Cloning And Expression Of ATP-citrate Lyase Gene From Different Fungi

ATP-citrate lyase is considered to be an essential cytoplasmic enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle, since it catalyzes the formation of oxaloacetate and acetyl-CoA from the cleavage of citrate. A survey of a wide variety of oil-producing microorganisms has found a correlation between those that accumulate high levels of lipids and the presence of ACL activity. In this study, The ATP-Citrate Lyase genes were obtained by GenomeWalker DNA walking and conventional PCR from Rhodotorula glutinis, Lipomyces starkeyi, Mortieralla alpine, Aspergillus oryzae and Sclerotinia sclerotiorum, and then were expressed in Escherichia coli and Pichiapastoris. The function of these products were verified by measuring enzyme activity and fatty acids content with the methods of ultraviolet spectrophotometry and GC, respectively. The main results are as follows:(1) Five fungal ael genes were successfully cloned, including the unsequenced acl genes cloned by genomic walking from Rhodotorula glutinis and Lipomyces starkeyi which were consistent and both contain two subunits acll (1953 bp) and acl2 (1494 bp).(2) Heterologous expression of these genes in Escherichia coli Rosetta-gami(DE3) and SDS-PAGE analysis showed that the relative molecular weight of the subunit polypeptides ACL1 and ACL2 from Rhodotorula glutinis, Lipomyces starkeyi, Aspergillus oryzae and Sclerotinia sclerotiorum were all about 66 kDa and 55 kDa, and the relative molecular weight of ACL protein from Mortieralla alpine was about 120 kDa.(3) Determination of enzymatic analysis of recombinant protein expression in Escherichia coli Rosetta-gami(DE3) showed that Rhodotorula glutinis and Lipomyces starkeyi acl gene products has the highest activity of 4.2 U/mg when the mass ratio of the two subunit proteins was 1:1, and both subunit proteins were essential for the ACL specific activity.(4) Coexpression of Rhodotorula glutinis acll and acl2 genes in Escherichia coli Rosetta-gami(DE3) and GC analysis showed that the recombinant strain had the highest lipid accumulation of 8.2% in the induction of 12 h, compared with the control strain increased the maximum amount of 24.8%. The effect is very significant.(5) Heterologous expression of Rhodotorula glutinis acll and acl2 genes in Pichia pastoris GS115 showed that the highest ACL specific activity was 6.5 U/mg, compared with the the enzyme activity of original expression increased by nearly 55%. And it also indicated the recombinant enzyme had optimal activity at pH8.5,37℃and 10 minutes. This study showed that the acl gene that from Rhodotorula glutinis and other oleaginous yeasts can be regulated by using genetic engineering to increase the ACL specific activity and enhance the fatty acid synthesis. Therefore, the oleaginous yeasts can provide a new genetic material that can be expressed in oil crops for substantial increase of the lipid accumulation.