In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes.

BACKGROUND: Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproduction of purine nucleosides by de novo metabolic engineering remains a great challenge due to their strict and complex regulatory machinery involved in biosynthetic pathways. RESULTS: In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on a genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes, Drm (purine nucleotides toward PPP) and YwjH (PPP-EMP), to resolve the competitive relationship between biomass and purine nucleotide synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides, which greatly increased the inosine production to 13.98-14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of the purine operon by promoter replacement enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, the deletion of the second backflow node YwjH and overexpression of Zwf were combined to increase inosine production to 22.01 ± 1.18 g/L by enhancing the metabolic flow of PPP. By switching on the flux node of the glucose-6-phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81 ± 1.23 g/L inosine by a pgi-based metabolic switch with a yield of 0.126 mol/mol glucose, a productivity of 0.358 g/L/h and a synthesis rate of 0.088 mmol/gDW/h, representing the highest yield in de novo engineered inosine bacteria. Under the guidance of this in silico-designed strategy, a general chassis bacterium was generated, for the first time, to efficiently synthesize inosine, adenosine, guanosine, IMP and GMP, which provides sufficient precursors for the synthesis of various purine intermediates. CONCLUSIONS: Our study reveals that in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring efficient targets, which could be applied as a superior strategy for efficient biosynthesis of biotechnological products.

LncRNA MIR99AHG mediated by FOXA1 modulates NOTCH2/Notch signaling pathway to accelerate pancreatic cancer through sponging miR-3129-5p and recruiting ELAVL1.

BACKGROUND: Pancreatic cancer (PCa) is a fatal malignancy with poor prognosis, high recurrence and mortality. Substantial reports have suggested long non-coding RNAs (lncRNAs) are implicated in development of numerous malignant tumors, and PCa is included. However, the correlation between novel lncRNA mir-99a-let-7c cluster host gene (MIR99AHG) and PCa remains elusive and needs to be deeply investigated. METHODS: In this study, we firstly used RT-qPCR to examine MIR99AHG expression. Functional assays were implemented for determination of the role of MIR99AHG in PCa cells. Mechanism experiments were designed and carried out for exploring the regulatory mechanism involving MIR99AHG. RESULTS: MIR99AHG was distinctly overexpressed in PCa cell lines. MIR99AHG deficiency abrogated PCa cell proliferation, migration and invasion. Moreover, MIR99AHG up-regulation was induced by transcription factor forkhead box A1 (FOXA1). Furthermore, MIR99AHG modulated notch receptor 2 (NOTCH2) expression and stimulated Notch signaling pathway through sequestering microRNA-3129-5p (miR-3129-5p) and recruiting ELAV like RNA binding protein 1 (ELAVL1). CONCLUSIONS: Altogether, the exploration of FOXA1/MIR99AHG/miR-3129-5p/ELAVL1/NOTCH2 axis in the progression of PCa might provide a meaningful revelation for PCa diagnosis and treatment.

Pancreatic Cancer Progression Is Regulated by IPO7/p53/LncRNA MALAT1/MiR-129-5p Positive Feedback Loop.

Background: Pancreatic cancer is a malignancy with poor prognosis. Importin 7 (IPO7) is a soluble nuclear transport factor, which has been linked to the pathogenesis of several human diseases. However, its role and underlying mechanism in pancreatic cancer are still obscure. Methods: Immunohistochemical staining and quantitative real-time polymerase chain reaction (qPCR) were performed to determine IPO7 expression in pancreatic cancer tissues and adjacent tissues. Western blot was used to measure IPO7 expression at the protein level in cell lines. Cell Counting Kit-8 (CCK-8), 5-bromo-2'-deoxyuridine (BrdU), flow cytometry, and Transwell assays were employed to explore the biological functions of IPO7. Subcutaneous xenograft transplanted tumor model and caudal vein injection model in mice were also established to validate the oncogenic role of IPO7. Western blot and qPCR were utilized to detect the regulatory function of IPO7 on p53 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), respectively. Interaction between MALAT1 and miR-129-5p and interaction between miR-129-5p and IPO7 were verified by bioinformatics prediction, qPCR, dual-luciferase reporter gene experiment, RNA immunoprecipitation (RIP), and pull-down assay. Results: Upregulation of IPO7 in pancreatic cancer tissues was associated with adverse prognosis of the patients with pancreatic cancer. Knocking down IPO7 remarkably suppressed cancer cell proliferation and metastasis, while it promoted apoptosis. Overexpression of IPO7 facilitated the malignant phenotypes of pancreatic cancer cells. Mechanistically, IPO7 could repress the expression of p53 and induce the expression of MALAT1 but reduce miR-129-5p expression. Furthermore, miR-129-5p was identified as a posttranscriptional regulator for IPO7, and its inhibition led to IPO7 overexpression in pancreatic cancer cells. Conclusion: IPO7 is a novel oncogene for pancreatic cancer, and IPO7/p53/MALAT1/miR-129-5p positive feedback loop facilitates the progression of this deadly disease.

LncRNA HCG11/miR-579-3p/MDM2 axis modulates malignant biological properties in pancreatic carcinoma via Notch/Hes1 signaling pathway.

BACKGROUND: Increasing reports have revealed that dysregulated expression of long non-coding RNAs (lncRNAs) is involved in pancreatic carcinoma progression. This study intends to explore the function and molecular mechanism of lncRNA HLA complex group 11 (HCG11) in pancreatic carcinoma. METHODS: The expression profiles of HCG11 in pancreatic carcinoma samples were detected by qPCR. Bioinformatics analysis was applied to detect the associations among HCG11/miR-579-3p/MDM2. The malignant properties of pancreatic carcinoma cells were measured by numerous biological assays. Xenograft model was exploited to detect the effect of HCG11 on tumor growth. RESULTS: A significant increase of HCG11 was occurred in pancreatic carcinoma samples. Knockdown of HCG11 suppressed the progression of pancreatic carcinoma cells. Bioinformatics analysis revealed that HCG11 upregulated MDM2 expression by competitively targeting miR-579-3p. The rescue assays showed that miR-579-3p reversed cell behaviors caused by HCG11, and MDM2 reversed cell properties induced by miR-579-3p. The Notch1 intracellular domain (NICD) and Hes1 protein levels were increased by overexpression of HCG11/MDM2. The tumor growth was suppressed after depletion of HCG11, followed by suppressing Ki67, PCNA and Vimentin expression, increasing TUNEL-positive cells and E-cadherin expression. CONCLUSIONS: Our observations highlighted that HCG11 contributed to the progression of pancreatic carcinoma by promoting growth and aggressiveness, and inhibiting apoptosis via miR-579-3p/MDM2/Notch/Hes1 axis.

In vitro assembly of the bacterial actin protein MamK from ' Candidatus Magnetobacterium casensis' in the phylum Nitrospirae.

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth's magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated 'Candidatus Magnetobacterium casensis' (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.