Clone And Expression Of ACC Gene From Rhodotorula Glutinis

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid synthesis. It catalyzes acetyl-CoA to form malonyl-CoA, which is used as a building block in two carbon increasements to extend the chain length of fatty acids. Therefore, ACC is also a rate-limiting enzyme of fatty acid synthesis. The main results are as follows in the study:Using the methods of degenerate PCR and chromosome walking, this paper obtained the full sequence information of ACC gene from Rhodotorula glutinis for the first time. Sequence analysis showed that ACC gene from genomic DNA have two introns, ranging from 42 to 147 and 315 to 677 nucleotides, and the coding region is 6801bp in length. Secondary structure analysis of the deduced amino acid sequence revealed that it contains three typical domains of ACC:biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT).ACC from Rhodotorula glutinis is an multi-domain enzyme, and its monomer molecular weight is above 200 kD. It's very difficult to express full-length cDNA of ACC, so according to methods for studing multi-subunit ACC, this paper did research on three domains of ACC from Rhodotorula glutinis. This study had used two prokaryotic expression systems pET-28a/BL21(DE3) and pETDuet-1/Transetta (DE3) to separatedly express and coexpress three domain genes. Firstly, nine expreesion vectors were successfully constructed. SDS-PAGE analysis revealed that each domain gene was successfully expressed in two expression systems. Moreover, it had achievd to coexpress BC and CT domains, as well as three domains.Fatty acid of recombinant strains was extracted by bath, and analyzed by Gas chromatography. Results suggested that the fatty acid content of recombinant strains in the system pETDuet-1/Transetta (DE3), which had separatedly expressed BC or CT, coexpressed BC and CT, as well as three domains, respectively increased 15.4%,27.07%, 35%,70.62% compared with the control group. However, it descreased 5.05% for expression of BCCP domain. In pET-28a/BL21(DE3) expression system, fatty acid content of recombinant strains compared with the control without any increase. It demonstrated that the genes had achievd functional expression in pETDuet-1/Transetta (DE3). For separately expression and coexpression of BC and CT, fatty acid content was increased to different degrees, when coexpression of three domains, it significantly promoted the accumulation of fatty acids.This study also used SOE-PCR to knock out two introns of ACC gene of Rhodotorula glutinis, and cDNA of ACC was devided into three sequences EK, KS, SX for PCR amplification, and they were connected into Pichia expression vector pPICZaA in turn to construct pPICZaA-acc. This work had layed the foundation for eukaryotic expression of ACC and functional study.