A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (CH), a plant sesquiterpene with versatile industrial applications, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.

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Protein gene product (PGP) 9.5 is a new brain-specific protein originally detected by high-resolution two-dimensional electrophoresis of the soluble proteins of human brain and other organs. We have purified this protein from human brain and raised a rabbit antihuman PGP 9.5 antiserum. The protein has a monomer molecular weight of approximately 27,000 and is present in brain at concentrations at least 50 times greater than in other organs. Immunoperoxidase labelling has localised PGP 9.5 to neurones in the human cerebral cortex with no evidence of staining of glial elements. PGP 9.5 is estimated to be present in brain at concentrations of 200-500 micrograms/g wet weight and represents a major protein component of neuronal cytoplasm. This new neurone-specific cytoplasmic marker may prove useful in studies of neuronal development and in the detection of neuronal damage in disease of the nervous system.

Various monosaccharides, oligosaccharides and small polysaccharides were labelled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and the resulting fluorescent derivatives were separated by high-resolution PAGE. The electrophoretic mobilities of the labelled saccharides are related largely to the compounds' Mr values, but they are also influenced by the individual chemical structures of the saccharides. Various positional isomers and some epimers, for instance galactose and glucose, were resolved. Oligosaccharide and small polysaccharide derivatives, prepared from an enzymic digest of starch, each differing in size by a single hexose residue and with a range of degrees of polymerization from 2 to 26, were all resolved in a single gel. The method was relatively rapid and simple to perform. It enabled multiple samples to be analysed in parallel with high sensitivity. The fluorescent-labelling procedure was virtually quantitative. As little as 1 pmol of ANTS-labelled saccharide was detected photographically when the gels were illuminated by u.v. light. When the gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrate moieties of glycoconjugates and other naturally occurring oligosaccharides.

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An antiserum to human 14-3-3 protein has been produced in rabbits. The protein was a poor antigen and attempts to improve immunogenicity were unsuccessful. A radioimmunoassay was developed using the antiserum, 125I-14-3-3-2, and unlabelled 14-3-3-2 as standards. The assay had a sensitivity limit of 2.5 ng.ml-1. The minor component of human 14-3-3 protein (14-3-3-1 protein) cross-reacted to approximately 10% in the assay. Human tissues were surveyed for 14-3- protein by two-dimensional electrophoresis and by radioimmunoassay. Two-dimensional electrophoresis showed a 14-3-3 protein complex in brain, intestine, and testis, but not in other tissues. Radioimmunoassay showed that although brain had the highest concentration of 14-3-3 (13.3 microgram.mg-1 soluble protein), immunoreactivity was present in all tissues, with the concentration in intestine and testis approaching 50% of the brain level. Lower levels (less than 1.0 microgram.mg-1 soluble protein) were seen in liver, kidney, skeletal muscle, and erythrocytes. The immunoreactivity present in tissues other than brain showed the same molecular weight and charge characteristics and authentic 14-3-3 protein. The radioimmunoassay also detected 14-3-3 protein in serum (50 ng.ml-1) and in CSF (5-130 ng.ml). The immunoreactivity present in CSF appeared to be intact 14-3-3 protein. CSF 14-3-3 levels were measured in 82 patients with various neurological disorders. Measurements of this protein did not appear sufficiently discriminating to be of diagnostic value.

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