1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l(-1) of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.

SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

The cDNA for porcine mitochondrial NADP-specific isocitrate dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction. Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide. Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence. The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific isocitrate dehydrogenase and to be immunoreactive with an antiserum against the yeast protein. Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme. The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent isocitrate dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-isocitrate site of the procaryotic enzyme.

During vertebrate embryogenesis retinoic acid (RA) synthesis must be spatiotemporally regulated in order to appropriately stimulate various retinoid signaling pathways. Various forms of mammalian aldehyde dehydrogenase (ALDH) have been shown to oxidize the vitamin A precursor retinal to RA in vitro. Here we show that injection of Xenopus embryos with mRNAs for either mouse Aldh1 or mouse Raldh2 stimulates RA synthesis at low and high levels, respectively, while injection of human ALDH3 mRNA is unable to stimulate any detectable level of RA synthesis. This provides evidence that some members of the ALDH gene family can indeed perform RA synthesis in vivo. Whole-mount immunohistochemical analyses of mouse embryos indicate that ALDH1 and RALDH2 proteins are localized in distinct tissues. RALDH2 is detected at E7.5-E10.5 primarily in trunk tissue (paraxial mesoderm, somites, pericardium, midgut, mesonephros) plus transiently from E8.5-E9.5 in the ventral optic vesicle and surrounding frontonasal region. ALDH1 is first detected at E9.0-E10. 5 primarily in cranial tissues (ventral mesencephalon, dorsal retina, thymic primordia, otic vesicles) and in the mesonephros. As previous findings indicate that embryonic RA is more abundant in trunk rather than cranial tissues, our findings suggest that Raldh2 and Aldh1 control distinct retinoid signaling pathways by stimulating high and low RA biosynthetic activities, respectively, in various trunk and cranial tissues.

The gastrointestinal tract is a major site of alcohol dehydrogenase (ADH) activity in humans and rodents. Because class I ADH (ADH-I) and class IV ADH (ADH-IV), but not class III ADH (ADH-III), function as retinol dehydrogenases in vitro and may thus participate in retinoid signaling needed for epithelial differentiation, the aim of this study was to determine the localization of these enzymes along the gastrointestinal tract. Specific antibodies were used to examine the tissue distribution of all three known classes of mouse ADH by Western blotting, and cellular localization was determined by immunohistochemistry. ADH-I was detected primarily in the intestine, liver, kidney, adrenal, and uterus, with detection of ADH-III in all tissues examined, and detection of ADH-IV primarily in the esophagus, stomach, adrenal, skin, ovary, and epididymis. Along the gastrointestinal tract, ADH-III was not specifically localized, whereas ADH-I was localized exclusively in the villus epithelium of the small intestine and absorptive epithelium of the large intestine, with ADH-IV being localized exclusively in the basal and suprabasal epithelial cells of the esophagus and gastric pit surface epithelium of the stomach. The ADH localization patterns observed are consistent with ADH-I and ADH-IV, but not ADH-III, functioning physiologically in retinol metabolism needed for epithelial differentiation. Our results further suggest that the functions of ADH-I and ADH-IV are regionally restricted to the lower and upper components, respectively, of the gastrointestinal epithelium, a finding that may relate to the different efficiencies of these two enzymes for retinol oxidation, as well as to the different susceptibilities of the upper and lower digestive tracts for ethanol-induced cancers.