Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Molecular properties of two proteins homologous to PduO-type ATP:cob(I)alamin adenosyltransferase from Sulfolobus tokodaii

In the thermophilic archaeon Sulfolobus tokodaii, there are two genes homologous to PduO-type ATP:cob(I)alamin adenosyltransferase, ST1454 and ST2180. To address the structure and function of these two sequence-related proteins from one organism, we prepared them by using the Escherichia coli expression system and analyzed them by immunoblotting, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy, circular dichroism spectrometry, ATP:cobalamin adenosyltransferase assay, and X-ray crystallography. Immunoblotting and matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy analyses showed that both these proteins are expressed in S. tokodaii cells as soluble proteins and are spontaneously digested at the N-terminal region. ATP:cob(I)alamin adenosyltransferase activity was detected for ST1454 but not for ST2180. ST2180 reduced the concentration of cob(I)alamin, suggesting that ST2180 might recognize cob(I)alamin as a ligand. The secondary structure of ST1454 was retained even in 7 M guanidine hydrochroride, whereas that of ST2180 was melted in 4.5 M guanidine hydrochloride. The X-ray crystal structural analysis revealed that the proteins shared a common structure: a trimer of five-helix bundles with a clockwise kink. There is a pocket surrounded by highly conserved residues, in which a polypropylene glycol 400 in the crystal structure of ST1454 was captured, suggesting that it is an active site. Structural comparison between these two proteins showed the difference in the number of ion pairs around the proposed active site. On the basis of these results, we propose that ST1454 and ST2180 have related but distinct functions.     

The complete mitochondrial genome of the wild silkworm moth, Actias selene

The complete mitochondrial genome (mitogenome) of Actias selene (Lepidoptera: Saturniidae) was determined to be 15,236 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. The AT skew of the mitogenome of A. selene was slightly negative, indicating a higher number of T compared to A nucleotides. The nucleotide composition of the mitogenome of A. selene was also biased toward A+T nucleotides (78.91%). All PCGs were initiated by ATN codons, except for the gene encoding cytochrome c oxidase subunit 1 (cox1), which may be initiated by the TTAG, as observed in other lepidopterans. Three genes, including cox1, cox2, and nad5, had incomplete stop codons consisting of just a T. With an exception for trnS1(AGN), all the other tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A+T-rich region of the mitogenome of A. selene was 339 bp in length, and contains several features common to the Lepidopteras, including non-repetitive sequences, a conserved structure combining the motif ATAGA and an 18-bp poly-T stretch and a poly-A element upstream of trnM gene. Phylogenetic analysis showed that A. selene was close to Saturniidae.     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

Probing recalcitrant problems in polyclad evolution and systematics with novel mitochondrial genome resources

For their apparent morphological simplicity, the Platyhelminthes or “flatworms” are a diverse clade found in a broad range of habitats. Their body plans have however made them difficult to robustly classify. Molecular evidence is only beginning to uncover the true evolutionary history of this clade. Here we present nine novel mitochondrial genomes from the still undersampled orders Polycladida and Rhabdocoela, assembled from short Illumina reads. In particular we present for the first time in the literature the mitochondrial sequence of a Rhabdocoel, Bothromesostoma personatum (Typhloplanidae, Mesostominae). The novel mitochondrial genomes examined generally contained the 36 genes expected in the Platyhelminthes, with all possessing 12 of the 13 protein-coding genes normally found in metazoan mitochondrial genomes (ATP8 being absent from all Platyhelminth mtDNA sequenced to date), along with two ribosomal RNA genes. The majority presented possess 22 transfer RNA genes, and a single tRNA gene was absent from two of the nine assembled genomes. By comparison of mitochondrial gene order and phylogenetic analysis of the protein coding and ribosomal RNA genes contained within these sequences with those of previously sequenced species we are able to gain a firm molecular phylogeny for the inter-relationships within this clade.Our phylogenetic reconstructions, using both nucleotide and amino acid sequences under several models and both Bayesian and Maximum Likelihood methods, strongly support the monophyly of Polycladida, and the monophyly of Acotylea and Cotylea within that clade. They also allow us to speculate on the early emergence of Macrostomida, the monophyly of a “Turbellarian-like” clade, the placement of Rhabditophora, and that of Platyhelminthes relative to the Lophotrochozoa (=Spiralia). The data presented here therefore represent a significant advance in our understanding of platyhelminth phylogeny, and will form the basis of a range of future research in the still-disputed classifications within this taxon.     

玉米芯木聚糖硫酸酯抗凝血活性及其机制的研究

采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和凝血酶原时间(PT)检测了玉米芯木聚糖硫酸酯(wisX-SB)的抗凝活性,结果表明wisX-SB能明显延长APTT和TT,而不影响PT,提示wisX-SB是通过内源性和/或共同途径发挥抗凝血作用的。采用发色底物法及纤维蛋白原转化实验分别考察了wisX-SB对凝血酶及纤维蛋白原的作用,结果提示wisX-SB的抗凝机制包括:直接抑制纤维蛋白原的转化;通过增强抗凝血酶III(AT-III)的活性,抑制凝血酶活性,从而达到抗凝目的。较低浓度时以前者为主,较高浓度下两者皆起作用。     

Development of New Oomycete Taxon‐specific Mitochondrial Cytochrome b Region Primers for Use in Phylogenetic and Phylogeographic Studies

Here, we describe the development of an oomycete‐specific primer pair for amplification of the cytochrome b region in plant pathogenic species that span the order Peronosporales (Phytophthora spp., downy mildews). Because of the high number of variable sites at both inter‐ and intra‐specific levels this marker provides a powerful tool for population genetics and phylogenetic studies in this taxa. We also demonstrate its potential compared with other oomycete‐specific mitochondrial markers currently available.     

POTENTIAL UTILITY OF MITOCHONDRIAL CYTOCHROME B AND ITS MRNA EDITING IN RESOLVING CLOSELY RELATED DINOFLAGELLATES: A CASE STUDY OF PROROCENTRUM (DINOPHYCEAE)1

Difficulties often occur in separating closely related dinoflagellate species. In this study, the potential utility of mitochondrial cytochrome b (cob) gene sequence and mRNA editing characteristics was assessed using Prorocentrum Ehrenberg as a model. The cob sequences and the patterns of their mRNA editing were analyzed for several Prorocentrum taxa. Results revealed little difference in cob sequence and mRNA editing characteristics between geographic populations of P. minimum (Pavillard) Schiller, while a notable difference was detected between different species (P. minimum and P. micans Ehrenberg). Furthermore, these P. minimum populations consistently formed a tight cluster on phylogenetic trees inferred from cob sequences as well as mRNA editing characteristics, whereas different Prorocentrum species were well separated, with a genetic distance of 0.0042±0.0024 for the former and 0.0141±0.0012 for the latter (P<0.01; two‐tailed t‐test). When the analysis was applied to the case of P. donghaiense Lu et Goebel and CCMP1517 strain of P. dentatum Stein, no differences were detected between these two taxa with respect to cob mRNA editing pattern and only small differences equivalent to those between P. minimum populations were detected in terms of cob sequence. On the cob sequence‐ and editing‐based phylogenetic trees, P. donghaiense and P. dentatum CCMP1517 consistently clustered together at a position sister to P. minimum. The results suggest that cob, combined with its mRNA editing, can potentially be a useful delineator of Prorocentrum species, and that P. donghaiense and P. dentatum CCMP1517 are most likely the same species and both are closely related to P. minimum.     

Fungicide control of Stenocarpella Maydis in the Nigerian Savanna

A survey of farmers' fields in the Savanna zone of Nigeria in 1999 indicated the presence of stalk and cob rots of maize at incidence rates of 15?–?43% and disease severity of 2.0?–?6.7. The causal organism was identified as Stenocarpella maydis (?=?Diplodia maydis). S. maydis was found to reduce seed germination by up to 29.2%. Laboratory and screen house experiments were used to evaluate the efficacy of six seed treatment fungicides indicated that Luxan (a local fungicide of unknown composition), benomyl (Benlate) and mancozeb (Dithane M-45) were more effective than metalaxyl?+?carboxin?+?furathiocarp (Apron-plus), carbendazin?+?maneb (Delsene M) and tetramethylthiuram disulphide?+?hexachlorobenzene (thiram?+?HCB) in controlling S. maydis. Stalk rot severity increased with increasing fertilization rates.     

Valorization of corn cob for the obtention and purification of endoglucanase produced by SSF

Agro-food by-products contain valuable nutrients which are wasted. In this work, solid-state fermentation (SSF) was carried out using corn cob as substrate for endoglucanase production. The radial growth of three fungal strains -Trichoderma harzianum T104, Aspergillus niger GH1 and Aspergillus niger NRRL3- was analyzed in order to select the most appropriate. Radial growth data were analyzed with a mixed linear model for longitudinal data and no statistically significant differences were found between both A. niger strains.Endoglucanase was separated from the extract of A. niger GH1 by fast protein liquid chromatography (FPLC).The highest endoglucanase activity was detected in fraction number three collected from FPLC corresponding to 72 h of SSF. Seven bands in a range from 24 to 50 kDa, which correspond to endoglucanase from fungal extract, were detected by zymogram analysis. According to protein quantification performed by the ImageJ software, 85% of the proteins present in the samples collected by FPLC corresponded to endoglucanase proteins. The purified endoglucanase retained about 100% of its catalytic activity at 30 °C and 50 °C and was stable in a pH range between 4.00-6.00. These properties make this isolated enzyme suitable for industrial applications such as the saccharification process for bioethanol production.