Establishment Of Oleaginous Fungal Transformation System And Engineering Strain Of Producing CLA

Conjugated linoleic acid(CLA)is a collective term used to describe the mixture of positional and geometric isomers of linoleic acid(LA),there are the health benefits of anti-carcinogen,anti-adipogenic,anti-atherogenic,immune modulation,t10,cl2-CLA is one of the most physiologically active isomers.Currently,conjugated linoleic acid is produced by chemical isomerization in industry,but the product is mixture of isomers,separation and purification is difficult and costly.The linoleic acid isomerase from Propionibacterium acnes(PAI)can convert LA to t10,c12-CLA specifically.Mortierella isabellina,Mortierella alpina ATCC16266,Mortierella ramanniana can accumulate plenty of free LA after fermentation,therefore they are the potential host strain of heterologous expression of pai.This study constructed the heterologous expression vector of pai,then attempted to introduce it to the three oil-producting fungus genomes by Agrobacterium-mediated transformation method and get can accumulate t10,c12-CLA engineering strain.The main results are as follows:(1)Observed the colony morphology of three oil-producting fungus and detected abundant free LA in mycelia after fermentation by gas chromatography.(2)The sensitivity experiment results show that there were no antibiotics can be used as selection marker of Mortierella isabellina,but the chlorimuron-ethyl(500 ?g/ml)and hygromycin(500 ?g/ml)can completely inhibite spore germination of Mortierella alpina ATCC 16266 and Mortierella ramanniana respectively.(3)Based on the pFGL59,in which connected gpdA promoter and trpc terminator of Aspergillus nidulans in turn resulting pLH51,then pai was connected between the promoter and terminator,building the gene expression vector pLH55.The hygromycin resistance gene of pLH55 was replaced by chlorimuron-ethyl resistance gene of pFGL820-1,building the gene expression vector pLH107.(4)The Ti area of plasmid pLH107 was integrated into the genome of M.alpina ATCC 16266 by Agrobacterium-mediated transformation method.Only one positive transformant was screened after a lot of work as the low transformation efficiency.The target product t10,c12-CLA was not detected in mycilium after fermentation.(5)68.75% transformants of M.ramanniana were stable tranformants afte co-culture.Co-culture temperature 28?,co-culture time 3 d,the ratio of Agrobacterium and spores 2:1 are the optimal co-culture conditions of M.ramanniana.The target product t10,c12-CLA can be detected in mycilium of transformants after fermentation,indicated that pai received successful expression in M.ramanniana.