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[en] Sequence control is of crucial significance in biology. There are great prospects in the application of information storage and transmission by controlling the sequence structure so as to control sequenced compounds. At present, although there are effective synthetic methods for both biosynthesis and chemical synthesis, the severest challenge may be that there is currently no general strategy for synthesizing sequence-controlled polymers. (paper)
[en] Highlights: • Overexpression of SlH6H or HnH6H significantly promoted the anisodamine production at similar level. • Overexpression of SlH6H or HnH6H markedly enhanced the scopolamine biosynthesis at different level. • HnH6H exhibited better effects on the enhancement of scopolamine biosynthesis than SlH6H. • Overexpression of HnH6H led to the increased production of total tropane alkaloids. Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.
[en] The structural characterization of CalO1 is reported. The X-ray structure determination at 2.4 Å resolution of the putative orsellinic acid C3 O-methyltransferase (CalO1) involved in calicheamicin biosynthesis is reported. Comparison of CalO1 with a homology model of the functionally related calicheamicin orsellinic acid C2 O-methyltransferase (CalO6) implicates several residues that are likely to contribute to the regiospecificity of alkylation. Consistent with the proposed requirement of an acyl-carrier-protein-bound substrate, this structural study also reveals structural determinants within CalO1 that are anticipated to accommodate an association with an acyl carrier protein
[en] The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies. - Highlights: • Sesquiterpene lactones are characteristic metabolites in Asteraceae family. • Barnadesioideae is the basal lineage of all Asteraceae plants, producing sesquiterpene lactones. • Two germacrene A synthases (GASs) were identified and characterized from Barnadesia spinosa. • A phylogenetic analysis showed two sub-clades of GASs have evolved in Asteraceae. • GAS enzymes evolved prior to the divergence and global dispersal of the modern Asteraceae.
[en] The inherent non-linearity of citric acid fermentation from Aspergillus niger renders its control difficult, so there is a need to fine-tune the bioreactor performance for maximum production of citric acid in batch culture. For this, fuzzy logic is becoming a popular tool to handle non-linearity of a batch process. The present manuscript deals with fuzzy logic control of citric acid accretion by A. niger in a stirred tank reactor using blackstrap sugarcane molasses as a basal fermentation medium. The customary batches were termed as control while those under fuzzy logic were experimental. The performance of fuzzy logic control of stirred tank reactor was found to be very encouraging for enhanced production of citric acid. The comparison of kinetic parameters showed improved citrate synthase ability of experimental culture (Yp/x = 7.042 g/g). When the culture grown on 150 g/l carbohydrates was monitored for Qp, Qs and Yp/s, there was significant enhancement in these variables over the control. Specific productivity of culture (qp = 0.070 g/g cells/h) was several fold increased. The enthalpy (HD = 70.5 kJ/mol) and entropy of activation (S = -144 J/mol/K) of enzyme for citric acid biosynthesis, free energies for transition state formation and substrate binding for sucrose hydrolysis of experimental were substantially improved. (author)
[en] In this study, green synthesis of silver nanoparticles (AgNPs) and silver-montmorillonite (MMT) nanocomposites (Ag/MMT) were carried out using two local plants from Iran (Ocimum basilicum L and Teucrium Polium L). The biomolecules presenting in the plant extracts acted as the reducing and capping agents. The average size of the synthesized AgNPs, using OB and TP plant extracts were around 80 nm and 20 nm, respectively. In addition, the nanoparticle sizes were further decreased using MMT as substrate, to 70 nm and 15 nm for OB and TP plant extracts, respectively. Finally, high antibacterial efficiency against E. coli and S. aureus pathogens as well as the appropriate cytotoxicity against HEP G2 cells were observed from the biosynthesized AgNPs. The present work is eco-friendly benign and can be used for large-scale fabrication of AgNPs. (paper)
[en] Highlights: • A CCA1-like GmMYB133 was functionally characterized as a new positive regulator in isoflavonoid synthesis. • GmMYB133 might form hetero- and homodimers with an isoflavonoid regulator GmMYB176 and itself, respectively. • The subcellular localization of GmMYB133 can be altered by its interaction with 14-3-3 protein. MYB transcription factors play important roles in the regulation of phenylpropanoid biosynthesis. However, the knowledge regarding the roles of CCA1-like MYBs in phenylpropanoid pathway is limited in plants. Previously, we identified 54 CCA1-like proteins in soybean. In the study, a CCA1-like MYB (GmMYB133) was functionally characterized as a positive regulator in isoflavonoid synthesis. GmMYB133 encodes a 330 aa protein featured with one CCA1 conserved motif. Further analysis indicated that the expression pattern of GmMYB133 was near-perfectly correlated with isoflavonoid accumulation as soybean embryos develop. GmMYB133 over-expression promoted the expression of two key isoflavonoid biosynthetic genes (GmCHS8 and GmIFS2) and increased total isoflavonoid content in hairy roots. Protein-protein interaction assays indicated that GmMYB133 might form hetero- and homodimers with an isoflavonoid regulator GmMYB176 and itself, respectively, while the subcellular localization of GmMYB133 can be altered by its interaction with 14-3-3 protein. These findings provided new insights into the functional roles of CCA1-like MYB proteins in the regulation of phenylpropanoid pathway, and will contribute to the future genetic engineering in the improvement of soybean seed quality.
[en] Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major intestinal pathogen that can infect both humans and a variety of animals. LoiA, a novel virulence-regulating protein encoded in Salmonella pathogenicity island (SPI)-14, has been shown to be induced under low oxygen conditions and contribute to S. Typhimurium invasion into intestinal epithetical cells by activating the SPI-1 invasion genes. However, the global regulatory network of LoiA remains unknown. Here, we used high-throughput RNA sequencing (RNA-seq) technology to investigate the regulatory function of LoiA in S. Typhimurium under low oxygen conditions. A total of 1250 genes were differentially expressed between the loiA mutant and the wild-type strain; 413 genes were up-regulated and 837 were down-regulated. SPI-1 gene expression was down-regulated in the loiA mutant, consistent with previous results. SPI-2 gene expression was not affected by deletion of loiA; the expression of most genes involved in flagellar basal body and hook biosynthesis was up-regulated in the loiA mutant, while the expression of genes associated with flagellin, motility, and chemotaxis was down-regulated; the expression of lon, encoding an ATP-dependent protease, was up-regulated in the mutant. This study indicates that LoiA regulates a variety of virulence-associated genes in S. Typhimurium. The negative regulation of Lon protease by LoiA indicates that LoiA can regulates several virulence-associated genes in S. Typhimurium via the Lon protease.