Gamma Linolenic Acid Production By Cunninghamella Sp And Cloning Of â–³6 Desaturase Gene

Gamma-linolenic acid(GLA), the essential fatty acid in human body, has been come tofront due to its biting quantities of important physiological activities.GLA productionthrough microbial fermentation is the hotspot in recent years. Firstly, the fundamental production medium suitable for Cunninghamella sp. C2producing GLA was optimized, and it was found that the optimal medium contained 10%glucose, soy flour liquor cotaining 6 g·L-1 protein and 3 g·L-1 K2HPO4, 2 g·L-1 NaH2PO4 ;respectively. Secondly the effects of pH and inoculum on production of GLA byCunninghamella sp. C2 were investigated, pH 6.0 and an amount of 10% (v/v) were optimalfor GLA production. The time courses of consumption of main nutrition composition,fungal biomass, lipid and gamma-linolenic acid accumulation were investigated on a shakerflask scale. When residual glucose concentration was under 15g·L-1,The fungus stoppedgrowing because of inefficient energy source and carbon source needed for their growth.Great increase of gamma-linolenic acid content in lipids occurred after the glucose wasexhausted. To obtain high gamma-linolenic acid yield, a fed-batch process was developedaccording to the analysis of consumption of substrates and accumulation of lipids andgamma-linolenic acid. Glucose was included in the feed medium. Initial glucoseconcentration was 100g·L-1 and initial nitrate concentration was 6g·L-1. 15 g·L-1·d-1 glucosewas fed at the 5th , 6th ,7th days of cultivation. A gamma-linolenic acid yield (1.86 g·L-1) in10 days were obtained, which was 1.7 folds of the highest yield of batch cultures. Key enzyme in biosynthetic pathway of gamma-linolenic acid ,△6 desaturase, wascloned via RT-PCR using a pair primer designed according to the known sequence of △6fatty acyl desaturase. The cDNA consisted of 1071 nucleotides. There were 357 aminoacids of the deduced protein, which showed 97% identity to reported △6 fatty acyldesaturase . The deduced protein had conserved domains of fatty acyl desaturases including IIa cytochrome b5 domain in the N terminal and 3 conserved histidine boxes.