Gottfried Oesterhelt

Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations

Utilising whole cells of recombinant Escherichia coli K27 (pCYP102, pGEc47) containing active cytochrome P450BM-3 monooxygenase [E.C. 1. 14.14.1], multiple oxidations of saturated and unsaturated fatty acids were performed by the enzyme under conditions of excess oxygen. The amount of oxygen dissolved in the culture medium strongly influenced the regioselectivity of the reaction, as reflected in the distribution and amount of oxidised products. We have verified by gas chromatography/mass spectrometry that the products of in vivo biotransformation of pentadecanoic acid by cytochrome P450BM-3 are identical to those formed in cell-free extracts containing the enzyme. The formation of keto- and dihydroxy acids, side products which are characteristic for in vitro conversions with purified cytochrome P450BM-3 in the presence of excess oxygen, has been observed as well. Thus, by varying the oxygen concentration, we could control the regioselectivity of oxidation and the number of products made. Under oxygen limiting conditions, only monooxidised 12-, 13-, and 14-hydroxy-pentadecanoic acids were obtained. Consequently, unwanted side products could be excluded by modulating the amount of oxygen used in the bioconversion. Furthermore, whole cell oxidation of two unsaturated long-chain fatty acids, cis-pentadec-10-enoic and cis-hexadec-9-enoic acid, resulted in the production of epoxides, various subterminal hydroxyalkenoic acids and keto- and hydroxyalkanoic acids. Although we obtained higher activities of C15:0 conversion in vitro, the whole cell biocatalyst proved to be useful for specific oxidations of long-chain fatty acids since there is no need to add the costly cofactor NADPH. This biooxidation by E. coli K27 (pCYP102, pGEc47) under oxygen limitation has been demonstrated at the 2-L scale, showing that 12-, 13-, and 14-hydroxypentadecanoic acids can be produced in the g L-1 range.

Fenpropimorph: A three site inhibitor of ergosterol biosynthesis in Nectria haematococca var. cucurbitae

Abstract The effect of fenpropimorph, fenpropidin, tridemorph, pyrifenox, triadimenol, and tebuconazole on growth and sterol synthesis of a wild-type strain of Nectria haematococca var. cucurbitae was studied. Fenpropimorph-treated mycelia showed a dose dependent drop in ergosterol content with a parallel accumulation of Δ 8 -sterols, Δ 14 -sterols, and squalene, indicative of inhibition of three different enzymes in the sterol biosynthetic pathway: Δ 8 -Δ 7 -isomerase, Δ 14 -reductase, and squalene epoxidase. Untreated mycelia incorporated [ 14 C]acetate into 4-desmethyl sterols, composed primarily of ergosterol, whereas in fenpropimorph-treated mycelia the label accumulated in squalene and a reduction of C-4 desmethyl sterols was observed. Fenpropidin was found to be a potent inhibitor of Δ 8 -Δ 7 -isomerase and Δ 14 -reductase, while tridemorph specifically inhibited Δ 8 -Δ 7 -isomerase. Triadimenol, pyrifenox, and tebuconazole inhibited the C-14 demethylation step, which resulted in a decrease in ergosterol and an increase in C-14 methyl sterols.

2‐Alkylimidazoline derivative to control fatty acid fragmentation upon electron impact and electrospray ionization

A new derivative of the carboxyl group, N-methyl-2-alkylimidazoline, was introduced with the aim of improving the control of fatty acid fragmentation. Owing to the strongly basic character and the cyclic structure of this derivative, strong stabilization of the charge on the ionized group is obtained so that extensive radical and charge-remote fragmentation of the chain can be achieved with electron impact and electrospray ionization.

FREE FATTY ACID UTILIZATION OF DIFFERENT HYBRIDOMA CELL LINES AND ITS EFFECT ON CELL GROWTH, METABOLISM AND ANTIBODY FORMATION

Cell growth and product formation as a function of various lipidic supplements were studied in serum-free medium for 2 hybridoma lines grown in small scale batch cultures. The influence of different combinations and concentrations of free fatty acids bound to albumin or of a commercially available growth enhancement media supplement on growth and productivity was found to be dependent on the cell line. The consumption and release of free and total fatty acids and cholesterol from the cells was recorded as a function of cultivation time.