at a dilution rate, d = 0.2 h^-1, fermentations resulted in 13.30 ± 0.01 and 4.06 ± 0.01 mg L^-1 h^-1 of PHA respectively.

Efficiently managing agricultural waste while innovating to derive value-added products is a significant challenge in the 21st century. In recent decades, these by-products have been increasingly explored as alternative sources for materials such as biosilica. Biosilica is renowned for its high surface area, biocompatibility, chemical stability, and modifiable surface, which makes it suitable for various applications. Additionally, the biomineralization process-biosilicification-in living organisms like diatoms offers an eco-friendly pathway for silica production. Despite the potential applications of biosilica, research on its use in sensor technology remains limited. This review aims to address this gap by covering the primary methodologies for extracting silica from biomass, discussing key techniques for its characterization, and highlighting its potential for functionalization in diverse applications. Special emphasis is given to the utility of diatom-derived biosilicas in developing sensors for detecting gaseous molecules and biomolecules.