Improved pinocembrin production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by engineering fatty acid synthesis

The development of efficient microbial processes for pinocembrin production has attracted considerable attention. However, pinocembrin biosynthetic efficiency is greatly limited by the low availability of the malonyl-CoA cofactor in Escherichia coli. Fatty acid biosynthesis is the only metabolic process in E. coli that consumes malonyl-CoA; therefore, we overexpressed the fatty acid biosynthetic pathway enzymes β-ketoacyl-ACP synthase III (FabH) and β-ketoacyl-ACP synthase II (FabF) alone and in combination, and investigated the effect on malonyl-CoA. Interestingly, overexpressing FabH, FabF or both enzymes in E. coli BL21 (DE3) decreased fatty acid synthesis and increased cellular malonyl-CoA levels 1.4-, 1.6-, and 1.2-fold, respectively. Furthermore, pinocembrin production was increased 10.6-, 31.8-, and 5.87-fold in recombinant strains overexpressing FabH, FabF and both enzymes, respectively. Overexpression of FabF, therefore, triggered the highest pinocembrin production and malonyl-CoA levels. The addition of cerulenin further increased pinocembrin production in the FabF-overexpressing strain, from 25.8 to 29.9 mg/L. These results demonstrated that overexpressing fatty acid synthases can increase malonyl-CoA availability and improve pinocembrin production in a recombinant E. coli host. This strategy may hold promise for the production of other important natural products in which cellular malonyl-CoA is rate limiting.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

Sensilla basiconica (grooved pegs) on the antennae of female mosquitoes: electrophysiology and morphology

The sensilla basiconica or grooved pegs of mosquitoes consist of two morphologically and functionally distinct classes. Short grooved pegs house sensory cells that are excited by lactic acid whereas lactic acid-excited cells are absent in long grooved pegs. This correlation was found in four species of mosquito examined:Aedes aegypti, Aedes atropalpus, Aedes epactius, andCulex pipiens. InAedes epactius, both short and long grooved pegs house receptors that are sensitive to butyric acid.     

Testing the covarion hypothesis of molecular evolution

The covarion hypothesis of molecular evolution states that the fixation of mutations may alter the probability that any given position will fix the next change. Tests of this hypothesis using the divergence of real sequences are compromised because models of rate variation among sites (e.g., the gamma version of the one-parameter equation) predict sequence divergence values similar to those for the covarion process. This study therefore focuses on the extent to which the varied and unvaried codons of two well-diverged taxa are the same, because fewer are expected by the covarion hypothesis than by the gamma model. The data for these tests are the protein sequences of Cu, Zn superoxide dismutase (SOD) for mammals and plants. Simulation analyses show that the covarion hypothesis makes better predictions about the frequencies of varied and unhit positions in common between these two taxa than does the gamma version of the one-parameter model. Furthermore, the analysis of SOD tertiary structure demonstrates that mammal and plant variabilities are distributed differently on the protein. These results support the conclusions that the variable and invariable codons of mammal and plant SODs are different and that the covarion model explains the evolution of this protein better than the gamma version of the one-parameter process. Unlike other models, the covarion hypothesis accounts for rate fluctuations among positions over time, which is an important parameter of molecular evolution.      

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo

Summary A cell-free system prepared from immature seed of Cucurbita pepo incorporates the label from mevalonate-2-¹⁴C into ent-kaur-16-en-19-oic acid (I), ent-7-hydroxy-kaur-16-en-19-oic acid (II), and ent-gibberell-16-en-7-al-19-oic acid (III) (gibberellin A₁₂-aldehyde). The products were identified by gas liquid chromatography and by combined gas chromatography-mass spectrometry of the methylated and trimethylsilylated fractions. The radioactivity of the compounds was established by recrystallisation to constant specific radioactivity. Gibberellin A₁₂ (IV), also detected in the system after incubation by combined gas chromatographymass spectrometry may be an artefact, derived from gibberellin A₁₂-aldehyde by a non-enzymatic conversion.With gibberellin A₁₂-aldehyde, the cell-free biosynthesis of an ent-gibberellane has been achieved for the first time.