Impact of heat processing on the nutritional content of Gryllus bimaculatus (black cricket).

Insects are increasingly suggested as a potential novel solution to global nutrition challenges. However, limited research is available on the impact of processing methods on the nutritional content of edible insects. This trial examines the effect of heat processing on the nutritional profile of the black cricket, Gryllus bimaculatus. Adult black crickets were killed by freezing and then dried at either a low (45°C) or high (120°C) temperature followed by nutritional analysis of protein and micronutrient content. An additional set of samples was either freeze-dried or dried at 32, 45, 72 or 120°C followed by nutritional analysis of lipid content. Analysis showed that percentage protein content was significantly higher in crickets dried at 45°C, a difference of roughly 1% of the total weight. Similarly, calcium content was also significantly higher in crickets dried at 45°C, although no other measured micronutrients were affected. Additionally, the fatty acid content was significantly influenced by higher temperature processing. Freeze-drying black crickets conserved significantly more of the long-chain polyunsaturated fatty acids than drying at 120°C. Insects hold potential as a source of essential nutrients and fatty acids; however, consideration must be given to heat processing at high temperatures as this may affect the nutritional profile.

Isolation and characterization of a cDNA encoding a Delta8 sphingolipid desaturase from Aquilegia vulgaris.

We have isolated a cDNA encoding the Delta(8) sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis - and trans -desaturated sphingoid bases which were not present in the wild-type yeast cells. The Delta(8) desaturated products co-eluted with known Delta(8)-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Delta(8) long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.

Genomic and functional characterization of polyunsaturated fatty acid biosynthesis in Caenorhabditis elegans.

The biosynthetic pathway for polyunsaturated fatty acids in the model animal Caenorhabditis elegans was examined in the context of the completed genome sequence. The genomic organization and location of seven desaturase genes and one elongase activity, all previously identified by functional characterization, were elucidated. A pathway for the biosynthesis of polyunsaturated fatty acids in C. elegans was proposed based on these genes. The role of gene duplication in enzyme evolution and proliferation is discussed.

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from alpha-linolenic acid. These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways. The C. elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates.

Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.

The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.

An unusual desaturase in Aquilegia vulgaris.

Aquilegia vulgaris seed oil contains high levels of the rare fatty acid columbinic acid (18:3 Delta(5,9,12)), which is unusual in having the double bond at the Delta(5) carbon in the trans configuration. Columbinic acid was found to be a seed-specific fatty acid not only present in the storage oil but also in membrane lipids. Several putative gene fragments have been isolated from plant RNA with sequences similar to previously characterized 'front-end' desaturases. Functional characterization of the Aquilegia cDNA is underway.

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans.

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.

Two fatty acid delta9-desaturase genes, ole1 and ole2, from Mortierella alpina complement the yeast ole1 mutation.

Genes encoding two distinct fatty acid delta9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, delta9-1 and delta9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The delta9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40-60% identity to the delta9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound delta9-desaturases. LM9 and delta9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina delta9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone delta9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the delta9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.

Plant desaturases: harvesting the fat of the land.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.

Functional identification of a fatty acid delta5 desaturase gene from Caenorhabditis elegans.

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid delta5 desaturase. Saccharomyces cerevisiae expressing the full-length cDNA was able to convert di-homo-gamma-linolenic acid to arachidonic acid, thus confirming delta5 desaturation. The 1341 bp delta5 desaturase sequence contained an N-terminal cytochrome b5 domain and was located within a kilobase of the C. elegans delta6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a delta5 desaturase gene isolated from an animal.

Isolation of a Delta5-fatty acid desaturase gene from Mortierella alpina.

Arachidonic acid (C20:4 Delta5,8,11,14) is a polyunsaturated fatty acid synthesized by the Delta5-fatty acid desaturation of di-homo-gamma-linolenic acid (C20:3 Delta8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Delta5-fatty acid desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Delta5-desaturase is the first example of a cloned Delta5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b5.