P. Böger

Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids

Abstract Carotenoids are of great commercial interest and attempts are made to produce different carotenoids in transgenic bacteria and yeasts. Development of appropriate systems and optimization of carotenoid yield involves transformation with several new genes on suitable plasmids. Therefore, the non-carotenogenic bacterium Escherichia coli JM101 was transformed in our study with several genes that mediated the biosynthetic production of the carotenoid zeaxanthin in this host. Selection of plasmids for the introduction of five essential genes for zeaxanthin formation showed that a pACYC-derived plasmid was the best. Multiplasmid transformation generally decreased production of zeaxanthin. By cotransformation with different plasmids, limitations in the biosynthetic pathway were found at the level of geranylgeranyl-pyrophosphate synthase and β-carotene hydroxylase. In our study a maximum zeaxanthin content of 289u2009μg/g dry weight was obtained. This involved the construction of a plasmid that mediated high-level expression of β-carotene hydroxylase. The level of expression was demonstrated on protein gels and solubilization by the mild detergent Brij 78 revealed that a significant portion of the expressed enzyme is located in the E. coli membranes where it can exert its catalytic function. Based on the results obtained, new strategies for vector construction and strain selection were proposed which could increase the present concentrations drastically. Optimal growth conditions of the transfomed E. coli strains for carotenoid formation were found at a temperature of 28u2009°C and a cultivation period of 2 days.

Synthesis of atypical cyclic and acyclic hydroxy carotenoids in Escherichia coli transformants

A total of eight different hydroxy carotenoids were produced in transformants of the non-carotenogenic bacterium Escherichia coli. They include the acyclic 1-hydroxyneurosporene, 1-hydroxylycopene, 1,1-dihydroxylycopene and demethylspheroidene as well as the cyclic 3-hydroxy-beta-zeacarotene, 7,8-dihydrozeaxanthin, 3 or 3-7,8-dihydro-beta-carotene and 1-hydroxy-gamma-carotene. Most of these uncommon carotenoids are found only in trace amounts in natural sources. For the synthesis of all the carotenoids mentioned above, E. coli was transformed with a combination of up to three compatible plasmids, which contained several carotenogenic genes from Erwinia uredovora and two Rhodobacter species. Their function in the pathway leading to the individual carotenoids was outlined. Finally, growth conditions were optimized for production of the hydroxy carotenoids in amounts which are suitable for their isolation and purification.

Correlation between structure and phytotoxic activities of nitrodiphenyl ethers

Abstract Various nitrodiphenyl ethers were assayed using a cell-free photosynthetic system and intact microalgae to approach a correlation between chemical structure and their effect upon three biochemical processes: (1) linear photosynthetic electron transport (Hill reaction); (2) photophosphorylation (energy-transfer inhibition by affecting the ATP synthetase); and (3) light-induced peroxidative degradation of thylakoid lipids (formation of short-chain hydrocarbons). Diphenyl ethers with a p -nitro group at one phenyl ring and one or two chloro substituents at the other exhibit energy-transfer inhibition. Analogs with different substituents are ineffective. A moderate electron-transport inhibition is seen with almost all derivatives. The most obvious phytotoxic effect is the occurrence of light-induced peroxidation (of polyunsaturated fatty acids) by p -nitro- and p -nitrosodiphenyl ethers. Substituents such as -OCH 3 , -OC 2 H 5 , -NHC 2 H 5 , or -COOCH 3 , -CONHCH 3 next to the p -nitro group enhance peroxidative activity and are apparently important for stabilization and/or reactivity of a diphenyl ether radical originating through photosynthetic electron flow. Activity is further enhanced by substituents with positive Hammett parameters in ortho or para positions at the phenyl ring not carrying the nitro group.

Modification of ferredoxin-NADP+ reductase from the alga Bumilleriopsis with butanedione and dansyl chloride.

Abstract Modification of ferredoxin-NADP + reductase from the alga Bumilleriopsis with butanedione (diacetyl) and dansyl chloride results in loss of enzymatic activity. Under pseudo-first order conditions the rate of inactivation by butanedione is directly proportional to the concentration of the modifying reagent with a slope of unity. The protective effect of pyridine nucleotides, as well as their analogs against inactivation by butanedione indicates involvement of arginine in the binding of pyridine nucleotides at the active site. Inactivation by dansyl chloride suggests that a further amino acid is involved, possibly lysine. Amino acid analyses of the butanedione-treated reductase show that the degree of inactivation correlates well with the decrease in arginine. Furthermore, two arginine residues are modified concomitant with complete inactivation of the enzyme, although this does not imply that both residues participate necessarily in the binding of pyridine nucleotides. Fingerprint analysis of the carboxymethylated, trypsin-digested enzyme indicates loss of one arginine-containing peptide when the protein had been modified by butanedione. There was no change in cysteine-containing peptides.

Bleaching activity and chemical constitution of phenylpyridazinones

Abstract The bleaching effect of 2-phenylpyridazinones substituted at the 4 or 5 position of the pyridazinone moiety or at the phenyl ring (position 9) was assayed using a greening Scenedesmus mutant after its transfer from heterotrophic to autotrophic growth. The following relationship between bleaching activities and Hammett electronic parameters of the various substituents could be demonstrated. The biological activity of the pyridazinone skeleton was enhanced with substituents showing (a) increasing σ m values in position 4, (b) increasing σ m or σ p values in position 9, and (c) decreasing σ p values in position 5. These findings could be corroborated by data on pigment bleaching and decrease of photosynthetic oxygen evolution of autotrophic (green) wild-type Scenedesmus after growth in the presence of sublethal concentrations of pyridazinones. There is no structure/activity relationship with direct inhibition of photosynthetic electron transport. Based on electronic parameters, the construction of phenylpyridazinone derivatives with bleaching activity is proposed.

PHOTOSYNTHETIC NITRITE REDUCTION BY DITHIOERYTHRITOL AND THE EFFECT OF NITRITE ON ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS

Abstract. Photosynthetic reduction of nitrite to ammonia with type C chloroplasts from the heterocont alga Bumilleriopsis filiformis was investigated using 3,6‐diaminodurene/ascorbate and 3,6‐diaminodurene/dithioerythritol (DAD/DTE) as electron donor couple. Rates approach 6–10 μmol NO‐2 reduced/mg chlorophyll/h and are steady for up to 30 min. The presence of oxygen or NADP+ only slightly diminished the rates of nitrite reduction obtained with DAD/DTE. Illuminated chloroplasts reduce oxygen in the presence of DAD/DTE at 135 μmol/mg chlorophyll/h without acceptor supplied. Photosynthetic oxygen uptake by this system in the presence of ferredoxin and NO‐2, however, is inhibited to 42% by nitrite reductase with concurrent nitrite reduction. NO‐3 and NO‐2 have no effect on photosystem I‐mediated NADP+ reduction, NO‐2 (10 mM) inhibits ferricyanide‐mediated oxygen evolution to 72%. Also photosystem II reactions assayed e.g. with silicomolybdate are inhibited significantly by NO‐2 (1 mM), but only slightly by NO‐3. Nitrite reductase is inhibited by p‐chloromercuribenzoate (pCMB), and this inhibition is prevented by DTE. Results suggest that photosynthetic nitrite reduction can cope with low concentrations of either compound, provided relevant thiol groups are protected.

Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin

Abstract The plastidic ferredoxin-NADP + reductase from the xanthophycean alga Bumilleriopsis forms a stoichiometric 1:1 complex with ferredoxin and NADP + which is demonstrated by difference spectra of both complexes. Butanedione modification of the flavoprotein results in loss of its enzymatic activities (transhydrogenase and diaphorase) concurrently with its capability to form a complex with NADP + , whereas the ferredoxin-binding site is practically not influenced by the modifying reagent and complex formation is still possible. It is assumed, therefore, that butanedione specifically reacts with the arginine residue of the protein involved in binding of pyridine nucleotides at the active site. Further, the data presented strongly support the previous proposal of different binding sites for ferredoxin and pyridine nucleotides at the reductase.

Nitrogen fixation of Azorhizobium in artificially induced root para-nodules in wheat

Nodule-like structures(para-nodules) can be induced in wheat roots by low concentrations of plant growth hormone (2,4-D).Growth of nodulated wheat plants was not affected by 2,4-D treatment. Infection of these para-nodules by adding oxygen-tolerant Azorhizobium caulincdans resulted in massive proliferation of bacterial cells in the intercellular spaces as well as inside the para-nodular cells. Para-nodules colonized by A.caulinadans not only reduced acetylene (about 2–4 n mol C2H4/plant/h), but transferred fixed nitrogen to the plant, contributing up to 16–23% of the nitrogen budget of infected wheat.

General Aspects of Location and Interaction of Respiratory and Photosynthetic Electron Transport in Blue-Green Algae

Blue-green algae (cyanobacteria) are photoautotrophic oragnisms with oxygenic photosynthesis (for review see Krogmann 1977). Their main difference to other algae is their prokaryotic organization — they lack particular organelles such as chloroplasts or mitochondria, but have different types of membranes (Lang 1968, Golecki and Drews 1982). Nevertheless, some filamentous species can diffentiate special cells known as heterocysts which serve for reductive fixation of atmospheric nitrogen (Haselkorn 1978).

Binding and Peroxidative Action of Oxyfluorfen in Sensitive and Tolerant Algal Species

Peroxidative activity of oxyfluorfen and binding of this nitrodiphenyl ether to cell fractions was investigated with the susceptible alga Scenedesmus acutus and the resistant alga Bumilleriopsis filiformis. Although a 10-fold higher concentration of oxyfluorfen was applied to Bumilleriopsis, the lag phase for initiation of peroxidative evolution of short-chain hydrocarbons from fatty acids was much longer than found with Scenedesmus. Oxyfluorfen was predominantly recovered after homogenization from the pellet which was separated into a lipid and a chloroform/methanol insoluble fraction. Parts of the oxyfluorfen which is present in the insoluble pellet fraction during the lag phase before the onset of peroxidation can be found in the lipid fraction when measurable peroxidative activities have started. This was observed with Scenedesmus as well as with Bumil-leriopsis. During peroxidation initiated by oxyfluorfen acyl lipids are degradated as monitored by the disappearance of the plastidic sulfolipid. Analysis of bound fatty acids showed that they are targets for peroxidative reactions in acyl lipids. Destruction of polyunsaturated fatty acids was higher than for saturated ones.