Elisabetta Franchi

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.

A putative new peptide synthase operon in Bacillus subtilis: partial characterization.

A large operon-type structure has been located between the gltA and citB loci on the Bacillus subtilis chromosome. On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases. The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.

A new human growth hormone production process using a recombinant Bacillus subtilis strain

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.

Assisted phytoremediation of a multi-contaminated soil: Investigation on arsenic and lead combined mobilization and removal

The removal of contaminants from an earthy matrix by phytoremediation requires the selection of appropriate plant species and a suitable strategy to be effective. In order to set up an assisted phytoremediation intervention related to a disused industrial site affected by an arsenic and lead complex contamination, an extensive experimental investigation on micro and mesocosm scale has been conducted. Particular attention was given to the choice of plant species: using crop plants (Lupinus albus, Helianthus annuus and Brassica juncea) a series of parallel test campaigns have been realized to investigate different scenarios for the reclamation. With regard to the arsenic contamination, which is certainly the most worrying, the possibility of employing a hyper-accumulator species (Pteris vittata) has also been investigated, highlighting advantages and difficulties associated with such an approach. The application of various mobilizing agents in different concentrations was tested, in order to maximize the extraction efficiency of plants in respect of both contaminants, showing the necessity of a chemically assisted approach to promote their uptake and translocation in the shoots. Phosphate addition appears to produce the desired results, positively affecting As phyto-extraction for both hyper-accumulator and crop plants, while minimizing its toxic effects at the investigated concentrations. With regard to Pb, although tests with EDDS have been encouraging, EDTA should be preferred at present due to lower uncertainties about its effectiveness. The performed tests also improved the addition of mobilizing agents, allowing the simultaneous removal of the two metals despite their great diversity (which in general discourages such approach), with significant saving of time and an obvious improvement of the overall process.

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.