Isolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae.

Abstract

In recent times, demands for bio-oils, such as biolipids and biofuels, have progressed, since they are very significant biomolecules for human health and for the ecological preservation of the earth's environment. Nevertheless, the supplements of bio-oils were not always stable because the harvests of oleaginous plants, animals, and fishes were influenced by climates and marine conditions. Various research and developments were carried out on microbial production of valuable oils, such as polyunsaturated fatty acids, from the viewpoint of factory-controllable fermentative formation of specified products. So far, it has been reported on the production of eicosapentaenoic acid (Shimizu et al., 1988), docosahexaenoic acid (Li and Ward, 1994), and g-linolenic acid (Hansson and Dostalek, 1988) using various oleaginous fungi. These products, however, were entirely localized at the inner space of cell. Therefore processes for cell disruption and oil extraction were essential to recover such products as other oleaginous plants or animals. The development of extracellular accumulation of fats (triacylglycerols, TGs) must be promising for an improvement in the cost performance of microbial lipid fermentation.Previously we reported the breeding of mutant strains of yeast Candida lipolytica that secrete palmitic acid into glucose medium (Miyakawa et al., 1984), and of the yeast Trichosporon sp., which accumulates TGs in the ethylpalmitate (Yagi et al., 1994) or glucose (Nojima et al., 1995) medium. As far as we know, no other reports exist concerning the extracellular production of useful lipids by microorganisms. We could exhibit the construction of lipid-secretable microorganisms, though their productivities were economically insufficient. For further improvement of the capacity to secrete lipid, it would be necessary to have not only a screening of a superior producer, but also an analysis of the molecular mechanism for the outward flow of lipids. We would advocate that the extracellular accumulation of hydrophobic biomaterials by the fermentation process has a higher probability to be realized if the equilibrium of membrane transport for hydrophobic compounds could be made up to incline toward pumping out. It is widely known that the yeast Saccharomyces cerevisiae is one of the most widely studied microorganisms in molecular genetics. Studies on lipid-excreting mechanisms using a mutant strain of S. cerevisiae should have advantages to breed useful microorganisms for lipid-accumulating fermentation. In this paper we describe isolation and characterization of the S. cerevisiae mutant strain that secretes TG into glucose medium. Moreover, we represent compleIsolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae Department of Bioscience and Biotechnology, Faculty of Engineering, Fukuyama University, Fukuyama 729-0292, Japan (Received September 11, 1998; Accepted January 7, 1999) To establish the molecular bases for development of a microbiological system approach...

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“…These genes were identified to have a function in fatty acid secretion through the use of a screening method wherein mutated yeast colonies were overlaid with an agar containing a fatty acid auxotrophic yeast strain that requires free fatty acids in order to proliferate. Mutant colonies that secrete fatty acids were thereby identified by the formation of a halo in the overlaid agar (120,132). Similar screening methods could be utilized to identify mi-croalgae that have the ability to secrete fatty acids.…”

“…In one of the yeast studies, random mutagenesis resulted in the secretion of TAGs. Unfortunately, neither the genes involved nor the mechanism has been described (132). S. cerevisiae has five genes with fatty acyl-CoA synthetase activity, including those encoding FAA1 and FAA4.…”

“…Several publications have shown that knocking out genes involved in ␤-oxidation in S. cerevisiae not only can lead to increased amounts of intracellular free fatty acids but also results in extracellular fatty acid secretion in some instances (120,132,162). The lipid catabolism genes that have been implicated in fatty acid secretion in S. cerevisiae include acyl-CoA oxidase and several acyl-CoA synthetases (see below).…”

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