Production of kojic acid by Aspergillus species: Trends and applications

Kojic acid (KA), produced mainly by Aspergillus species, is a product of fungal secondary metabolism and has great potential in biotechnological applications. The use of KA has steadily increased, chiefly in the pharmaceutical industry, where KA is used for skin lightning. The market for KA has grown considerably in recent years and is expected to reach $39 million by 2026. In this review, we summarise the relevant information regarding the application of KA, describe the optimal cultivation conditions for Aspergillus species used in the production of KA, and assess the prospects for the KA market. Based on our findings, we established that the highest yields of KA can be achieved using submerged fermentation with glucose and yeast extract as the primary sources of carbon and nitrogen, respectively. Furthermore, according to literature, the main species/strains reported as the best producers of KA are Aspergillus flavus (44-L), Aspergillus oryzae (AR-47 and NRRL 484), and Aspergillus terreus (C5-10 mutant of the strain PTCC 5283). Given the commercial importance of KA and the growing demand for this natural product, further studies are needed to identify novel strains of Aspergillus as potential high producers of this acid. Similarly, it will be desirable to identify novel sources of substrate for the low-cost production of KA, thereby promoting its production for use in pharmaceutical, healthcare, and other potential industrial applications. In addition, given the current limited knowledge regarding the biosynthetic pathway of KA, further studies are required to elucidate that biosynthetic pathway.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly ¹³C-/¹⁵N-enriched protein as well as the protein containing selectively ¹⁵N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, ¹H, ¹³C, ¹³C and ¹³CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk viral p47^gag-crk - EGF epidermal growth factor - GAP GTPase-activating protein - PI3K phosphatidylinositol-3-kinase - PLC- phospholipase-C-, shc, src homologous and collagen - src sarcoma family of nonreceptor tyrosine kinase     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

Colonization in an abandoned East-Mediterranean vineyard

Abstract. Previous studies on secondary succession in abandoned agricultural land in the Mediterranean area were carried out by the chronosequence method, including data from different sites. A unique opportunity to study secondary succession arose from a situation in which different parts of one homogeneous East-Mediterranean vineyard were abandoned for 5, 8, 15 and 35 yr, and did not suffer from any disturbance subsequently. Most of the perennial species that colonized the abandoned vineyard were fleshy fruited species, which apparently were dispersed by birds from the surrounding maquis into the vineyard. These bird-dispersed species were the first to be established, and were the dominant plant group according to dispersal modes. The abandoned vine plants and their supporting columns provided the birds with perching and feeding sites, enhancing the arrival of bird-dispersed species regardless of their life forms. Under these conditions the most important attribute that affected vegetation dynamics was seed dispersal mode. Trees were among the first to colonize in the vineyard, implying that no facilitation was needed for their establishment. Annual plant species were the only species to disappear during succession. Almost all perennial species which had arrived persisted in the vineyard, and no replacement of perennial species was found. The rate of succession was rapid, as expressed by the short time (8–15 yr) needed for the stabilization of species composition, for growth to average height of late succession trees, and for reaching high cover of the invading perennial species in the abandoned vineyard. The secondary succession described above differs from that in the western Mediterranean by the absence of perennial species replacement and its rapid rate. The possible causes are discussed.     

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed. Received: 4 September 1996 / Accepted: 7 April 1997     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

The relationship between amide proton chemical shifts and secondary structure in proteins

Summary The parameters for HN chemical shift calculations of proteins have been determined using data from high-resolution crystal structures of 15 proteins. Employing these chemical shift calculations for HN protons, the observed secondary structure chemical shift trends of HN protons, i.e., upfield shifts on helix formation and downfield shifts on -sheet formation, are discussed. Our calculations suggest that the main reason for the difference in NH chemical shifts in helices and sheets is not an effect from the directly hydrogen-bonded carbonyl, which gives rise to downfield shifts in both cases, but arises from an additional upfield shift predicted in helices and originating in residues i-2 and i-3. The calculations also explain the well-known relationship between amide proton shifts and hydrogen-bond lengths. In addition, the HN chemical shifts of the distorted amphipathic helices of the GCN4 leucine zipper are calculated and used to characterise the solution structure of the helices. By comparing the calculated and experimental shifts, it is shown that in general the agreement is good between residues 15 and 28. The most interesting observation is that in the N-terminal half of the zipper, although both calculated and experimental shifts show clear periodicity, they are no longer in phase. This suggests that for the N-terminal half, in the true average solution structure the period of the helix coil is longer by roughly one residue compared to the NMR structures.