The efficient production of L-glutamate is dependent on the product's rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on L-glutamate synthesis and transport in SCgGC7, a constitutive L-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of L-glutamate producers. Next, temperature-sensitive L-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for L-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of L-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the L-glutamate excretion and metabolic flux for both L-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of L-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high L-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for L-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.
Corynebacterium glutamicum/genetics* ; Glutamic Acid/biosynthesis* ; Fermentation ; Metabolic Engineering ; Bacterial Proteins/metabolism* ; Bioreactors/microbiology* ; Gene Knockout Techniques

The high content of sucrose and raffinose reduces the prebiotic value of soybean oligosaccharides. Fructan sucrases can catalyze the conversion of sucrose and raffinose to high-value products such as fructooligosaccharides and melibiose. To obtain a fructan sucrase that can efficiently convert soybean oligosaccharides, we first mined the fructan sucrase gene from microorganisms in the coastal areas of Xisha Islands and Bohai Bay and then characterized the enzymatic and catalytic properties of the enzyme. Finally, recombinant extracellular expression of this gene was carried out in Bacillus subtilis. The results showed that a novel fructan sucrase, BhLS 39, was mined from Bacillus halotolerans. With sucrose and raffinose as substrates, BhLS 39 showed the optimal temperatures of 50 ℃ and 55 ℃, optimal pH 5.5 for both, and K_cat/K_m ratio of 3.4 and 6.6 L/(mmol·s), respectively. When 400 g/L raffinose was used as the substrate, the melibiose conversion rate was 84.6% after 30 min treatment with 5 U BhLS 39. Furthermore, BhLS 39 catalyzed the conversion of sucrose to produce levan-type-fructooligosaccharide and levan. Then, the recombinant extracellular expression of BhLS 39 in B. subtilis was achieved. The co-expression of the intracellular chaperone DnaK and the extracellular chaperone PrsA increased the extracellular activity of the recombinant BhLS 39 by 5.2 folds to 17 U/mL compared with that of the control strain. BhLS 39 obtained in this study is conducive to improving the quality and economic benefits of soybean oligosaccharides. At the same time, the strategy used here to enhance the extracellular expression of BhLS 39 will also promote the efficient recombinant expression of other proteins in B. subtilis.
Oligosaccharides/metabolism* ; Glycine max/metabolism* ; Bacillus subtilis/metabolism* ; Sucrase/biosynthesis* ; Raffinose/metabolism* ; Fructans/metabolism* ; Sucrose/metabolism* ; Bacillus/genetics* ; Recombinant Proteins/biosynthesis* ; Bacterial Proteins/biosynthesis*

To screen and identify a chitosanase with high stability, we cloned the chitosanase gene from Bacillus atrophaeus with a high protease yield from the barren saline-alkali soil and expressed this gene in Escherichia coli. The expressed chitosanase of B. atrophaeus (BA-CSN) was purified by nickel-affinity column chromatography. The properties including optimal temperature, optimal pH, substrate specificity, and kinetic parameters of BA-CSN were characterized. The results showed that BA-CSN had the molecular weight of 31.13 kDa, the optimal temperature of 55 ℃, the optimal pH 5.5, and good stability at temperatures below 45 ℃ and pH 4.0-9.0. BA-CSN also had good stability within 4 h of pH 3.0 and 10.0, be activated by K⁺, Na⁺, Mn²⁺, Ca²⁺, Mg²⁺, and Co²⁺, (especially by Mn²⁺), and be inhibited by Fe³⁺, Cu²⁺, and Ag⁺. BA-CSN showcased the highest relative activity in the hydrolysis of colloidal chitosan, and it had good hydrolysis ability for colloidal chitin. Under the optimal catalytic conditions, BA-CSN demonstrated the Michaelis constant K_m and maximum reaction rate V_max of 9.94 mg/mL and 26.624 μmoL/(mL·min), respectively, for colloidal chitosan. In short, BA-CSN has strong tolerance to acids and alkali, possessing broad industrial application prospects.
Bacillus/genetics* ; Hydrogen-Ion Concentration ; Escherichia coli/metabolism* ; Glycoside Hydrolases/biosynthesis* ; Substrate Specificity ; Enzyme Stability ; Chitosan/metabolism* ; Temperature ; Kinetics ; Cloning, Molecular ; Bacterial Proteins/biosynthesis* ; Recombinant Proteins/genetics*

The Split-Cre system consists of two inactive polypeptides: NCre and CCre, which can be recombined into an active full-length Cre under certain conditions. This system is typically used with LoxP. To develop an efficient Split-Cre system, this study used Rma intein from Rhodothermus marinus to split Cre and screened out the split site S102 which could efficiently mediate the recombination of Cre in the "Traffic Light" reporter cell line. Moreover, the S102 Split-Cre system was delivered to mice by dual-adeno-associated virus (AAV), and it was demonstrated that the efficiency of the Rma intein-mediated S102 Split-Cre system was comparable to the full-length Cre in mice. This system lays a foundation for both basic and applied research on Split-Cre.
Inteins/genetics* ; Animals ; Integrases/biosynthesis* ; Mice ; Dependovirus/metabolism* ; Bacterial Proteins/genetics* ; Recombination, Genetic ; Humans

Phenylpyruvic acid (PPA) is used as a food and feed additive and has a wide range of applications in the pharmaceutical, chemical and other fields. At present, PPA is mainly produced by chemical synthesis. With the green transformation of the manufacturing industry, biotransformation will be a good alternative for PPA production. The L-amino acid deaminase (PmiLAAD) from Proteus mirabilis has been widely studied for the production of PPA. However, the low yield limits its industrial production. To further enhance the production of PPA and better meet industrial demands, a more efficient synthesis method for PPA was established. In this study, PmiLAAD was heterologously expressed in Escherichia coli. Subsequently, a colorimetric reaction method was established to screen the strains with high PPA production. The semi-rational design of PmiLAAD was carried out, and the obtained triple-site mutant V18 (V437I/S93C/E417A) showed a 35% increase in catalytic activity compared with the wild type. Meanwhile, the effect of N-terminal truncation on the catalytic activity of the V18 mutant was investigated. After the optimization of the whole-cell conditions for the obtained mutant V18-N7, fed-batch conversion was carried out in a 5-L fermenter, and 44.13 g/L of PPA was synthesized with a conversion rate of 88%, which showed certain potential for industrial application. This study lays foundation for the industrial production of phenylpyruvic acid and also offers insights into the biosynthesis of other chemicals.
Escherichia coli/metabolism* ; Proteus mirabilis/genetics* ; Phenylpyruvic Acids/metabolism* ; Protein Engineering/methods* ; Recombinant Proteins/biosynthesis* ; Bacterial Proteins/metabolism*

Bacillus is a class of spore-producing Gram-positive bacteria that produce a variety of antimicrobial substances with different structures and functions. The application of the antimicrobial substances produced by Bacillus can effectively inhibit the activity of harmful bacteria and fungi and promote the sustainable development of green agriculture. The antimicrobial substances produced by Bacillus mainly include proteins, lipopeptides, polyketones, and polypeptides. This paper reviews the synthesis gene clusters, synthesis pathways, structures, and mechanisms of various antimicrobial substances produced by Bacillus and discusses the challenges in the industrial application of these antimicrobial substances. Furthermore, this paper clarifies the future research and development focuses and prospects the application prospects, and provides comprehensive theoretical support for the in-depth research and wide application of the antimicrobial substances produced by Bacillus.
Bacillus/genetics* ; Anti-Infective Agents/metabolism* ; Bacterial Proteins/genetics* ; Antimicrobial Peptides/biosynthesis* ; Lipopeptides/biosynthesis*

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L HPO at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C (5 μm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 μg·mL. The intra- and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.
Bacterial Proteins ; genetics ; metabolism ; Biosynthetic Pathways ; Chromatography, Reverse-Phase ; methods ; Cyclohexenes ; analysis ; Hexosamines ; analysis ; biosynthesis ; Metabolic Engineering ; Streptomyces ; genetics ; metabolism

As a novel cofactor of oxidoreductase, pyrroloquinoline quinone (PQQ) has a great potential of application in medicine, food industries. In order to improve the efficiency of the PQQ production by Methylobacterium extorquens AM1, the strain was treated by atmospheric and room temperature plasma (ARTP). Positive mutants with changes in PQQ yield were obtained based on a high-throughput screening approach. After ARTP treatment, analysis data show that the positive mutation rate was 31.6%. Furthermore, we obtained an excellent positive mutant M. extorquens AM1 (E-F3) with the yield of 54.0 mg/L PQQ, which was approximately 3 times as much compared with that of the wild-type strain. The robust high-throughput screening method for mutagenesis by ARTP improves PQQ production. In addition, this method also provides a new strategy for further strain improvement.
Bacterial Proteins ; biosynthesis ; High-Throughput Screening Assays ; Methylobacterium extorquens ; enzymology ; genetics ; Mutagenesis ; PQQ Cofactor ; biosynthesis ; Plasma Gases ; Temperature

Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward β-phenylalanine (β-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of β-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to β-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-β-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.
Bacterial Proteins ; biosynthesis ; genetics ; Burkholderia ; enzymology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Transaminases ; biosynthesis ; genetics

PURPOSE: In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. MATERIALS AND METHODS: Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. RESULTS: H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. CONCLUSION: H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells.
Blotting, Western ; Caffeic Acids ; Cell Line, Tumor ; Cell Proliferation ; DNA, Bacterial/analysis/genetics ; DNA-Binding Proteins/*metabolism ; Epithelial Cells/*metabolism ; Gastric Mucosa/*metabolism/pathology ; Gastritis/pathology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/metabolism/pathology/physiopathology ; Helicobacter pylori/pathogenicity/physiology ; Humans ; NF-kappa B/antagonists & inhibitors/*biosynthesis/metabolism ; Peptide Fragments ; Phenylethyl Alcohol/analogs & derivatives ; Proto-Oncogene Proteins c-jun ; Repressor Proteins ; Transcription Factor AP-1/*biosynthesis ; Transcription Factors/*metabolism ; beta Catenin/*metabolism