Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

O dealkylation and aliphatic and aromatic hydroxylation of 3-methoxy-17 beta-estradiol by Aspergillus alliaceus

Aspergillus alliaceus UI 315 was examined for its ability to metabolize 3-methoxy-17 beta-estradiol. Preparative-scale incubations with this substrate afforded good yields of 6 beta-hydroxy-17 beta-estradiol, 4-hydroxy-17 beta-estradiol, and 4,6 beta-dihydroxy-17 beta-estradiol, which were identified by high-pressure liquid chromatography, 1H and 13C nuclear magnetic resonance, and high-resolution mass spectrometry.     

Differential expression in male and female mouse liver of very similar mRNAs specified by two group 1 major urinary protein genes.

The major urinary proteins of the mouse are encoded by a large multigene family composed of several distinct groups of genes distinguished by differences in sequence and expression characteristics. The genes in the largest group (group 1) show greater than 99% pairwise similarity in their exons. By hybridization between RNA and a specifically designed oligonucleotide, we confirmed that genes of this group are expressed mainly in the liver. By using additional gene-specific oligonucleotide probes, we have been able to distinguish between the species of mRNA corresponding to two of these genes and to measure their abundance in male and female liver. Both mRNAs are present in male liver at high but different levels. Both are also present in female liver, one at a much lower level than in the male and the second at a very low level indeed. Both are present at male levels in the livers of females induced with testosterone. These results show unequivocally that the expression of different group 1 Mup genes is differentially influenced by the hormonal status of the mouse.     

The 5'' noncoding region of the human leukemia-associated oncogene BCR/ABL is a potent inhibitor of in vitro translation.

The mRNA encoding the chimeric BCR/ABL oncogene, which is transcribed from the Philadelphia chromosome in human chronic myelogenous leukemia, has a 5' noncoding sequence greater than 500 bases in length which is highly GC rich and contains a short open reading frame. This untranslated sequence has a dramatic inhibitory effect upon translational efficiency in vitro. However, when BCR/ABL message is expressed in certain cell types such as the NIH 3T3 cell line, the 5' noncoding region has little inhibitory effect on translational efficiency.     

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.     

The effect of alkaline borohydride treatment onN-linked carbohydrates of glycoproteins

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc(1-N) Asn with alkalil alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field¹H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing theN-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing theN-linked carbohydrates as oligosaccharide-alditols.Abbreviation RNase ribonuclease     

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.     

Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation.

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.