Effects Of Polyunsaturated Fatty Acids Biosynthesis Inhibition On The Cold Adaptation Of Rhodosporidium Kratochvilovae

Polyunsaturated fatty acids(PUFAs)are unbranched fatty acids containing more than one double bond.They are essential components of human health including LA,EPA,ALA,DHA and so on.Many microorganisms capable of producing PUFAs have been isolated and characterized so far,but the exact molecular and biochemical role of PUFAs accumulated in these oleaginous microbes for themselves remains unclear.Market demand for PUFAs is growing continuously but current sources are insufficient to satisfy this demand.Therefore,seeking for alternative sources is demanding.Rhodosoridium kratochvilovae is one of the most important strains in endomycetales fungi which can produce many kinds of PUFAs,especially linoleic acid(18:2n-6,LA)and a-linolenic acid(18:3n-3,ALA).The fatty acid ?12-desaturase is a key enzyme for producing LA in filamentous fungi ·Over 75%of the Earth's biosphere experiences temperatures of less than 5? throughout the year,where various low-temperature adapted microorganisms inhabit.Due to long-term natural selection,these microorganisms have evolved some adaptive mechanisms at cellular and molecular levels to adapt to their living environments.One of the mechanisms is to increase PUFAs content of membrane lipids to maintain optimum membrane fluidity at low temperatures.Based on the effects of PUFAs biosynthesis on microbial growth,the membrane fluidity and composition and expression levels of membrane proteins,this paper discusses the contribution of PUFAs to the cold adaptation of microorganisms and summarize the research progress in the regulatory mechanisms of PUFAs biosynthesis at low temperatures,providing a reference for relevant fundamental and application research in this field.Based on the sequence information of the known yeast ?12-fatty acid desaturase genes,a pair of specific primers was designed and used for the amplification of YM25235 ?12-fatty acid desaturase gene.A full-length cDNA sequence of 1341 bp was amplified from Rhodosporidium kratochvilovae strain YM25235.Sequence analysis showed this sequence comprised an open reading frame encoding 446 amino acids of 50.6KD.The deduced amino-acid sequence showed similarity to the known ?12-fatty acid desaturases which comprise three characteristic conserved histidine-rich regions for the membrane-bound desaturases,indicating this cDNA sequence is a novel ?12-fatty acid desaturase gene.The sequence was further cloned into the expression vector pYES3.0/CT to generate a recombinant plasmid pY3RKD12,which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression.Total fatty acids analysis of the transformed yeast cells by gas chromatography(GC)showed that a novel peak corresponding to the standards of linoleic acid methyl ester was detected with the same retention time,which was absent in the cell transformed with empty vector.The ratio of this new fatty acid to total fatty acids was 3.76%.So the cDNA sequence we obgained was a novel ?12-fatty acid desaturase gene(GenBank accession No.502671).In this study,we successfully constructed a disruption vector pRH-D12D.Following transformation of pRH-D12D into Rhodosporidium kratochvilovae YM25235.One fatty acid?12-desaturase gene-defective mutant strain designated as RKD12D-5 was selected on the medium plate containing 150?g HmB/mL as determined by molecular analysis.By analysis with gas chromatography,RKD12D-5 could produce LA and ALA lowlier but accumulate a significantly high percentage content of SA,compared with those in the case of Rhodosporidium kratochvilovae YM25235 respectively.The morphology and physiology of RKD12D-5,such as colonial shape,color and growth rate,were changed dramatically compared with that of strain Rhodosporidium kratochvilovae YM25235.We assessed mechanisms of regulation of fatty acid desaturase gene expression through gene knock-out in Rhodosporidium kratochvilovae YM25235 for the first time.We wished to make it possible to obtain a better understanding of the mechanisms and got some theoretical knowledge to offer some guidance to the industrial production of PUFAs by transgenic technology and microbial fermentation technology.