β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production.
Basidiomycota ; enzymology ; Farnesyltranstransferase ; genetics ; metabolism ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Metabolic Engineering ; Polyisoprenyl Phosphates ; Saccharomyces cerevisiae ; metabolism ; beta Carotene ; biosynthesis

A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Azulenes ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Open Reading Frames ; Polyisoprenyl Phosphates ; Recombinant Proteins ; biosynthesis ; Sesquiterpenes ; metabolism ; Sesquiterpenes, Guaiane ; Thymelaeaceae ; enzymology ; genetics

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a disease entity which is rare, but is a serious side effect of bisphosphonate therapy. Several professional associations have published position papers on BRONJ; in 2009, the Korean position statement was reported as a collaborative effect between the Korean Endocrine Society, Korean Society of Bone Metabolism, Korean Society of Osteoporosis, and Korean Association of Oral and Maxillofacial Surgeons. Diagnostic criteria and treatment strategies for BRONJ are now being established through a thorough investigation and cooperation amongst numerous specialties. Still obscure, it is suggested that the pathogenesis of BRONJ is due to the inhibition of farnesyl pyrophosphate synthase of the osteoclastic mevalonate pathway, thus disturbing the cytoskeletal motility for the fusion of mononuclear cells into a multinucleated giant cell or the establishment of a ruffled border. Eventually, such changes will be followed by inactivation and apoptosis of osteoclasts, leading to decreased bone resorption. The incidence of BRONJ is known to be as low as 0.01~0.001% of the entire population, but BRONJ is as high as 1 in 300 in the case of dental intervention of patients on bisphosphonate therapy. It is important for clinicians to remember in requesting a dental consultation for a patient on bisphosphonate therapy that oral cavity has a special environment for wound healing. Routine minor trauma caused by foreignbodies, such as hard food, is compensated by an appropriate wound healing mechanism involving rapid bone turnover due to the rich vascular supply of the oral mucosa. Bisphosphonate will disturb this normal wound healing as a consequence of decreased bone turnover. It should also be kept in mind that the disturbed wound healing is further complicated by the presence of normal microflora in the oral cavity and by the unique anatomic condition of the thin oral mucosa covering the the mandible, most mobile skeleton in the head and neck area. The potency of the bisphosphonates (intravenous bisphosphonate), local factors, such as local dental intervention (especially dental extraction), and systemic factors, such as patient age (old age), have statistical significance for all BRONJ risk factors. Although the recognition of BRONJ by clinicians has been inadequate until now, the growing body of evidence is unveiling the detailed aspects of BRONJ. Continued investigation and extensive cooperation of related specialties will elucidate the nature of the disease, thus enhancing the quality of life of patients on bisphosphonate therapy.
Apoptosis ; Bisphosphonate-Associated Osteonecrosis of the Jaw ; Bone Resorption ; Diphosphonates ; Giant Cells ; Head ; Humans ; Incidence ; Jaw ; Mandible ; Mevalonic Acid ; Mouth ; Mouth Mucosa ; Neck ; Osteoclasts ; Osteonecrosis ; Osteoporosis ; Polyisoprenyl Phosphates ; Quality of Life ; Risk Factors ; Sesquiterpenes ; Skeleton ; Wound Healing

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.
1-Phosphatidylinositol 3-Kinase/metabolism ; Alkyl and Aryl Transferases/metabolism ; Animals ; Anticholesteremic Agents/pharmacology ; Cells, Cultured ; Enzyme Inhibitors/metabolism/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Gene Expression Regulation, Enzymologic/*drug effects ; I-kappa B Kinase/antagonists & inhibitors/metabolism ; Macrophages, Alveolar/cytology/*drug effects/*enzymology ; Matrix Metalloproteinase 9/genetics/*metabolism ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Polyisoprenyl Phosphates/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Sesquiterpenes/metabolism ; Signal Transduction/physiology ; Simvastatin/*pharmacology ; Smoke/*adverse effects ; *Tobacco/adverse effects/chemistry