Mikrobna proizvodnja limunske i izolimunske kiseline na podlozi od suncokretovog ulja

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated with the simultaneous utilization of glycerol and free fatty acids produced during oil hydrolysis. The activities of enzymes of glycerol metabolism (glycerol kinase), fatty acid assimilation enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) and citric acid cycle were comparatively assayed in Y. lipolytica grown on sunflower oil, glycerol and oleic acid. Glycerol kinase and enzymes of glyoxylate cycle were active during the whole period of cell cultivation on sunflower oil. Citric acid production and a ratio between citric and isocitric acids depended on both the strain used and the medium composition. It was revealed that wild type strain Y. lipolytica VKM Y-2373 produced almost equal amounts of citric and isocitric acids at pH=4.5 and predominantly accumulated isocitric acid at pH=6.0. The mutant Y. lipolytica N 15 produced only citric acid (150 g/L with mass yield (YCA) of 1.32 g/g). Biochemical characteristics of mutant strain Y. lipolytica N 15 were discussed.U radu je ispitan rast divljeg soja kvasca Yarrowia lipolytica VKM Y-2373 i mutantnog soja Yarrowia lipolytica N 15, te biosinteza limunske i izolimunske kiseline na podlozi od suncokretovog ulja. Utvrđeno je da je rast stanica povezan s istodobnim iskorištenjem glicerola i slobodnih masnih kiselina nastalih tijekom hidrolize ulja. Usporedno je ispitana aktivnost enzima koji sudjeluju u metabolizmu glicerola (glicerol kinaze), glioksilatnom ciklusu asimilacije masnih kiselina (izocitrat liaze i malat sintaze) i ciklusu limunske kiseline u kvascu Y. lipolytica uzgojenom na podlozi od suncokretovog ulja, glicerola i oleinske kiseline. Glicerol kinaza i enzimi glioksilatnog ciklusa bili su aktivni cijelo vrijeme uzgoja stanica na podlozi od suncokretovog ulja. Količina proizvedene limunske kiseline i omjer limunske i izolimunske kiseline ovise o upotrijebljenom soju i sastavu podloge. Otkriveno je da je divlji soj Y. lipolytica VKM Y-2373 proizveo skoro jednaku količinu limunske i izolimunske kiseline pri pH=4,5, a pretežno izolimunsku kiselinu pri pH=6.0. Mutantni soj Y. lipolytica N 15 proizveo je samo limunsku kiselinu (150 g/L, s masenim stupnjem pretvorbe YCA=1,32 g/g). U radu su također razmotrene biokemijske značajke mutantnog soja Y. lipolytica N 15

Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation

BACKGROUND: The glyoxylate cycle is thought to be present in bacteria, protists, plants, fungi, and nematodes, but not in other Metazoa. However, activity of the glyoxylate cycle enzymes, malate synthase (MS) and isocitrate lyase (ICL), in animal tissues has been reported. In order to clarify the status of the MS and ICL genes in animals and get an insight into their evolution, we undertook a comparative-genomic study. RESULTS: Using sequence similarity searches, we identified MS genes in arthropods, echinoderms, and vertebrates, including platypus and opossum, but not in the numerous sequenced genomes of placental mammals. The regions of the placental mammals' genomes expected to code for malate synthase, as determined by comparison of the gene orders in vertebrate genomes, show clear similarity to the opossum MS sequence but contain stop codons, indicating that the MS gene became a pseudogene in placental mammals. By contrast, the ICL gene is undetectable in animals other than the nematodes that possess a bifunctional, fused ICL-MS gene. Examination of phylogenetic trees of MS and ICL suggests multiple horizontal gene transfer events that probably went in both directions between several bacterial and eukaryotic lineages. The strongest evidence was obtained for the acquisition of the bifunctional ICL-MS gene from an as yet unknown bacterial source with the corresponding operonic organization by the common ancestor of the nematodes. CONCLUSION: The distribution of the MS and ICL genes in animals suggests that either they encode alternative enzymes of the glyoxylate cycle that are not orthologous to the known MS and ICL or the animal MS acquired a new function that remains to be characterized. Regardless of the ultimate solution to this conundrum, the genes for the glyoxylate cycle enzymes present a remarkable variety of evolutionary events including unusual horizontal gene transfer from bacteria to animals. REVIEWERS: Arcady Mushegian (Stowers Institute for Medical Research), Andrey Osterman (Burnham Institute for Medical Research), Chris Ponting (Oxford University)

Lipase Secretion and Citric Acid Production in Yarrowia lipolytica Yeast Grown on Animal and Vegetable Fat

The aim of the study was to investigate the potentiality of the utilization of raw agro- -industrial fat for the biotechnological production of valuable products (lipase and citric acid) by the yeast Yarrowia (Candida) lipolytica. Thirty strains of the aforementioned species were investigated for their capability of lipase secretion and citric acid production on media containing animal fat or rapeseed oil as a sole carbon and energy source. Strain Y. lipolytica 704, exhibiting the highest lipase activity on rapeseed oil (2760 U/mL), was selected for the study of biochemical peculiarities of cell growth, and strain Y. lipolytica 187/1, exhibiting the maximum citric acid synthesis, was selected for the subsequent studies on citric acid production. A relationship between lipase production and residual rapeseed oil concentration was studied. The essential factor for lipase production was found to be the concentration of rapeseed oil in the medium, which should be no less than 5 g/L. Under optimal conditions of cultivation, citric acid production by rapeseed-oil-grown yeast Yarrowia lipolytica 187/1 amounted to 135 g/L; specific rate of citric acid production reached m(CA)/m(cell)=127 mg/(g·h); mass yield (YCA) and energy yield (hCA) were 1.55 and 0.41, respectively

Microbiological Production of Citric and Isocitric Acids from Sunflower Oil

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Yarrowia lipolytica N 15 as well the biosynthesis of citric and isocitric acids on sunflower oil were studied. It was indicated that cell growth was associated with the simultaneous utilization of glycerol and free fatty acids produced during oil hydrolysis. The activities of enzymes of glycerol metabolism (glycerol kinase), fatty acid assimilation enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) and citric acid cycle were comparatively assayed in Y. lipolytica grown on sunflower oil, glycerol and oleic acid. Glycerol kinase and enzymes of glyoxylate cycle were active during the whole period of cell cultivation on sunflower oil. Citric acid production and a ratio between citric and isocitric acids depended on both the strain used and the medium composition. It was revealed that wild type strain Y. lipolytica VKM Y-2373 produced almost equal amounts of citric and isocitric acids at pH=4.5 and predominantly accumulated isocitric acid at pH=6.0. The mutant Y. lipolytica N 15 produced only citric acid (150 g/L with mass yield (YCA) of 1.32 g/g). Biochemical characteristics of mutant strain Y. lipolytica N 15 were discussed