Screening Of Marine Oleaginous Microorganisim And Cloning Of Acyl Carrier Protein Gene

The bacteria,yeast,mould and algae microorganisms can turn carbohydrates into oils in the appropriate conditions and stored in the cell which called microbial oils or single cell oil.Marine microorganisms in special conditions can produce various bioactive substances including fatty acids,antitumor,antibacterial,and unsaturated fatty acids.Acyl carrier proteins,one kind of highly conservative transporters,usually used as acyl donor in fatty acid metabolism,are indispensable cofactors in fatty acid synthesis pathway.Therefore,studies on the structure and function of acyl carrier protein will provide guideline for the researches on the fatty acid synthesis pathways and the novel synthetic pathway design.One oleaginous yeast was screened from sea mud and algae surface by Sudan Black B staining and identified as Meyerozyma guilliermondii T-1.Its acid-heating extraction was optimized by orthogonal experiment as follows:0.5 g of dried cells was hydrolyzed with 10 mL 4 mol/L HCl in a boiling water bath for 10 min,and then 25 mL chloroform:methanol(2:1)were added.After mixing the lipids were separated and washed by 10 mL 0.15%(m/v)NaCl.The total lipid extraction rate could reach to 40.12%.The ACP gene was amplified from Meyerozyma guilliermondii T-1 genomic DNA.The bioinformatics analysis results showed that the ACP gene was full-length 384 bp containing a complete ORF which encoding 127 amino acids with one phosphopantetheine moiety.It was a non-secreted hydrophilic protein without signal peptide,had nine existence potential phosphorylation sites,its secondary and tertiary structures were mainly composed of a helix and coli.The recombinant plasmid of pET21a-mgACP was constructed and transformed into E.coli BL21(DE3)and the expression of mgACP gene was induced by IPTG.The growth kinetics results showed that the optimal IPTG concentration was 0.1mmol/L and the molecular weight of expressed fusion protein was 22.0 KDa.