The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

Three differently metabolically engineered strains, 2 single PHA ) and Hup ) mutants and one double PHA ) /Hup ) mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H 2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H 2 recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup ) and the double PHA ) / Hup ) mutants, but not the single PHA ) mutant, exhibited increased rates of H 2 photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup ) mutant showed higher rates of H 2 production, but all 3 mutants sustained a longer-term H 2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H 2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H 2 photoproduction using actual waste-derived substrates.

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Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.Two mechanisms for the biosynthesis of peptides are known to exist in bacteria and fungi, ribosomal synthesis and production by peptide synthetases. These are large enzymatic complexes responsible for the synthesis of hundreds of types of peptides, some of which have immunoactive, antibiotic, antifungal, or surfactant properties. Whereas polypeptides produced by ribosomal synthesis typically contain only the amino acids directly specified by the triplets of the genetic code, peptides built on synthetases often contain unusual amino or hydroxy acids as building blocks that are not present in proteins. The amino acids can be modified by peptide synthetases either through methylation, hydroxylation, or enantiomerization. The peptides are typically short (up to about 20 residues) and can be linear, circular, or branched (1, 2). Peptide synthesis proceeds by the "multiple carrier thiotemplate mechanism" (3, 4), and many of the details of these systems remain to be investigated experimentally. According to this mechanism each domain recognizes a specific amino (or hydroxy) acid that is covalently bound to the cofactor via a thioester bond after activation to the corresponding acyladenylate derivative. The growth of the polypeptide chain thus occurs through a series of thioester bond cleavages and the simultaneous formation of amide or ester bonds in the peptide. At the end of synthesis of each peptide, the chain is thought to be released from the enzyme by a thioesterase (TE) 1 activity encoded in the synthetase gene (1). Peptide synthetases are interesting not only from a scientific and evolutionary point of view but also for their biotechnological potential. Many enzymatically synthesized peptides are in fact biologically active, and some of them are industrially produced, among which are cyclosporins, surfactin, and fungicides. A growing number of laboratories are involved in applied research projects focused on the isolation and characterization of new peptide synthetases and on the genetic manipulation of peptide synthetase genes to optimize peptide production and to genetically modify the sequence of the peptides produced. In fact, the structural organization...