N-Terminal Sequences of Signal Peptides Assuming Critical Roles in Expression of Heterologous Proteins in Bacillus subtilis.

The N-terminal sequences of proteins and their corresponding encoding sequences may play crucial roles in the heterologous expression. In this study, the secretory expression of alkaline pectin lyase APL in B. subtilis was investigated to explore the effects of the N-terminal 5-7 amino acid sequences of different signal peptides on the protein expression and secretion. It was identified for the first time that the first five amino acid sequences of the N-terminal of the signal peptide (SP-LipA) from Bacillus subtilis lipase A play an important role in promoting the expression of APL. Furthermore, it was revealed that SP-LipA resulted in higher secretory expression compared to other signal peptides in this study primarily due to its encoding of N-terminal amino acids with relatively higher transcription levels and its efficient secretion capacity. Based on this foundation, the recombinant strain constructed in this work achieved a new record for the highest extracellular yields of APL in B. subtilis, reaching 12,295 U/mL, which was 1.9-times higher than that expressed in the recombinant Escherichia coli strain previously reported. The novel theories uncovered in this study are expected to play significant roles in enhancing the expression of foreign proteins both inside and outside of cells.

Extracellular Overexpression of a Neutral Pullulanase in Bacillus subtilis through Multiple Copy Genome Integration and Atypical Secretion Pathway Enhancement.

Neutral pullulanases, having a good application prospect in trehalose production, showed a limited expression level. In order to address this issue, two approaches were utilized to enhance the yield of a new neutral pullulanase variant (PulA3E) in B. subtilis. One involved using multiple copies of genome integration to increase its expression level and fermentation stability. The other focused on enhancing the PulA-type atypical secretion pathway to further improve the secretory expression of PulA3E. Several strains with different numbers of genome integrations, ranging from one to four copies, were constructed. The four-copy genome integration strain PD showed the highest extracellular pullulanase activity. Additionally, the integration sites ytxE, ytrF, and trpP were selected based on their ability to enhance the PulA-type atypical secretion pathway. Furthermore, overexpressing the predicated regulatory genes comEA and yvbW of the PulA-type atypical secretion pathway in PD further improved its extracellular expression. Three-liter fermenter scale-up production of PD and PD-ARY yielded extracellular pullulanase activity of 1767.1 U/mL at 54 h and 2465.1 U/mL at 78 h, respectively. Finally, supplementing PulA3E with 40 U/g maltodextrin in the multi-enzyme catalyzed system resulted in the highest trehalose production of 166 g/L and the substrate conversion rate of 83%, indicating its potential for industrial application.

Insights into the impact of complex phosphates on acid-induced milk fan gel properties: Texture, rheological, microstructure, and molecular forces.

Milk fan cheese, a type of stretched -cheese, presents challenges in its stretch-forming. This study investigated the impacts of complex phosphates (sodium tripolyphosphate and sodium dihydrogen phosphate, STPP-DSP) on the gelling properties of acid-induced milk fan gel and the mechanisms contributing to its stretch-forming. The treatment of milk fan gel with STPP-DSP resulted in improved functional and textural properties compared with the control group. In particular, drawing length increased significantly from 69.67 nm to 80.33 nm, and adhesiveness increased from 1737.89 g/mm to 1969.79 g/mm. The addition of STPP-DSP also led to increased viscosity, elastic modulus (G'), and viscous modulus (G"). Microstructural analysis revealed the formation of a fibrous structure within the gel after STPP-DSP treatment, facilitating uniform embedding of fat globules and emulsification. Structural analysis showed that the addition of STPP-DSP increased ß-fold and decreased random coiling of the gel, facilitating the unfolding of protein structures. Additionally, UV absorption spectroscopy and excitation-emission matrix spectroscopy results indicated the formation of a chelate between STPP-DSP and milk fan gel, increasing protein-protein molecular interactions. Evidence from differential scanning calorimetry and x-ray diffraction demonstrated the formation of sodium caseinate chelate. Fourier transform infrared spectroscopy and zeta potential analysis revealed that the sodium caseinate chelate formed through hydrophobicity, hydrogen bonding, and electrostatic forces. These findings provided theoretical insights into how phosphates can improve the stretch-forming of milk fan gel, facilitating the application of phosphate additives in stretched -cheese processing.

Long non-coding RNA LOXL1-AS1: a potential biomarker and therapeutic target in human malignant tumors.

Long non-coding RNAs (lncRNAs) are transcripts that contain more than 200 nucleotides. Despite their inability to code proteins, multiple studies have identified their important role in human cancer through different mechanisms. LncRNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1), a newly discovered lncRNA located on human chromosome 15q24.1, has recently been shown to be involved in the occurrence and progression of various malignancies, such as colorectal cancer, gastric cancer, hepatocellular carcinoma, prostate cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, breast cancer, glioma, thymic carcinoma, pancreatic carcinoma. LOXL1-AS1 acts as competitive endogenous RNA (ceRNA) and via sponging various miRNAs, including miR-374b-5p, miR-21, miR-423-5p, miR-589-5p, miR-28-5p, miR-324-3p, miR-708-5p, miR-143-3p, miR-18b-5p, miR-761, miR-525-5p, miR-541-3p, miR-let-7a-5p, miR-3128, miR-3614-5p, miR-377-3p and miR-1224-5p to promote tumor cell proliferation, invasion, migration, apoptosis, cell cycle, and epithelial-mesenchymal transformation (EMT). In addition, LOXL1-AS1 is involved in the regulation of P13K/AKT and MAPK signaling pathways. This article reviews the current understanding of the biological function and clinical significance of LOXL1-AS1 in human cancers. These findings suggest that LOXL1-AS1 may be both a reliable biomarker and a potential therapeutic target for cancers.

Enhancing Poly-γ-glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the γ-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 ± 0.69 g/L γ-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry. RESULTS: A total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase. CONCLUSION: The Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.