Elizabeth C. Verbree

Optimization of lipid production in the oleaginous yeastApiotrichum curvatum in wheypermeate

SummaryLipid production of the oleaginous yeastApiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h-1. At C/N-ratios higher than 25–30 lipid content gradually increased to nearly 50% at C/N=70 and the maximal obtainable dilution rate decreased to 0.02 h-1 at C/N=70. From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30–35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates ofA. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes with high cell densities. A lipid production rate of nearly 1 g/l/h was reached in a partial recycling culture. It was calculated that by using this cultivation technique lipid production rates of even 2.9 g/l/h may be reached when the supply of oxygen can be optimized.

Isolation and characterization of fatty acid auxotrophs from the oleaginous yeast Apiotrichum curvatum

SummaryIn order to improve the economic value of lipids produced by the oleaginous yeast strain Apiotrichum curvatum ATCC 20509, a search was made for mutants defective in the conversion of stearic acid to oleic acid. Mutants could be selected as unsaturated fatty acid auxotrophs, since unsaturated fatty acids are essential componenets in membrane lipids. After treatment of A. curvatum wild-type with N-methyl-N′-nitro-N⇔-nitrosoguanidine, 58 fatty-acid-requiring mutants were isolated. On the basis of (1) the growth response to saturated and unsaturated fatty acids and (2) the fatty acid composition of lipids produced by these mutants, it was concluded that only 18 of them were real unsaturated fatty acid (Ufa) mutants, while the other 40 were designated as fatty acid synthetase (Fas) mutants. It is further shown that Ufa mutants of A. curvatum are able to produce high amounts of lipids consisting of more than 90% triacylglycerols with a percentage of saturated fatty acids resembling that of cocoa butter, when grown in the presence of relatively small amounts of oleic acid in the growth medium. This may offer an economically favourable alternative in comparison with other methods that have been developed for the production of cocoa butter equivalents by microorganisms.

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.

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.

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.

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.