Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in Streptomyces albus B4 chassis.

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. IMPORTANCE: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

LncRNA-ANRIL inhibits cell senescence of vascular smooth muscle cells by regulating miR-181a/Sirt1.

BACKGROUND: Cardiovascular disease is one of the major threats to human life and health, and vascular aging is an important cause of its occurrence. Antisense non-coding RNA in the INK4 locus (ANRIL) is a kind of long non-coding RNA (lncRNA) that plays important roles in cell senescence. However, the role and mechanism of ANRIL in senescence of vascular smooth muscle cells (VSMCs) are unclear. METHODS: Cell viability and cell cycle were evaluated using an MTT assay and flow cytometry analysis, respectively. Senescence-associated (SA)-ß-galactosidase (gal) staining was used to determine cell senescence. Dual luciferase reporter assays were conducted to confirm the binding of ANRIL and miR-181a, as well as miR-181a and Sirt1. The expression of ANRIL, miR-181a, and Sirt1 was determined using qRT-PCR and protein levels of SA-ß-gal and p53-p21 pathway-related proteins were evaluated by Western blotting. RESULTS: ANRIL and Sirt1 were down-regulated, whereas miR-181a was up-regulated in aging VSMCs. In young and aging VSMCs, over-expression of ANRIL could down-regulate miR-181a and up-regulate Sirt1. MTT and SA-ß-gal staining assays showed that over-expression of ANRIL and inhibition of miR-181a promoted cell viability and inhibited VSMC senescence. The dual-luciferase reporter assay determined that miR-181a directly targets ANRIL and the 3'-UTR of Sirt1. Furthermore, over-expression of ANRIL inhibited cell cycle arrest and the p53-p21 pathway. CONCLUSION: ANRIL promotes cell viability and inhibits senescence in VSMCs, possibly by regulating miR-181a/Sirt1, and alleviating cell cycle arrest by inhibiting the p53-p21 pathway. This study provides novel insights for the role of ANRIL in the development of cell senescence.

Heterologous activation and metabolites identification of the pks7 gene cluster from Saccharopolyspora erythraea.

The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.

Engineering Streptomyces albus B4 for Enhanced Production of (R)-Mellein: A High-Titer Heterologous Biosynthesis Approach.

The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in ß-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Adiponectin attenuates the premature senescence of vascular smooth muscle cells induced by high glucose through mTOR signaling pathway.

OBJECTIVE: Cardiovascular diseases and vascular aging are common in patients with diabetes. High glucose is a major cause of vascular aging and cardiovascular diseases. Premature senescence of vascular smooth muscle cells (VSMCs) is one of the main contributors to vascular aging. Adiponectin has been demonstrated to have an anti-aging effect. The present study explored the mechanisms by which adiponectin protects VSMCs against high-glucose-induced senescence. METHODS: Senescence-associated ß-galactosidase (SA-ß-gal) staining was used to detect senescence cells. Western blot was used for measuring protein levels. Flow cytometry was carried out to detect the cell cycle and telomeric repeat amplification protocol (TRAP)-polymerase chain reaction (PCR) silver staining was selected to measure the telomerase activity. RESULTS: Premature senescence of VSMCs was induced by high glucose (30 mM) in a time-dependent manner, which was verified by an increased number of senescence cells, p21 and p53 expression, as well as the decreased proliferation index. High glucose reduced telomerase activity of VSMCs via inhibition of the AMPK/TSC2/mTOR/S6K1 pathway and activation of the PI3K/Akt/mTOR/S6K1 pathway, while adiponectin treatment significantly increased telomerase activity of VSMCs through activation of AMPK/TSC2/mTOR/S6K1 signaling and inhibition of PI3K/Akt/mTOR/S6K1 signaling. CONCLUSION: Adiponectin attenuated the high-glucose-induced premature senescence of VSMCs via increasing telomerase activity of VSMCs, which was achieved by activation of AMPK/TSC2/mTOR/S6K1 signaling and inhibition of PI3K/Akt/mTOR/S6K1 signaling.

LncRNA-ES3 inhibition by Bhlhe40 is involved in high glucose-induced calcification/senescence of vascular smooth muscle cells.

Long noncoding RNAs (lncRNAs) have been investigated as novel regulatory molecules involved in diverse biological processes. Our previous study demonstrated that lncRNA-ES3 is associated with the high glucose-induced calcification/senescence of human aortic vascular smooth muscle cells (HA-VSMCs). However, the mechanism of lncRNA-ES3 in vascular calcification/aging remained largely unknown. Here, we report that the expression of basic helix-loop-helix family member e40 (Bhlhe40) was decreased significantly in HA-VSMCs treated with high glucose, whereas the expression of basic leucine zipper transcription factor (BATF) was increased. Overexpression of Bhlhe40 and inhibition of BATF alleviated calcification/senescence of HA-VSMCs, as confirmed by Alizarin Red S staining and the presence of senescence-associated ß-galactosidase-positive cells. Moreover, we identified that Bhlhe40 regulates lncRNA-ES3 in HA-VSMCs by binding to the promoter region of the lncRNA-ES3 gene (LINC00458). Upregulation or inhibition of lncRNA-ES3 expression significantly promoted or reduced calcification/senescence of HA-VSMCs, respectively. Additionally, we identified that lncRNA-ES3 functions in this process by suppressing the expression of miR-95-5p, miR-6776-5p, miR-3620-5p, and miR-4747-5p. The results demonstrate that lncRNA-ES3 triggers gene silencing of multiple miRNAs by binding to Bhlhe40, leading to calcification/senescence of VSMCs. Our findings suggest that pharmacological interventions targeting lncRNA-ES3 may be therapeutically beneficial in ameliorating vascular calcification/aging.

Rapamycin­induced miR­30a downregulation inhibits senescence of VSMCs by targeting Beclin1.

Vascular senescence is considered to be an independent risk factor for cardiovascular diseases. The present study aimed to investigate the effects of rapamycin on miR­30a and its relationship with autophagy and senescence in vascular smooth muscle cells (VSMCs). Young and aging VSMCs were treated with rapamycin or transfected with miR­30a mimics. Measurement of cellular senescence was conducted using senescence­associated (SA)­ß­Galactosidase (gal) staining. Dual luciferase reporter assay was used to confirm binding for miR­30a and Beclin1. The expression levels of miR­30a and Beclin1 were determined with reverse transcription­quantitative polymerase chain reaction analysis. Autophagy­related protein levels were determined using immunofluorescence or western blot assays. The results demonstrated that rapamycin treatment significantly decreased miR­30a expression and increased Beclin1 expression in both young and aging cells, as well as promoted autophagy in VSMCs. In addition, rapamycin inhibited senescence in VSMCs and could also alleviate the aging VSMC cycle arrest. Dual luciferase reporter assay confirmed that miR­30a could directly bind the 3'untranslated region of Beclin1 and inhibit its expression. Furthermore, miR­30a inhibited autophagy and promoted senescence of VSMCs. In conclusion, the present results indicated that rapamycin could inhibit the senescence of VSMCs by downregulating miR­30a, which resulted in upregulation of Beclin1 and activation of autophagy. The current study is the first to demonstrate an inhibitory role of rapamycin on VSMC senescence and might provide novel insights and potential new molecular targets in senescence treatment.

Exosomes from hyperglycemia-stimulated vascular endothelial cells contain versican that regulate calcification/senescence in vascular smooth muscle cells.

BACKGROUND: To determine whether and how exosomes from human umbilical vein endothelial cells (HUVEC-Exos) regulates vascular smooth muscle cells (VSMCs) calcification/senescence in high glucose condition. METHODS: HUVEC-Exos were isolated from normal glucose (NG) and high glucose (HG) stimulated HUVECs (NG/HG-HUVEC-Exos) by super speed centrifugation. HUVEC-Exos were identified by transmission electron microscopy and Western blot of CD63. Protein profile in HUVEC-Exos was examined to screen the candidate molecules that mediate HUVEC-Exos function. VSMCs were incubated with HUVEC-Exos. A series of functional assays in vitro were performed to assess the effects of HUVEC-Exos on the calcification/senescence of VSMCs. The role of the candidate protein in HUVEC-Exos-induced VSMCs dysfunction was assessed. RESULTS: Exosomes isolated from HG-HUVEC-Exos induced calcification/senescence in VSMCs as assessed by Alizarin Red Staining, senescence-associated ß-galactosidase (SA-ß-gal) staining, and the expression of ALP and p21. HG-HUVEC-Exos significantly increased LDH activity, as well as the product of lipid peroxidation (MDA content), and decreased oxidative stress marker activity, as compared with NG-HUVEC-Exos. Moreover, mechanism studies showed that mitochondrial membrane potential and the expression levels of mitochondrial function related protein HADHA and Cox-4 were significantly decreased in HG-HUVEC-Exos compared to controls. Proteomic analysis showed that HG-HUVEC-Exos consisted of higher level of versican (VCAN), as compared with NG-HUVEC-Exos. Observation under laser confocal microscopy revealed that most green fluorescence of VCAN could overlap with the red fluorescence came from mitochondria, indicating VCAN is mainly localized to the mitochondria of VSMCs. Knockdown of VCAN with siRNA in HUVECs, inhibited HG-HUVEC-Exos-induced mitochondrial dysfunction and calcification/senescence of VSMCs. CONCLUSIONS: Our data indicate an intracellular role for VCAN in VSMCs. VCAN participates in hyperglycemia-induced calcification/senescence via modulation of mitochondrial function in VSMCs.

Exosomal Notch3 from high glucose-stimulated endothelial cells regulates vascular smooth muscle cells calcification/aging.

AIMS: Vascular calcification/aging can cause different kind of serious diabetic vascular complications. High glucose could induce vascular smooth muscle cells (VSMCs) calcification/aging and then lead to diabetes-related vascular calcification/aging. In this study, we investigated how information in the blood is transmitted to VSMCs and the mechanisms of VSMCs calcification/aging under hyperglycaemic conditions. MATERIALS AND METHODS: Transmission electron microscopy and molecular size analysis were used to assess the morphology and size of exosomes. Alizarin Red S staining and senescence-associated ß galactosidase (SA-ß-gal) staining were carried out to detect calcification and senescence in VSMCs, respectively. Proteomics analysis was carried out to detect the different expression of exosomal proteins. Protein levels were measured by western blot analysis. KEY FINDINGS: The results show that exosomes isolated from high glucose stimulated human umbilical vein endothelial cell (HG-HUVEC-Exo) exhibited a bilayer structure morphology with a mean diameter of 63.63 ±â€¯2.96 nm. The presence of exosome markers including CD9, CD63 and TSG101 were also detected in HG-HUVEC-Exo. High glucose could induce VSMCs calcification/aging by increasing the expression of osteocalcin (OC) and p21 as well as the formation of mineralised nodules and SA-ß-gal positive cells. Fluorescence microscopy verified that the exosomes were taken up by VSMCs and Notch3 protein was enriched in HG-HUVEC-Exo. Most importantly, mTOR signalling was closely related to Notch3 protein and was involved in regulating HG-HUVEC-Exo-induced VSMCs calcification/aging. SIGNIFICANCE: The data demonstrate that Notch3 is required for HG-HUVEC-Exo promoted VSMCs calcification/aging and regulates VSMCs calcification/aging through the mTOR signalling pathway.

lncRNA-ES3/miR-34c-5p/BMF axis is involved in regulating high-glucose-induced calcification/senescence of VSMCs.

Vascular calcification/aging is common in diabetes and is associated with increased morbidity and mortality of patients. MiR-34c-5p, not miR-34c-3p, was suppressed significantly in calcification/senescence of human aorta vascular smooth muscle cells (HA-VSMCs) induced by high glucose, which was proven by the formation of mineralized nodules and staining of senescence associated-ß-galactosidase staining (SA ß-gal) positive cells. Overexpression of miR-34c-5p alleviated calcification/senescence of HA-VSMCs, whereas inhibition of miR-34c-5p received the opposite results. Bcl-2 modifying factor (BMF) was a functional target of miR-34c-5p and it was involved in the process of calcification/senescence of HA-VSMCs. Besides, lncRNA-ES3 acted as a competing endogenous RNAs (ceRNA) of miR-34c-5p to enhance BMF expression. Further, lncRNA-ES3 inhibited miR-34c-5p expression by direct interaction and its knockdown suppressed the calcification/senescence of HA-VSMCs. Our results showed for the first time that the calcification/senescence of VSMCs was regulated by lncRNA-ES3 /miR-34c-5p/BMF axis.