Francesco Rodriguez

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilo‐bases 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.

Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes.

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 H2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H2 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 H2 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 H2 production, but all 3 mutants sustained a longer-term H2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H2 photoproduction using actual waste-derived substrates.

Engineering of Peptide Synthetases KEY ROLE OF THE THIOESTERASE-LIKE DOMAIN FOR EFFICIENT PRODUCTION OF RECOMBINANT PEPTIDES

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 functionalsfp gene.

Characterization of the surfactin synthetase multi-enzyme complex

Three subunits (srfAORF1, srfAORF2 and srfAORF3) of the Bacillus subtilis surfactin synthetase multi-enzyme complex have been identified by SDS-PAGE and Western blot analyses. In accordance with the sequence analysis of the surfactin (srfA) operon, the protein subunits have a molecular mass of 402,000 Da, 401,000 Da and 144,000 Da, respectively. Confirmation of the identity of the proteins was obtained by analysing the total protein content of a number of mutant strains which harbour deletions or insertions either in the srfA promoter or in different positions within the srfA operon. The three subunits were partially purified by means of a series of chromatographic steps including ion-exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The partially purified proteins were used in activity assays to establish their amino-acid recognition specificity. In agreement with previously published results, this analysis showed that srfAORF1 recognizes glutamic acid and Leu, srfAORF2 recognizes Val, aspartic acid and Leu and srfAORF3 recognizes Leu. In addition, the subunits can activate and bind other amino acids, although with lower specificity. In particular, srfAORF1 binds Val, Ile and aspartic acid, srfAORF2 glutamic acid and Ile and srfAORF3 Ile and Val. Competition experiments as well as sequence comparison strongly suggest that the Leu binding sites of the three subunits can accept, beside Leu, Ile and Val. The kinetic parameters of srfAORF3 for Leu, Ile and Val have been determined.

An operon encoding a novel ABC-type transport system in Bacillus subtilis

Downstream from the surfactin synthetase operon in Bacillus subtilis a new operon-type structure has been localized which, on the basis of sequence determination, potentially encodes an ABC-type transport system. The 268 amino acid protein, the product of orf1, represents the solute-binding component of the system whereas the orf2 product, a 234 amino acid protein, is the transmembrane component. Finally orf3 potentially encodes a typical 241 amino acid ATP-binding protein involved in energy supply. Comparison of the three proteins with the subunits of other ABC-type systems suggests that this new system is involved in amino acid transport.

In vivo evolution of the Rhodococcus sp. strain DS7: selection of recombinants able to desulfurize both dibenzothiophene and benzothiophene

Rhodococcus sp. DS7, isolated from a polluted soil, has shown good desulfurizing activity towards dibenzothiophene (DBT) and its derivatives, but is not able to desulfurize benzothiophene (BT), the other thiophenic molecule recalcitrant to the chemical hydrodesulfurization (HDS) process, and most abundant in gasoline. To select a Rhodococcus DS7 derivative strain able to desulfurize both DBT and BT, we took advantage of the verified capacity of this strain to integrate exogenous DNA randomly, with a good efficiency. Heterologous chromosomal DNA, digested with restriction enzymes, from two BT but not DBT desulfurizing strains, Rhodococcus sp. ATCC 27778 and Gordonia sp. ATCC 19067, was electroporated into Rhodococcus DS7. Selection on minimal medium with BT as sole sulfur source allowed us to isolate several DS7 derivatives with the capacity to desulfurize both thiophenic molecules. Two strains, one derived from the integration and recombination of DNA from ATCC 27778, and the other from ATCC 19067, have been partially characterized. These recombinant microorganisms are an interesting starting point to develop new biodesulfurization processes.

Molecular Biology of Hydrogenases

Hydrogenases, found in a wide variety of organisms, catalyze either the consumption or the production of H2 in response to different physiological conditions. In recent years, a large body of biochemical and genetic data on enzymes isolated from different sources has contributed to elucidating fundamental aspects about their catalytic properties and gene organization. In addition, the recently obtained crystal structure of a [Ni-Fe] hydrogenase sheds light on structure-function relationships in these enzymes. With the ultimate goal of engineering photosynthetic strains to improve their light-dependent hydrogen evolution capacity, we have characterized at the molecular level hydrogenases from different microbial sources. In particular, we have focused our attention on the H2-evolving hydrogenases from Pyrococcus furiosus and Acetomicrobium flavidum and on the uptake hydrogenase system from Rhodobacter sphaeroides RV. This microorganism represents the species selected for use in a photobioreactor, as planned in the Japanese hydrogen production project in which we are involved.