Ana C. Adam

Cloning and molecular characterization of the Δ6-desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco

The synthesis of GLA (Δ6, 9, 12-18:3) is carried out in a number of plant taxa by introducing a double bond at the Δ6 position of its precursor, linoleic acid (Δ9, 12-18:2), through a reaction catalyzed by a Δ6-desaturase enzyme. We have cloned genes encoding the Δ6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b5 domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other Δ6-desaturases in a different cluster from that of the highly related Δ8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

Lactose: The Milk Sugar from a Biotechnological Perspective

Abstract Lactose is a very important sugar because of its abundance in the milk of humans and domestic animals. Lactose is a valuable asset as a basic nutrient and the main substrate in fermentative processes that led to the production of fermented milk products, such as yogurt and kefir. In some instances, lactose also can be a problem as the causative agent of some diseases, such as lactose intolerance and galactosemia, or for being a by-product generated in huge amounts by the cheese industry. The study of the biochemical reactions leading to the synthesis and assimilation of lactose has provided valuable models for the understanding of biosynthetic and catabolic processes. Lactose-hydrolyzing enzymes are structurally and phylogenetically related to different types of beta-galactosidases and bacterial cellobiases involved in the enzymatic degradation of cellulose. Biotransformation of lactose, by either enzymatic or fermentative procedures, is important for different types of industrial applications in dairy and pharmaceutical industries.

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae.

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β‐galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose‐inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non‐selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild‐type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast‐growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro‐fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield.

Construction of a lactose-assimilating strain of baker's yeast.

A recombinant strain of bakers yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese‐making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a β‐galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread‐making, the genetic transformation of the host bakers yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two‐fold: it affords a cheap alternative to the procedure generally used for the propagation of bakers yeast, and it offers a profitable use for cheese whey. Copyright

Structural analysis of glucoamylase encoded by the STA1 gene of Saccharomyces cerevisiae (var. diastaticus).

The sequence of the STA1‐encoded glucoamylase of amylolytic Saccharomyces cerevisiae (var. diastaticus) strains shows two well‐defined regions: an amino‐terminal part rich in serine and threonine residues and a carboxy‐terminal part very similar to the catalytic domain of other fungal glucoamylases. A version of the enzyme in which most of the amino‐terminal region was deleted still has glucoamylase activity, indicating that the remaining carboxy‐terminal part forms a functional catalytic domain. Homology‐based models of the two parts of the protein have been obtained. As expected, the shortened form of the enzyme is very similar to the catalytic domain of related glucoamylases of known structure. However, the amino‐terminal part yielded a structure revealing an unexpected similarity to bacterial invasins, suggesting functional connections between several yeast proteins homologous to STA1‐encoded glucoamylase and invasins. A characteristic of Saccharomyces glucoamylase in its native form is its extreme degree of glycosylation. Despite its high molecular mass (about 300 kDa), and in contrast with what occurs with other extracellular glycoproteins produced by yeast, the enzyme does not remain attached to the cell wall, being fully and efficiently secreted into the medium, even when it is produced in large amounts by overexpression of its gene. Copyright

Construction of a Saccharomyces cerevisiae strain able to ferment cellobiose.

SummaryThe bglA gene, encoding a β-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the CYC-GAL promoter inducible by galactose. The expression of bglA-encoded activity in the strain used as a host was not sufficient to allow its growth with cellobiose as a carbon source. However, a recessive mutation in a gene designated cem1 has been obtained which, combined with the expression of β-glucosidase activity, allows the growth of S. cerevisiae on cellobiose. The expression of the bglA gene in a cemt strain confers on S. cerevisiae the capability for an efficient fermentation of cellobiose, as detected by the formation of CO2.

The ribosomal DNA of the Zygomycete Mucor miehei.

Abstract The ribosomal DNA from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70–80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi.