Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity.

A membrane-bound protease activity that specifically converts Big endothelin-1 has been purified from bovine endothelial cells (FBHE). The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT-PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture and oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE-1.

The unique properties of brain endothelial cells, which form the blood-brain barrier, are reflected by the expression of specific cell surface molecules. We report here the purification, cloning and expression of one such molecule which is recognized by HT7 monoclonal antibodies. The HT7 antigen is a highly glycosylated 45-52 kd protein localized in brain endothelial cells, kidney epithelial cells and erythroblasts. The protein was purified to homogeneity from plasma membrane proteins isolated from all three sources using immunoaffinity chromatography and reverse phase HPLC. The aminoterminal amino acid sequences of the proteins were found to be identical. Based on amino acid sequence information, specific primers were designed and the polymerase chain reaction was used to obtain a full length cDNA clone. The nucleotide sequence encoded a novel glycoprotein with two C2-like immunoglobulin related domains, one transmembrane domain and a cytoplasmic tail. Expression of the transfected cDNA in COS cells resulted in the appearance of the HT7 antigen on the surface of these cells. On the basis of our results we propose that the protein may be a receptor involved in cell surface recognition at the blood-brain barrier.

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