Identification and Characterisation of an Oleaginous Fungus Producing High g-Linoleneic Acid

摘要

Oleaginous fungi serve as good alternatives for the production of essential, polyunsaturated fatty acids (PUFA) such as g-linolenic acid (GLA; 18:3). In this study, a locally isolated fungus, isolate 2A1, was evaluated for its lipid and GLA production, and its identity was determined. The fungus was grown in nitrogen-limited media and the amount of lipid and GLA produced were determined every 12 hours. The highest percentage of lipid produced per cell dry weight was 38.64% (w/w) while the highest amount of GLA produced was 0.65 g/L. These observations indicate that isolate 2A1 is a good lipid producer and capable of accumulating high amounts of GLA. To identify isolate 2A1 to the species level, its morphology was observed under the light and electron microscope. Subsequently, its 18S rDNA and the internal transcribed spacer (ITS) sequences were cloned, sequenced and analysed phylogenetically to 18S rDNA and ITS1 sequences of related fungi. Microscopic observation showed that isolate 2A1 produced coenocytic hyphae and self-bearing globose sporangioles, with a diameter between 6-8 μm. Neighbour-joining tree built based on the 1.6 kb 18S rDNA region clustered isolate 2A1 with fungi of the genus Cunninghamella. Maximum parsimony tree analysis based on a 154 bp ITS1 sequence grouped isolate 2A1 together with Cunninghamella bainieri strain NRRL 1375 with 100% bootstrap value. Thus, based on morphological and molecular phylogenetic data, isolate 2A1 is designated as C. baineri strain 2A1. Introduction Gamma-linolenic acid (GLA: 18:3) is considered as an essential fatty acid in humans and acts as an important intermediate in the biosynthesis of biologically active prostaglandin from linolenic acid. It has been reported to be effective for the prevention or curing a variety of diseases including rheumatoid arthritis, cardiovascular diseases, hyper-cholestromia, atopic eczema and asthma [1-3]. Dietary supplement of GLA has shown a positive effect on the disorders related to the deficiencies of this fatty acid. As a result there is always considerable interest for the large scale production of GLA to support the demand of the industries.The principal sources of GLA are the seeds of evening primrose (Oenothera biennis) with 8-10% (w/w) GLA, borage seeds (Borago oficinalis) containing 24-25% (w/w) GLA and black currant seeds (Ribes nigrum), which consist of 16-17% (w/w) GLA [4]. However, the productivity of GLA from these seed oils is still considered low, since both long periods and huge areas for plant cultivation are required. The production of GLA from plants is also dependent on the seasonal and climatic changes, which could destabilise the price of the oil in the market. In addition, with the increase interest in growing plants such as canola and soybean for biodiesel production and the need to grow crops for essential food production, stiff competition for fertile agricultural land is expected in the near future. These factors could further contribute towards the fluctuation of the production cost of GLA from plants. To overcome these limitations, microorganisms may serve as an alternative source for GLA production. Microorganisms have several advantages over plants in the production of GLA including high growth rates, simple culture conditions, independence of seasonal and climatic changes, and can be readily grown under controlled conditions with nutritional regimes that may stimulate or repress the key steps of fatty acid formation [5]. Fungi, such as Zygomycetes have been widely reported for their competence in producing GLA. Genera of Mucor, Mortierella, Absidia and Cunninghamella had been extensively investigated as an alternative source for GLA production [6]. Research and development of GLA production from fungi is in progress and mostly aimed at improving the economic competitiveness of fungal GLA production. These include searching for high GLA producing fungi [7], optimising growth media for optimum lipid and GLA producti