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In this letter, an analytical method for the beampattern synthesis of symmetric nonuniform array is proposed. This method consists of two steps. In the first step, it acquires a real symmetric excitation by the convex optimization method to attain a pencil beam. In the second step, it superposes the pencil beams pointing in different directions to synthesize the prescribed beampattern. Numerical results are provided to verify the effectiveness of the proposed method. This letter proposes an analytical beampattern synthesis scheme for the symmetric nonuniform array based on superposition principle.

Background D-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 ( Bs 168) for efficient synthesis of D-allulose under the acidic conditions without browning. Results First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 ( DS dpe) and Clostridium cellulolyticum H10 ( RC dpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs 168/pMA5- DS dpe- RC dpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs 168/pMA5- DS dpe and Bs 168/pMA5- RC dpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168 /pMA5-P spoVG - DS dpe-P srfA - RC dpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst. Conclusion In this work, the engineered recombinant strain Bs168 /pMA5-P spoVG - DS dpe-P srfA - RC dpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.

Fermentation plays a pivotal role in shaping the flavor and overall quality of Pu-erh tea, a microbially fermented dark tea. Here, we monitored physicochemical properties, chemical constituents, and microbial succession at 15 fermentation time points. Amplicon sequencing identified Staphylococcus, Bacillus, Kocuria, Aspergillus, Blastobotrys, Thermomyces, and Rasamsonia as dominant genera, with prokaryotic communities showing greater richness and diversity than eukaryotic ones. Beta diversity and clustering analyses revealed stable microbial structures during late fermentation stages. Non-targeted metabolomics detected 347 metabolites, including 56 significantly differential compounds enriched in caffeine metabolism and unsaturated fatty acid biosynthesis. Fermentation phases exhibited distinct metabolic patterns, with volatile aroma compounds (2-acetyl-1-pyrroline, 2,5-dimethylpyrazine) and health-beneficial fatty acids (linoleic acid, arachidonic acid) accumulating in later stages. OPLS-DA and KEGG PATHWAY analyses confirmed significant shifts in metabolite profiles relevant to flavor and biofunctionality. RDA revealed strong correlations between microbial taxa, environmental parameters, and representative metabolites. To functionally verify microbial contributions, 17 bacterial and 10 fungal strains were isolated. Six representative strains, mainly Bacillus and Aspergillus, exhibited high enzymatic activity on macromolecules, confirming their roles in polysaccharide and protein degradation. This integrative multi-omics investigation provides mechanistic insights into Pu-erh tea fermentation and offers a scientific basis for microbial community optimization in tea processing.

The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To improve the robustness of the enzyme at different fermentation strategies, mutation LreuglsAH105K was built by a computer-aided design, and the recombinant protein expression level, an essential parameter in industrial applications, was increased 5.61-fold with the synthetic biology strategy by improving the mRNA stability. Finally, the LreuglsAH105K functional expression box was contributed to Bacillus subtilis 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 ± 20.83 U mL−1, the highest production ever reported. When the immobilized cells were applied to high-salt dilute-state soy sauce brewing, the L-glutamate level was increased by 45.9%. This work provides insight into the salt-tolerant enzyme for improving the efficiency of industrial applications.

Bacillus subtilis is a gram-positive bacterium, a promising microorganism due to its strong extracellular protein secretion ability, non-toxic, and relatively mature industrial fermentation technology. However, cell autolysis during fermentation restricts the industrial application of B. subtilis. With the fast advancement of molecular biology and genetic engineering technology, various advanced procedures and gene editing tools have been used to successfully construct autolysis-resistant B. subtilis chassis cells to manufacture various biological products. This paper first analyses the causes of autolysis in B. subtilis from a mechanistic perspective and outlines various strategies to address autolysis in B. subtilis. Finally, potential strategies for solving the autolysis problem of B. subtilis are foreseen.

d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

The production of androst-4-ene-3,17-dione (AD) by the steroidal microbial cell factory requires transcription factors (TFs) to participate in metabolic regulation. However, microbial cell factory lacks effective TFs that can respond to AD in its metabolic pathway. Additionally, finding and obtaining natural TFs that specifically respond to AD is a complex and onerous task. In this study, we devised an artificial TF that responds to AD, termed AdT, based on structure-guided molecular dynamics (MD) simulation. According to MD analysis of the conformational changes of AdT after binding to AD, an LBD in which the N- and C-termini exhibited convergence tendencies was used as a microswitch to guide the assembly of a DNA-binding domain lexA, a linker (GGGGS)2, and a transcription activation domain B42 into an artificial TF. As a proof of design, a AD biosensor was designed and constructed in yeast on the basis of the ligand-binding domain (LBD) of hormone receptor. In addition, the transcription factor activity of AdT was increased by 1.44-fold for its variant F320Y. Overall, we created non-natural TF elements for AD microbial cell factory, and expected that the design TF strategy will be applied to running in parallel to the signaling machinery of the host cell. Schematic of activation model for transcription factor AdT. [Display omitted]

Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS. In this study, we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation. Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells, resulting in improved cell growth and increased protein solubility of the protein. The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS (∆N27 PlaS), highlighting their binding capabilities. Furthermore, in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 and ∆N27 PlaS, exhibiting high affinity. Molecular docking analysis provides insights into the hydrogen bond interactions between ∆N27 PlaS and PlaA1, identifying key amino acid residues crucial for their binding. Finally, the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test. Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1, offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.

Corynebacterium glutamicum is a Gram-positive bacterium (non-spore-forming) that has been wildly used for amino acid production. Due to its stable protein secretion, low extracellular hydrolase activity, and non-toxicity, the application field of C. glutamicum has been greatly expanded. Currently, gene editing technology based on synthetic biology has great potential for synthetic biology research and genetic modification in C. glutamicum because of its ability to efficiently regulate the physiological and metabolic networks of the strain. Therefore, we summarize the gene editing tools and strategies of C. glutamicum from the aspects of genetic modification and expression elements, and we also describe the effects of gene editing techniques on a variety of products such as amino acids and their derivatives, recombinant proteins, and functional sugars, which provide a certain theoretical basis for the research on the modification of C. glutamicum strains and industrial applications. Finally, we prospect the design and industrial application of C. glutamicum genetic modification from multiple perspectives based on gene editing techniques.

α-glucosidase is an essential enzyme for the production of isomaltooligosaccharides (IMOs). Allowing α-glucosidase to operate at higher temperatures (above 60 °C) has many advantages, including reducing the viscosity of the reaction solution, enhancing the catalytic reaction rate, and achieving continuous production of IMOs. In the present study, the thermal stability of α-glucosidase was significantly improved by constructing cyclized proteins. We screened a thermotolerant α-glucosidase (AGL) with high transglycosylation activity from Thermoanaerobacter ethanolicus JW200 and heterologously expressed it in Bacillus subtilis 168. After forming the cyclized α-glucosidase by different isopeptide bonds (SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, SdyTag/SdyCatcher, RIAD/RIDD), we determined the enzymatic properties of cyclized AGL. The optimal temperature of all cyclized AGL was increased by 5 °C, and their thermal stability was generally improved, with SpyTag-AGL-SpyCatcher having a 1.74-fold increase compared to the wild-type. The results of molecular dynamics simulations showed that the RMSF values of cyclized AGL decreased, indicating that the rigidity of the cyclized protein increased. This study provides an efficient method for improving the thermal stability of α-glucosidase.