Ira I. G. S. Verwoert

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene, encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.

Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

SummaryFrom six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter.

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis.

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severly affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.

MOLECULAR CHARACTERIZATION OF AN ESCHERICHIA COLI MUTANT WITH A TEMPERATURE-SENSITIVE MALONYL COENZYME A-ACYL CARRIER PROTEIN TRANSACYLASE

The temperature‐sensitive malonyl CoA‐ACP transacylase found in the Escherichia coli strain LA2‐89, carrying the fabD89 allele, was shown to result from the presence of an amber mutation in the fabD gene, at codon position 257, in combination with the supE44 genotype of this strain. The truncated form of the protein produced as the result of the amber mutation was demonstrated to be enzymatically inactive, whereas amber suppression rendered the resulting enzyme temperature labile. Site‐directed mutagenesis of codon 257 revealed a requirement for an aromatic amino acid at this position in the polypeptide chain, to assure temperature stability of the enzyme.

A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase.

In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated. Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.

Modification of the fatty-acid composition in lipids of the oleaginous yeast Apiotrichum curvatum by intraspecific spheroplast fusion

SummaryModification of the fatty-acid composition in lipids of the oleaginous yeast Apiotrichum curvatum has been achieved by means of spheroplast fusion between a methionine auxotrophic mutant and an unsaturated fatty acid mutant. Fusion products were a result of nuclear hybridization as determined by flow-cytometric DNA-content analyses. A broad range of fatty acid composition was observed in lipids of different hybrids. In general the level of saturated fatty acids in lipids of hybrid strains is higher than in wild-type A. curvatum and in some hybrids even approaches cocoa butter. Intraspecific spheroplast fusion seems a promising approach for the production of cocoa butter equivalents.

Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds

In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.

The molecular basis of the high linoleic acid content in Petunia seed oil: analysis of a seed-specific linoleic acid mutant.

From a random transposon mutagenesis experiment, using Petunia line W138, a seed-specific linoleic acid mutant was isolated. The tagged gene was cloned and identified as a microsomal Delta(12) desaturase. Expression of the gene, however, was constitutive and not, as might have been expected, seed-specific. Moreover, self-fertilized homozygous mutants still contain 40% 18:2 in the seed lipid fraction. This suggests that at least two (seed-specific) Delta(12) desaturase genes are responsible for the high linoleic acid content in Petunia seed oil. Five members of the microsomal Delta(12) desaturase gene family have been identified and isolated. Data are presented on the molecular characterization and tissue-specific expression of these genes, which suggest that, in Petunia, the flux through the prokaryotic and eukaryotic pathways of lipid synthesis might be different from the situation found in Arabidopsis.

Transgenic Expression of Bacterial Fas Components in Rapeseed

Fatty acid biosynthesis in plants is catalysed by the so-called FAS II fatty acid synthetase complex, which very much resembles the bacterial FAS system. The close evolutionary relationship between the plant and bacterial FAS system, as exemplified by structural and functional similarities, was unambiguously demonstrated by Kater et al. [1]. Genereplacement studies showed that a single component of the plant FAS system (enoyl-ACP reductase) can functionally replace its bacterial counterpart. Recently, it was shown that the E. coli fabD gene, encoding malonylCoA-ACP transacylase (MCAT), under the control of a napin promoter, can be expressed at a high level and in a seed- and developmental specific manner, in transgenic plants [2]. Plastid targeting of the bacterial FAS component was successfully accomplished by the use of the B. napus enoyl-ACP reductase leader sequence [3] . In this study, data are presented on the transgenic expression of the 1) bacterial fabH, encoding 3-ketoacyl-ACP synthase III (KAS III), and 2) the bacterial fabA, encoding 3hydroxy decanoyl thioester dehydrase (HDD), in rapeseed. Overexpression of HDD did not result in the production of bacterial-type fatty acids (e.g. cis-vaccenic acid) in transgenic plants. Overexpression of bacterial KAS III, however, resulted in increased relative amounts of C 18:2 and C18:3, and a decrease in the C 18:1 content of the storage lipid fraction. From these data it seems evident that bacterial FAS proteins can interact with the plant FAS system, and that the expression of heterologous FAS components in transgenic plants can contribute to elucidate the regulatory steps in plant lipid biosynthesis.