Wilfried Lühs

New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry.

Oils and fats are the most important renewable raw materials for the chemical industry. Hitherto, industrial oleochemistry has concentrated predominantly on the carboxy functionality of fatty acids but, more recently, modern synthetic methods have been applied extensively to fatty compounds for the selective functionalization of the alkyl chain. Radical, electrophilic, nucleophilic, and pericyclic as well as transition metal catalyzed additions to the C-C double bond of, for example, oleic acid as the prototype of a readily accessible, unsaturated fatty acid have led to a large number of novel fatty compounds from which interesting properties are expected. Functionalization of C-H bonds in the alkyl chain is also feasible with remarkable selectivity. Effective and highly versatile catalysts for the metathesis of esters of unsaturated fatty acids have been developed, which lead to new and interesting omega-unsaturated fatty acids. The epoxidation of unsaturated fatty acids has been developed extensively. Enzymatic reactions allow syntheses with high selectivity and yield of mono- and diglycerides and esters of carbohydrates with a variety of surfactant properties. Regio- and enantioselective microbial hydrations and hydroxylations widen the spectrum of selective reactions. Of considerable significance is that, with the use of gene technology, natural oils and fats have been improved significantly and will be improved still further, insofar as they show a more uniform and often unusual fatty acid spectrum. Numerous fatty acids are now available in a purity which makes them attractive for synthesis and as raw materials for the chemical industry.

Analysis of Genetic Diversity in the Brassica napus L. Gene Pool Using SSR Markers

Genetic diversity throughout the rapeseed (Brassica napus ssp. napus) primary gene pool was examined by obtaining detailed molecular genetic information at simple sequence repeat (SSR) loci for a broad range of winter and spring oilseed, fodder and leaf rape gene bank accessions. The plant material investigated was selected from a preliminary B. napus core collection developed from European gene bank material, and was intended to cover as broadly as possible the diversity present in the species, excluding swedes (B. napus ssp. napobrassica (L.) Hanelt). A set of 96 genotypes was characterised using publicly available mapped SSR markers spread over the B. napus genome. Allelic information from 30 SSR primer combinations amplifying 220 alleles at 51 polymorphic loci provided unique genetic fingerprints for all genotypes. UPGMA clustering enabled identification of four general groups with increasing genetic diversity as follows (1) spring oilseed and fodder; (2) winter oilseed; (3) winter fodder; (4) vegetable genotypes. The most extreme allelic variation was observed in a spring kale from the United Kingdom and a Japanese spring vegetable genotype, and two winter rape accessions from Korea and Japan, respectively. Unexpectedly the next most distinct genotypes were two old winter oilseed varieties from Germany and Ukraine, respectively. A number of other accessions were also found to be genetically distinct from the other material of the same type. The molecular genetic information gained enables the identification of untapped genetic variability for rapeseed breeding and is potentially interesting with respect to increasing heterosis in oilseed rape hybrids.

Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.

Seed-specifically expressed β-ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type. The genes from the two cultivars were found to be nearly identical. They encode proteins of 507 amino acids, the sequences of which differ only at position 282. The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus. In yeast cells the β-ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products. A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants. Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil. On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil.

Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.)

In addition to the normal-type sunflower (Helianthus annuus L.) where linoleic acid is the major seed fatty acid, a dominant negative high-oleic mutant with oleic acid as the predominant fatty acid was previously obtained. We report the isolation and characterization of three different cDNA sequences, designated Ha89FAD2-1, Ha89FAD2-2, and Ha89FAD2-3, encoding sunflower microsomal oleate desaturases (FAD2), using a PCR strategy. All three deduced amino acid sequences showed significant homology to the known plant FAD2 sequences. Genomic Southern blot analysis revealed that at least one copy of each of these genes is present in the sunflower genome, except for the FAD2-1 gene from the high-oleic mutant, which might be duplicated. The FAD2-2 and FAD2-3 genes were weakly expressed in all tissues studied from both varieties. In contrast, the FAD2-1 gene was expressed strongly and exclusively in developing embryos of normal-type sunflower, whereas its expression in high-oleic developing embryos was drastically reduced. Functional expression of the corresponding cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Furthermore, the FAD2-1 gene from the high-oleic variety also expresses a fully active enzyme. These results suggest that the high-oleic mutation in sunflower interferes with the regulation of the transcription of the seed-specific FAD2 gene.

Characterisation of plant tocopherol cyclases and their overexpression in transgenic Brassica napus seeds

Tocopherols, collectively known as vitamin E, are only synthesised in photosynthetic organisms. Tocopherol cyclase (TC) catalyses the formation of the chromanol headgroup of the various tocopherol isoforms. TCs from Arabidopsis and maize (Zea mays) were expressed in Escherichia coli and purified. Analysis of the enzymatic properties revealed similarities but also differences between the two enzymes. Overexpression of chimeric TC gene constructs in developing seeds of transgenic rapeseed plants enhanced and modified the relative abundance of individual tocochromanol species in the seed oil, indicating a regulatory function of the enzyme in prenyllipid metabolism.

Neue Synthesen mit Ölen und Fetten als nachwachsende Rohstoffe für die chemische Industrie

Ole und Fette sind die wichtigsten nachwachsenden Rohstoffe der chemischen Industrie. Die industrielle Oleochemie konzentriert sich bisher uberwiegend auf die Carboxyfunktion der Fettsauren, doch wurden in letzter Zeit auch moderne Synthesemethoden zur selektiven Funktionalisierung in der Alkylkette auf Fettstoffe angewandt. Additionen an die C-C-Doppelbindung z. B. von Olsaure als Prototyp einer gut zuganglichen ungesattigten Fettsaure fuhrten zu einer Vielzahl neuartiger Fettstoffe mit interessanten Eigenschaften. Auch Funktionalisierungen von C-H-Bindungen in der Alkylkette sind bemerkenswert selektiv moglich. Es wurden effektive Katalysatoren fur die Metathese ungesattigter Fettsaureester entwickelt, die zu vielfaltig einsetzbaren ω-ungesattigten Fettsauren fuhrt. Die Epoxidierung ungesattigter Fettsauren wurde wesentlich weiterentwickelt. Enzymatische Reaktionen erlauben mit hoher Selektivitat und Ausbeute die Synthese von Mono- und Diglyceriden und von Kohlenhydratestern mit interessanten oberflachenaktiven Eigenschaften. Regio- und enantioselektive mikrobielle Hydratisierungen und Hydroxylierungen erweitern das Spektrum der selektiven Reaktionen. Die nativen Ole und Fette wurden durch Pflanzenzuchtung (auch gentechnisch) betrachtlich verbessert, und zahlreiche Fettsauren stehen heute in genugender Reinheit fur die Synthese und als Rohstoff fur die chemische Industrie zur Verfugung.

RECENT DEVELOPMENTS AND PERSPECTIVES OF INDUSTRIAL RAPESEED BREEDING

Due to substantial progress in breeding and cultivation practice rapeseed has become the worlds third most important source of vegetable oil. Modification of the fatty acid composition to make rapeseed oil more competitive in various segments of the food and industrial oil markets has been an important objective of plant breeding and molecular genetics in recent years. While making up the primary demand by food and animal feed industry furnished by “double-low” quality rapeseed, so-called “canola”, interest increased to produce “Biodiesel” feedstocks or special materials being directed to several industrial niche markets, because of their higher value than commodity oils. Rapeseed oil is unique in having a large spectrum of usability and good properties for non-food applications, such as relatively homogeneous composition, high degree of refinement, freedom from contaminants, and also biodegradability, giving it advantages over petrochemicals. Consequently, one of the most important objectives of rapeseed breeding is the genetic modification of the seed oil by maximizing the proportion of specific fatty acids, like laurate, erucate or functionalized acids, in order to obtain tailor-made raw materials suited for industrial purposes.

Increase of the tocochromanol content in transgenic Brassica napus seeds by overexpression of key enzymes involved in prenylquinone biosynthesis

Lipid soluble tocochromanols, only synthesised in photosynthetic organisms, are industrially interesting compounds because of their antioxidative properties and their essential function in nutrition. In order to increase the tocochromanol content in the seed oil of transgenic plants, approaches were undertaken to engineer the flux of substrates and intermediates through the pathway. To this end, we overexpressed genes encoding hydroxyphenylpyruvate dioxygenases, alone or in combination with chimeric homogentisate phytyltransferase and tocopherol cyclase genes, in seeds of transgenic Brassica napus plants and analysed total tocochromanol content and composition. Overexpression of chimeric hydroxyphenylpyruvate dioxygenase genes, both in the cytosol or in the plastids of developing seeds, yielded a slight although significant increase in total tocochromanol level. Coexpression of a hydroxyphenylpyruvate dioxygenase gene with both a homogentisate phytyltransferase gene and a tocopherol cyclase gene elevated this effect with maximum increases of up to two-fold in individual lines and this phenotype was found to be stably inherited. These data showed that the three enzymes are critical in determining the total tocochromanol content in the seed oil of Brassica napus plants, while the tocopherol cyclase, unlike hydroxyphenylpyruvate dioxygenase and homogentisate phytyltransferase, had additionally an effect on the relative abundance of individual tocochromanol species and resulted in an increase of δ-tocopherol and plastochromanol-8 in the seeds.

sn-1-Acylglycerol-3-phosphate acyltransferase of Escherichia coli causes insertion of cis-11 eicosenoic acid into the sn-2 position of transgenic rapeseed oil

The plsC gene of Escherichia coli encoding sn-1-acylglycerol-3-phosphate acyltransferase was modified by inserting an endoplasmic reticulum retrieval signal to its 3′ end and introduced into rapeseed (Brassica napus L.) plants under the control of a napin promotor. In developing seeds from transgenic plants an sn-1-acylglycerol-3-phosphate acyltransferase activity was detectable which showed substrate specificities typical of the E. coli enzyme. Moreover, seed oil from the transformants unlike that from untransformed plants contained substantial amounts of triacylglycerol species esterified with very-long-chain fatty acids at each glycerol position. Analysis of fatty acids at the sn-2 position of triacylglycerol showed hardly any very-long-chain fatty acids in untransformed plants, but in certain transformants these fatty acids were present, namely about 4% erucic acid and 9% eicosenoic acid. These data demonstrate that the bacterial acyltransferase can function in developing rapeseed and alters the stereochemical composition of transgenic rape seed oil by directing very-long-chain fatty acids, especially cis-11 eicosenoic acid, to its sn-2 position.

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.