SdrR, a LysR-type regulator, responds to the mycobacterial antioxidant defense.

Protection against oxidative stress is a vital defense mechanism for Mycobacterium tuberculosis within the host. However, few transcription factors that control bacterial antioxidant defense are known. Here, we present evidence that SdrR, encoded by the MSMEG_5712 (Ms5712) gene, functions as an oxidative stress response regulator in Mycobacterium smegmatis. SdrR recognizes an 11-bp motif sequence in the operon's upstream regulatory region and negatively regulates the expression of short-chain dehydrogenases/reductases (SDR). Overexpressing sdrR inhibited SDR expression, which rendered the strain oxidative more stress-sensitive. Conversely, sdrR knockout alleviates SDR repression, which increases its oxidative stress tolerance. Thus, SdrR responds to oxidative stress by negatively regulating sdr expression. Therefore, this study elucidated an underlying regulatory mechanism behind mycobacterial oxidative stress adaptation.

Preventive Effects of Ilex Cornuta Aqueous Extract on High-Fat Diet-Induced Fatty Liver of Mice.

Objective: To investigate the preventive effects of Ilex cornuta aqueous extract (ICAE) on high-fat diet (HFD)-induced fatty liver of mice and its mechanisms. Materials and Methods: Twenty-six male KM (Kunming) mice were divided into 3 groups, including the control group (n = 9), fed with normal diet; HFD group (n = 9), fed with HFD; ICAE + HFD group (n = 8), fed with HFD and administered with ICAE (3 g·kg-1·d-1) at the same time for 10 weeks. Body weight, liver weight, intra-abdominal and subcutaneous fat weight, serum triglyceride (TG), total cholesterol (TC), and blood glucose were determined to evaluate the preventive effects of ICAE on obesity. The average 24 h food consumption of the mice was monitored for 5 times in the 9th week of the experiment to investigate the effects of ICAE on food intake. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were assayed to observe the influences of HFD and ICAE on liver function. HE staining was adopted to observe the influence of ICAE on the morphology of adipose tissue and liver tissue. Hepatic TG and TC content assay and oil red O staining were used to evaluate the influences of ICAE on HFD-induced fatty liver, and the protein expression of peroxisome proliferator-activated receptors γ (PPARγ) and adipose differentiation-related protein (ADRP) in liver were examined by immunoblotting. Results: ICAE treatment significantly reduced the increase of body weight, intra-abdominal, and subcutaneous fat and liver weight induced by HFD (P < 0.001), but has no influence on food intake; ICAE treatment attenuated the elevation of serum TG, TC, and glucose, as well as serum ALT and AST (P < 0.01, P < 0.05, P < 0.001) and dramatically decreased the content of TG in liver (P < 0.01), but has no influence on hepatic TC content. HE staining and oil red O staining showed that ICAE significantly reduced HFD-induced white adipocyte hypertrophy and significantly inhibited lipid accumulation in liver. Immunoblotting showed that the protein levels of PPARγ and ADRP were significantly increased by HFD induction, which can be dramatically reduced by ICAE treatment (P < 0.05, P < 0.0001). Conclusion: ICAE has preventive effects on HFD-induced obesity and fatty liver in mice, exerted beneficial effects upon HFD-induced hepatic injury. The preventive effects of ICAE on fatty liver are concerned with the downregulation of PPARγ and ADRP protein expression in liver.

Design and optimization of E. coli artificial genetic circuits for detection of explosive composition 2,4-dinitrotoluene.

The detection of mine-based explosives poses a serious threat to the lives of deminers, and carcinogenic residues may cause severe environmental pollution. Whole-cell biosensors that can detect on-site in dangerous or inaccessible environments have great potential to replace conventional methods. Synthetic biology based on engineering modularity serves as a new tool that could be used to engineer microbes to acquire desired functions through artificial design and precise regulation. In this study, we designed artificial genetic circuits in Escherichia coli MG1655 by reconstructing the transcription factor YhaJ-based system to detect explosive composition 2,4-dinitrotoluene (2,4-DNT). These genetic circuits were optimized at the transcriptional, translational, and post-translational levels. The binding affinity of the transcription factor YhaJ with inducer 2,4-DNT metabolites was enhanced via directed evolution, and several activator binding sites were inserted in sensing yqjF promoter (PyqjF) to further improve the output level. The optimized biosensor PyqjF×2-TEV-(mYhaJ + GFP)-Ssr had a maximum induction ratio of 189 with green fluorescent signal output, and it could perceive at least 1 µg/mL 2,4-DNT. Its effective and robust performance was verified in different water samples. Our results demonstrate the use of synthetic biology tools to systematically optimize the performance of sensors for 2,4-DNT detection, that lay the foundation for practical applications.

Reconstruction of Secondary Metabolic Pathway to Synthesize Novel Metabolite in Saccharopolyspora erythraea.

Natural polyketides play important roles in clinical treatment, agriculture, and animal husbandry. Compared to natural hosts, heterologous chassis (especially Actinomycetes) have many advantages in production of polyketide compounds. As a widely studied model Actinomycete, Saccharopolyspora erythraea is an excellent host to produce valuable heterologous polyketide compounds. However, many host factors affect the expression efficiency of heterologous genes, and it is necessary to modify the host to adapt heterologous production. In this study, the CRISPR-Cas9 system was used to knock out the erythromycin biosynthesis gene cluster of Ab (erythromycin high producing stain). A fragment of 49491 bp in genome (from SACE_0715 to SACE_0733) was deleted, generating the recombinant strain AbΔery in which erythromycin synthesis was blocked and synthetic substrates methylmalonyl-CoA and propionyl-CoA accumulated enormously. Based on AbΔery as heterologous host, three genes, AsCHS, RgTAL, and Sc4CL, driven by strong promoters Pj23119, PermE, and PkasO, respectively, were introduced to produce novel polyketide by L-tyrosine and methylmalonyl-CoA. The product (E)-4-hydroxy-6-(4-hydroxystyryl)-3,5-dimethyl-2H-pyrone was identified in fermentation by LC-MS. High performance liquid chromatography analysis showed that knocking out ery BGC resulted in an increase of methylmalonyl-CoA by 142% and propionyl-CoA by 57.9% in AbΔery compared to WT, and the yield of heterologous product in AbΔery:AsCHS-RgTAL-Sc4CL was higher than WT:AsCHS-RgTAL-Sc4CL. In summary, this study showed that AbΔery could potentially serve as a precious heterologous host to boost the synthesis of other valuable polyketone compounds using methylmalonyl-CoA and propionyl-CoA in the future.

A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora erythraea Erythromycin Biosynthetic Gene Cluster with Knock-in Bidirectional Promoters.

The regulation of biosynthetic pathways is a universal strategy for industrial strains that overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were tested by reporter mCherry. The SACE_0720 ( eryBIV)-SACE_0721 ( eryAI) spacer was selected as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs) were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression increased 32 and 79 times, respectively. High performance liquid chromatography results showed that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in bidirectional promoter knock-in recombinant strains. On the basis of the activated strain Ab::Pj23119-PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study showed that bidirectional promoter knock-in and CRISPR interference could regulate gene expression in S. erythraea. This strategy has potential application for biosynthetic gene cluster activation and gene regulation in Actinobacteria.

Cross-talk between the three furA orthologs in Mycobacterium smegmatis and the contribution to isoniazid resistance.

The ferric uptake regulator A (FurA) plays an essential role in responding to oxidative stress in mycobacteria. The genome of Mycobacterium smegmatis harbours three FurA orthologs; however, the potential cross-talk and contribution to drug resistance of different furA operon remain underdetermined. In this study, we characterized the cross-regulation and effect in drug resistance of these orthologs from M. smegmatis. Cross-binding of FurA protein to furA promoter was observed. The binding of FurA1 to furA3p and FurA2 to furA1p or furA3p is even more pronounced than their self-binding. The three FurA proteins are all functional at repressing the expression of the peroxidase enzyme katG1/katG2 in vivo. When overexpressing any of the furA orthologs in M. smegmatis, the bacteria become more resistant to isoniazid (INH). This pattern is consistent with that in Mycobacterium bovis. However, the knockdown of furA does not affect the INH sensitivity. This is the first report of cross-talk and contribution to drug resistance of all three furA orthologs in M. smegmatis.

Optimization of ʟ-ornithine production in recombinant Corynebacterium glutamicum S9114 by cg3035 overexpression and manipulating the central metabolic pathway.

BACKGROUND: ʟ-Ornithine is an important amino acid with broad applications in pharmaceutical and food industries. Despite lagging ʟ-ornithine productivity and cost reduction, microbial fermentation is a promising route for sustainable ʟ-ornithine production and thus development of robust microbial strains with high stability and productivity is essential. RESULTS: Previously, we systematically developed a new strain, SO1 originate from Corynebacterium glutamicum S9114, for ʟ-ornithine production. In this work, overexpression of cg3035 encoding N-acetylglutamate synthase (NAGS) using a plasmid or by inserting a strong P tac promoter into the chromosome was found to increase ʟ-ornithine production in the engineered C. glutamicum SO1. The genome-based cg3035 modulated strain was further engineered by attenuating the expression of pta and cat, inserting a strong P eftu promoter in the upstream region of glycolytic enzymes such as pfkA, gap, and pyk, and redirecting carbon flux to the pentose phosphate pathway. The final strain with all the exploratory metabolic engineering manipulations produced 32.3 g/L of ʟ-ornithine, a yield of 0.395 g ornithine per g glucose, which was 35.7% higher than that produced by the original strain (23.8 g/L). CONCLUSION: These results clearly demonstrated that enhancing the expression of NAGS promoted ʟ-ornithine production and provide a promising alternative systematic blueprint for developing ʟ-ornithine-producing C. glutamicum strains.

High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea.

The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.

Ischemic or toxic injury: A challenging diagnosis and treatment of drug-induced stenosis of the sigmoid colon.

A 48-year-old woman was admitted with 15-mo history of abdominal pain, diarrhea and hematochezia, and 5-mo history of defecation difficulty. She had been successively admitted to nine hospitals, with an initial diagnosis of inflammatory bowel disease with stenotic sigmoid colon. Findings from computed tomography virtual colonoscopy, radiography with meglumine diatrizoate, endoscopic balloon dilatation, metallic stent implantation and later overall colonoscopy, coupled with the newfound knowledge of compound Qingdai pill-taking, led to a subsequent diagnosis of ischemic or toxic bowel disease with sigmoid colon stenosis. The patient was successfully treated by laparoscopic sigmoid colectomy, and postoperative pathological examination revealed ischemic or toxic injury of the sigmoid colon, providing a final diagnosis of drug-induced sigmoid colon stenosis. This case highlights that adequate awareness of drug-induced colon stenosis has a decisive role in avoiding misdiagnosis and mistreatment. The diagnostic and therapeutic experiences learnt from this case suggest that endoscopic balloon expansion and colonic metallic stent implantation as bridge treatments were demonstrated as crucial for the differential diagnosis of benign colonic stenosis. Skillful surgical technique and appropriate perioperative management helped to ensure the safety of our patient in subsequent surgery after long-term use of glucocorticoids.

Therapeutic experience of 289 elderly patients with biliary diseases.

AIM: To present clinical characteristics, diagnosis and treatment strategies in elderly patients with biliary diseases. METHODS: A total of 289 elderly patients with biliary diseases were enrolled in this study. The clinical data relating to these patients were collected in our hospital from June 2013 to May 2016. Patient age, disease type, coexisting diseases, laboratory examinations, surgical methods, postoperative complications and therapeutic outcomes were analyzed. RESULTS: The average age of the 289 patients with biliary diseases was 73.9 ± 8.5 years (range, 60-102 years). One hundred and thirty-one patients (45.3%) had one of 10 different biliary diseases, such as gallbladder stones, common bile duct stones, and cholangiocarcinoma. The remaining patients (54.7%) had two types of biliary diseases. One hundred and seventy-nine patients underwent 9 different surgical treatments, including pancreaticoduodenectomy, radical resection of hilar cholangiocarcinoma and laparoscopic cholecystectomy. Ten postoperative complications occurred with an incidence of 39.3% (68/173), and hypopotassemia showed the highest incidence (33.8%, 23/68). One hundred and sixteen patients underwent non-surgical treatments, including anti-infection, symptomatic and supportive treatments. The cure rate was 97.1% (168/173) in the surgical group and 87.1% (101/116) in the non-surgical group. The difference between these two groups was statistically significant (χ2 = 17.227, P < 0.05). CONCLUSION: Active treatment of coexisting diseases, management of indications and surgical opportunities, appropriate selection of surgical procedures, improvements in perioperative therapy, and timely management of postoperative complications are key factors in enhancing therapeutic efficacy in elderly patients with biliary diseases.