The Expression Of 10-HDA Biosynthesis Gene In Pichia Pastoris

According to the different age of bee production capacity of 10-HDA, to find out the differences of protein mass spectrometry, auxiliary identification shows that the key enzyme gene, beta electron transfer flavoprotein subunit gene and ketoacyl coenzyme A thioesterase gene of Apis mellifera, will integrate the two key enzyme gene into the Pichia genome and the expression, hope that the two the key enzyme gene expression in microorganisms can play a way of expression is similar to those of the bees in the body, so as to get to the production of engineered strains of 10-HDA. Thioesterase in organisms can catalyze the hydrolysis of acyl carrier protein and fatty acyl chain, of medium chain fatty acids (Medium chain fatty acids, MCFAs) plays a key role in the accumulation of. In order to obtain engineering bacteria with chain fatty acid production ability, using Arabidopsis cDNA as template, PCR amplified the acyl-ACP thioesterase gene AtFatA, plasmid EcoR I /Xba I digestion and then linked to the same double digestion, the rEcombinant plasmid pPICZ alpha AtFata. The rEcombinant plasmid was transferred into Pichia pastoris GS115 shock, by Zeocin resistance screening, selected positive clones and shake flask fermentation induced, obtained obvious target protein bands SDS-polyacrylamide gel electrophoresis, for the first time successfully established AtFatA fungi expression system. GC-MS detection of fermentation product of extracellular free fatty acids, found in Pichia pastoris rEombinant AtFatA strain free than the starting strain GS115 extracellular MCFAs, acid production increased by 51%, MCFAs accounted for extracellular total fatty acids yield 28.7%, while the wild mushroom in this ratio is only 18.1%, which explores a new way for the future production of safe non-toxic MCFAs.