Formation and degradation of lipid bodies found in the riboflavin-producing fungus Ashbya gossypii

Microdroplets in the hyphae of Ashbya gossypii were found to become stained by Nile red. Purification of the stainable substance showed that the yellow fluorescent bodies consisted of triacylglycerol. During growth on glucose as the carbon source 8%–12% of the mycelial dry weight was found to be neutral lipid. When glucose declined in the medium, the content decreased to 3%–4% and the respiration quotient shifted to 0.6 indicating a reserve function of the fat. The fatty acid composition of the storage lipid was found to be strongly influenced by the carbon source. Mycelia cultivated on glucose contained 5% linoleic acid and 20% palmitoleic acid in their neutral fat while linoleic acid made up 54% and palmitoleic acid was not detectable (< 0.1%) in soybean-oil-grown mycelia. When plant oil was given as the sole carbon source, the fatty acid composition of the storage lipid showed a high similarity to the fed fat. ¹⁴C-labelled free oleic acid added to a culture growing on soybean oil was immediately incorporated into the fungal lipid. A pulse of 0.9 g/l free palmitoleic acid, fed during growth on olive oil, increased the content of this particular fatty acid in the fungal triacylglycerol from 0.8% to 9.6%. In addition, a liberation of free fatty acid and diacylglyceride was found in the culture supernatant when pure triolein was given as the sole carbon source. Obviously, the fungus cleaved the lipid serving as the carbon source extracellularly and used the liberated fatty acids for its storage lipid formation.This paper is dedicated to Prof. Dr. F. Wagner on the occasion of his 65th birthday     

Threonine Aldolase Overexpression plus Threonine Supplementation Enhanced Riboflavin Production in Ashbya gossypii

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg⁻¹ was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.     

The potential for protein production from green plants

Data on protein yields show that forage crops, particularly alfalfa, produce several times more protein per acre than do seed crops. Amino acid analyses and estimations of biological values by enzymatic hydrolysis and feeding trials indicate that protein concentrates from green plants have high nutritive value. The protein concentrates from 10 plant species had a similar amino acid composition and biological value which indicates that good protein might be obtained from many plant species. It is suggested that the use of the fibrous residue as a feed for ruminants and the use of the protein concentrate as a high protein feed or base for processing into new protein foods may make it possible for the production of protein from green plants to compete with other sources of protein. This would markedly increase protein production per acre and allow the use of new plant species in our agriculture. The need for more research on protein production from different types of green plants and on ways to harvest, concentrate and process their proteins into edible forms is discussed.     

Modelling and parameter identification for batch fermentations with Streptomyces tendae under phosphate limitation

Summary A simple structured model for the dynamics of phosphate-limited batch fermentations with Streptomyces tendae is presented. The model describes the influence of intracellular phosphate storage upon the growth behav our of the culture. The development of the model takes into account the possible internal regulatory processes of phosphate metabolism. These complex biochemical pathways are summarized with regard to rate-limiting steps to obtain relatively simple model equations. The model parameters are fitted to the experimental data with an identification programme based on the sequential quadratic programming algorithm. Modifications in this algorithm yield a good performance for this application. With respect to the sensitivity of the model parameters, a feedback on the modelling is given. After several loops of modelling and identification, a model was achieved that fits to a set of batch fermentations. Furthermore the simulations show that RNA measurements of some recent fermentations can be interpreted by the simulated internal state variable and that there is evidence for RNA as an intracellular phosphate reserve. Offprint requests to: K.-P. Kuhn     

Gene for integrin-associated protein (IAP, CD47): physical mapping, genomic structure, and expression studies in skeletal muscle.

Integrin-associated protein (IAP) is a widely expressed membrane protein with multiple functions in immunological and neuronal processes. Having physically mapped the IAP gene into a BAC/PAC contig covering approximately 1 Mb on human chromosome 3ql3.1-q13.2, we determined the genomic organization of the gene, established its expression in skeletal muscle, and identified a novel splice variant. Our expression studies demonstrate expression of integrin-associated protein in the t-tubular system and the euchromatin of skeletal muscle cells where its function thus far is not known.     

Biliproteidkomposition und Pigmentverteilung in scheibchenförmigen Phycobilisomen von Rhodella violacea

Ein Modell der Anordnung von Phycoerythrin, Phycocyanin und Allophycocyanin in den scheibchenförmigen Phycobilisomen von Rhodella violacea wird vorgestellt; es steht in Einklang mit der quantitativen und qualitativen Pigmentkomposition, dem intermolekularen Energietransfer sowie der Feinstruktur isolierter Phycobilisomen. Hohe Lichtintensitäten und sechstägiger Nitratmangel führen zu einer Reduktion des gesamten Biliproteidgehalts der Zellen. Darüber hinaus ist eine Veränderung der Pigmentkomposition der isolierten Phycobilisomen nachweisbar. Der Phycocyanin- und der Allophycocyaningehalt sind davon unberührt, während der Phycoerythrinanteil auf wenigstens 78% verringert wird. Der Verlust von zwei bis drei Phycoerythrinaggregaten je Phycobilisom beeinträchtigt weder die Fähigkeit zum Energietransfer noch die Fluoreszenzausbeute bei 665 nm in Suspensionen gleichen Phycocyanin- und Allophycocyaningehalts. Wir danken der Deutschen Forschungsgemeinschaft (SFB 103) für die finanzielle Unterstützung.     

Leaf protein concentrates: a comparison of protein production per acre of forage with that from seed and animal crops

The essential amino acid production for ten crops harvested for forage and fifteen crops harvested for seed was calculated from average crop yields in the United States for the ten-year period of 1953 to 1962. Highest yields per acre of essential amino acids were calculated from forages which could be processed into leaf protein concentrates. Alfalfa produced the highest yield per acre of essential amino acids of the twenty major crops; soybean seed was second. The calculated yields of edible protein and essential amino acids of leaf protein from corn were equal to or greater than the protein from the corn seed and several times that from animals fed the corn grain. At the present time, leaf protein concentrate may not be able to compete in cost with the by-product protein concentrates such as soybean meal, cottonseed meal, peanut meal, linseed meal, or tankage for feeding farm animals. However, further study of the production of leaf protein concentrates should be made to assure protein supplies for those regions where other adequate protein sources are lacking.