Biosensors have become powerful tools for real-time monitoring of specific small molecules and precise control of gene expression in biological systems. High-throughput sensors for 1, 4-butanediamine biosynthesis can greatly improve the screening efficiency of high-yielding 1, 4-butanediamine strains. However, the strategies for adapting the characteristics of biosensors are still rarely studied, which limits the applicability of 1, 4-butanediamine biosensors. In this paper, we propose the development of a 1, 4-butanediamine biosensor based on the transcriptional regulator PuuR, whose homologous operator puuO is installed in the constitutive promoter P_gapA of Escherichia coli to control the expression of the downstream superfolder green fluorescent protein (sfGFP) as the reporter protein. Finally, the biosensor showed a stable linear relationship between the GFP/OD₆₀₀ value and the concentration of 1, 4-butanediamine when the concentration of 1, 4-butanediamine was 0-50 mmol/L. The promoters with different strengths in the E. coli genome were used to modify the 1, 4-butanediamine biosensor, and the functional properties of the PuuR-based 1, 4-butanediamine biosensor were explored and improved, which laid the groundwork for high-throughput screening of engineered strains highly producing 1, 4-butanediamine.
Biosensing Techniques/methods* ; Escherichia coli/metabolism* ; Promoter Regions, Genetic/genetics* ; Green Fluorescent Proteins/metabolism* ; Transcription Factors/genetics* ; Escherichia coli Proteins/genetics* ; Diamines/metabolism* ; Gene Expression Regulation, Bacterial

S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.
Schizosaccharomyces/genetics* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Cysteine/biosynthesis* ; Mixed Function Oxygenases/metabolism* ; Schizosaccharomyces pombe Proteins/metabolism* ; Oxygenases/metabolism* ; Kinetics

Objective:To determine the frequency and characteristics of AZF microdeletions of Y chromosome and karyotypic abnormalities among infertile male patients from southwest China. Methods:4 278 infertile male patients treated at West China Second University Hospital of Sichuan University from September 2018 to July 2023 were selected as the study subjects. Results of Y chromosome microdeletion detection and G-banded karyotyping analysis were retrospectively reviewed.Results:Clinical data of the patients were collected, which have included 2 048 patients with azoospermia, 1 536 patients with oligozoospermia, 310 patients with mild to moderate oligozoospermia, and 384 patients with infertility but normal sperm concentration. An abnormal karyotype was found in 213 (8.80%) of 2 421 patients who had undergone karyotyping analysis. The frequency of Y chromosome microdeletions was 9.86% (422/4 278), which had occurred in 10.4%, 13.28%, 0.97% and 0.52% of the cases with azoospermia, severe oligozoospermia, mild to moderate oligozoospermia, and infertility with normal sperm concentration, respectively.Conclusion:Y chromosome microdeletion detection and karyotyping analysis are crucial for assessing the cause of male infertility. Early diagnosis can facilitate the selection of reproductive methods.

Objective To perform genetic analysis in a family line of a pregnant couple with autosomal reces-sive non-syndromic deafness in order to identify its possible genetic etiology and provide prenatal diagnosis.Methods Whole-exome sequencing(WES)was used to analyze the genes of the proband,and Sanger sequencing was used to verify the suspected pathogenic loci.Prenatal genetic diagnosis was performed after amniotic fluid collection at 18 weeks of pregnancy.Results Autosomal recessive deafness type 3 related gene MYO15A c.10419_10423delCAGCT/c.10294_10308delCCTTGCATCCTTGCC compound heterozygous variant was found in the wife.A compound heterozygous variant of autosomal recessive deafness type 77-related gene LOXHD1:c.6388C＞T/ex-on 33-38 del.Maternal MYO15A c.10294_10308del CCTTGCATCCTTGCC heterozygous variant were detected in the husband and paternal LOXHD1 exon 33-38 del heterozygous variant were detected in the fetus.At the same time,the paternal CDH23 c.6693delT heterozygous mutation and the maternal PCDH15 c.5048_5051dupAGAA heterozygous mutation were detected in the fetus.These two heterozygous mutations lead to the possibility of the fe-tus suffering from ID/F Usher syndrome.Conclusion The deafness of the couple is caused by two different deaf gene mutations,and the probability of the fetus having the same deafness as the couple is very low.However,the fetus has a high possibility of having deafness caused by two gene mutations.Therefore,deafness caused by two gene mutations should be paid attention to in the prenatal diagnosis of families with both deaf parents.

ObjectiveTo explore the effect of Anmeidan on the sleep quality and serum levels of brain-derived neurotrophic factor （BDNF）， glial fibrillary acidic protein （GFAP）， and irisin in the patients with chronic insomnia. MethodA multicenter， randomized， double-blind， placebo-controlled clinical study was carried out， including 480 patients with chronic insomnia （deficiency syndrome） in Wuhan （Hubei）， Guangzhou （Guangdong）， and Lanzhou （Gansu）. They were randomized into an observation group and a control group at a ratio of 1∶1. The observation group was orally administered with Anmeidan granules at a dose of 11 g， 3 times per day， and the control group with Anmeidan simulant at a dose of 11 g， 3 times per day， Both groups of patients received sleep education after enrollment. After 4 weeks of medication， the Athens insomnia scale （AIS） scores， Spiegel scale scores， and serum levels of BDNF， GFAP， and irisin were compared between the two groups as well as between before and after treatment. ResultA total of 480 adult patients with chronic insomnia were enrolled in this study， with 64 patients falled off. Finally， the 415 patients were included in the analysis， including 213 patients in the observation group and 202 patients in the control group. There was no difference in age or sex between the two groups of patients. Compared with before treatment， the treatment in both groups decreased the AIS and Spiegel scores （P<0.01）. After treatment， the observation group had lower AIS and Spiegel scores than the control group （P<0.01）. The treatment in the observation group slightly lowered the level of BDNF， elevated the level of irisin （P<0.05）， and lowered the level of GFAP （P<0.05） in the serum. After treatment， the observation group showed higher level of irisin （P<0.05） and lower levels of BDNF and GFAP in the serum than the control group. ConclusionAnmeidan may improve the sleep quality of patients with chronic insomnia by elevating the irisin level and lowering the GFAP level in the serum.

Osteonecrosis of the femoral head （ONFH） is a painful and debilitating disease caused by impaired blood supply to the femoral head and cellular and tissue degeneration， leading to gradual destruction of the bone structure and progressive collapse of the femoral head. The main pathological mechanism of ONFH is the disruption of the balance between bone absorption and the reconstruction of new bone， resulting from microcirculation damage and decreased cellular tissue ability. This imbalance leads to biomechanical changes and accelerates the pathological progression of ONFH. In the early stages， clinical manifestations may not be obvious， mainly presenting as pain or discomfort in the hip or groin area， which can be relieved after rest. In the later stage of the disease， pain intensifies， and limb shortening， lower limb weakness， difficulty walking， or limping may occur. Currently， western medicine commonly uses osteogenic agents， anticoagulants， and artificial joint replacement for treatment， but there are also many issues such as prosthesis loosening and infection. Research has shown that traditional Chinese medicine （TCM） treatment of ONFH takes a holistic approach and employs multi-functional， multi-target， and multi-system Chinese medicine therapies， ensuring the safety and effectiveness of the treatment. The osteoprotegerin （OPG）/receptor activator of nuclear factor-κB （RANK）/RANK ligand （RANKL） signaling pathway plays a crucial role in maintaining the dynamic balance of bone remodeling. TCM treatments utilize this pathway to promote apoptosis of osteoclasts， reduce bone resorption， and accelerate bone formation， thereby playing an important role in the prevention and treatment of ONFH. This paper reviewed the role of OPG/RANK/RANKL signaling pathway and related cytokine expression in ONFH by reviewing relevant literature in China and abroad and research status of Chinese medicinal monomers， Chinese medicinal formulations， and combinations with physical therapy in increasing osteoblast secretion， promoting OPG expression， enhancing cytokine expression levels， and inhibiting osteoclast activity for the prevention and treatment of ONFH. This paper is expected to provide new ideas and directions for TCM in the prevention and treatment of ONFH.

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.
Gene Deletion ; Bacillus amyloliquefaciens/metabolism* ; Aminolevulinic Acid/metabolism* ; Heme/metabolism* ; Fermentation

Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter P_trc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter P_odhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.
Corynebacterium glutamicum/metabolism* ; Gene Library ; Metabolic Engineering ; Promoter Regions, Genetic/genetics*

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×10⁶ CFU/mL and 1.44×10⁶ CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.
Bacillus amyloliquefaciens/genetics* ; Bacterial Proteins ; Cell Count ; Endopeptidases/genetics* ; N-Acetylmuramoyl-L-alanine Amidase/genetics*