Advances and developments in strategies to improve strains of Saccharomyces cerevisiae and processes to obtain the lignocellulosic ethanol--a review.

The conversion of biomass into ethanol using fast, cheap, and efficient methodologies to disintegrate and hydrolyse the lignocellulosic biomass is the major challenge of the production of the second-generation ethanol. This revision describes the most relevant advances on the conversion process of lignocellulose materials into ethanol, development of new xylose-fermenting strains of Saccharomyces cerevisiae using classical and modern genetic tools and strategies, elucidation of the expression of some complex industrial phenotypes, tolerance mechanisms of S. cerevisiae to lignocellulosic inhibitors, monitoring and strategies to improve fermentation processes. In the last decade, numerous engineered pentose-fermenting yeasts have been developed using molecular biology tools. The increase in the tolerance of S. cerevisiae to inhibitors is still an important issue to be exploited. As the industrial systems of ethanol production operate under non-sterile conditions, microbial subpopulations are generated, depending on the operational conditions and the levels of contaminants. Among the most critical requirements for production of the second-generation ethanol is the reduction in the levels of toxic by-products of the lignocellulosic hydrolysates and the production of low-cost and efficient cellulosic enzymes. A number of procedures have been established for the conversion of lignocellulosic materials into ethanol, but none of them are completely satisfactory when process time, costs, and efficiency are considered.

The role played by endogenous and exogenous electric fields in DNA signaling and repair.

Comments are made and new insights are provided on the key role played by endogenous and exogenous electric fields, where the former starts and conducts the repairing chain, while the latter is able to scramble the completion of the repair process and, as a consequence, may have important potential as a radiation sensitizer for clinical application.

The role played by endogenous and exogenous electric fields in DNA signaling and repair

Comments are made and new insights are provided on the key role played by endogenous and exogenouselectric fields, where the former starts and conducts the repairing chain, while the latter is able to scramblethe completion of the repair process and, as a consequence, may have important potential as a radiationsensitizer for clinical application.

In vitro effects of gamma radiation from 60Co and 137Cs on plasmid DNA.

The effects of gamma radiation from (60)Co and (137)Cs on DNA in aqueous solution are studied experimentally. Using an improved plasmid purification protocol and improved electrophoretic gel analysis techniques provided results with relatively small uncertainties. The results are compared with both theoretical and experimental results. In particular, the results obtained here are discussed in the light of recent discussion on supposed differences of the effects induced by gamma radiation from (60)Co and (137)Cs. We find that the effects of both types of gamma radiation are similar.

Enhancement of Ty transposition at the ADH4 and ADH2 loci in meiotic yeast cells.

Genome polymorphism in the yeast Saccharomyces cerevisiae is frequently the result of transposition and recombination events involving Ty elements. The activity of these retrotransposons is closely integrated with the life cycle of the host. Ty transcription is repressed in diploid, but not haploid, cells and is induced by certain stress conditions. We have found that Ty transposition at the ADH4 and ADH2 loci is not only active, but 50-fold more frequent in meiotic yeast than in mitotic cells. These data provide a further example of the success of Ty elements in maximising their own chances of spread and survival while minimising the risks to the host yeast population.

Further characterization of the yeast pso4-1 mutant: interaction with rad51 and rad52 mutants after photoinduced psoralen lesions.

The pso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction between pso4-1 and rad51 and between pso4-1 and rad52 mutants indicated that the PSO4 gene belongs to the RAD52 epistasis group for strand-break repair. Moreover, the presence of the pso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of the rad51 and rad52 mutants. Complementation tests using heterozygous diploid strains showed that the pso4 protein might interact with the rad52 protein during repair of 8-mop photolesions. The pso4-1 mutant, even though defective in inter- and intra-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to the rad51 mutant, pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that the PSO4 gene belongs to the RAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.

Immunogenic properties of the M. leprae recombinant 18-kDa antigen purified from Saccharomyces cerevisiae; enhancement of delayed-type hypersensitivity after gamma-irradiation.

In this paper we report the purification and study of the immunogenic properties of the Mycobacterium leprae 18-kDa protein antigen produced and secreted by the yeast Saccharomyces cerevisiae, using an expression system we recently described [Biotech. Lett. 16 (1994) 1241-1246]. The 18-kDa protein was purified from the yeast culture media by precipitation, ion exchange chromatography (MonoQ) and exclusion size chromatography (Sephacryl S-100). The biological properties of the recombinant protein, previously irradiated with gamma rays, were assayed by immunization of mice. Humoral and cellular responses, monitored by antibody production and delayed-type hypersensitivity, respectively, were obtained. Furthermore, gamma-irradiation of the recombinant protein prior to the administration was shown to significantly potentiate the T-cell response. The data suggest that this irradiated recombinant antigen could be used in a more sensitive standardized skin test to monitor M. leprae infection.

A new promoter-probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene.

A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp-type was constructed so as to carry multiple unique cloning sites at the 5' end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.

The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.

The alpha-amylase gene as a marker for gene cloning: direct screening of recombinant clones.

We report the construction and use of a new system for the direct screening of recombinant clones after transformation. The system uses a Bacillus subtilis-Escherichia coli shuttle vector that carries the B. subtilis structural gene for alpha-amylase. Insertion of foreign DNA into this gene results in a loss of amylolytic activity in the host cells that can be assayed using a simple and inexpensive staining procedure.

PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae.

The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle. As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1. Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS). Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays. In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2. Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E. coli SOS repair pathway.

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae.

The induction of mitotic gene conversion and crossing-over in Saccharomyces cerevisiae diploid cells homozygous for the pso4-1 mutation was examined in comparison to the corresponding wild-type strain. The pso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for the pso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of the PSO4 gene. On the other hand, the pso4-1 mutant is mutationally defective for all agents used. Therefore, the pso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that the PSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between the pso4-1 mutation and the RecA or LexA mutation of Escherichia coli.