Mutagenesis And Transcriptional Analysis Of Lipase Strains For Catalyzing The Biosynthesis Of Vitamin A Palmitate

Vitamin A palmitate is a more maturely studied vitamin A derivative,which is used in pharmaceutical,cosmetic,feed and other industries.The production of commercially available vitamin A palmitate mainly relies on chemical synthesis and enzymatic synthesis,while enzymatic synthesis with mild conditions and good environment is the focus and hot spot of current research.The type and activity of catalyst lipase and esterification reaction conditions are the limiting factors for the enzymatic synthesis of vitamin A palmitate.In order to improve the conversion rate of vitamin A palmitate,a mutant strain with improved lipase 2(Yl Lip2)activity was obtained by Genome shuffling of Yarrowia lipolytica(YLL).Transcriptome sequencing was used to analyze the genetic differences between mutant and original strain,and to find the related genes that control lipase activity.Finally,the mutant strain was applied to produce Yl Lip2,catalyze the synthesis of vitamin A palmitate,optimize the esterification reaction conditions,and improve the catalytic yield.The main findings were as follows.(1)Two kinds of mutagenesis,nitroguanidine(NTG)and Atmospheric Room Temperature Plasma(ARTP),were used as the basis for Genome shuffling to obtain four primary mutagenic strains N1,N3,A2-60 and A3-60.Three rounds of recursive fusion were performed to obtain a strain with excellent Yl Lip2 enzyme activity named YLL-S33 as a positive mutation library.The lipase enzyme activity of this strain was13.96 U/m L,which was 2.2 times higher than that of the starting strain YLL,and the trait could be inherited stably.(2)Based on RNA-Seq technology,421 differentially expressed genes were screened,and 106 genes were transcriptionally up-regulated and 315 were down-regulated in YLL-S33 relative to YLL.The analysis yielded genes that may affect the enzymatic activity of Yl Lip2 mainly involved in biological processes such as peroxisome,glycine,serine and threonine metabolism,protein processing in the endoplasmic reticulum,MAPK signaling pathway and fatty acid degradation.(3)The enzymatic synthesis of vitamin A palmitate was carried out using Yl Lip2 produced by mutant strain YLL-S33 as a catalyst and vitamin A acetate(2%)and palmitic acid(4.6%)in organic solvent hexane as substrates,and the esterification reaction conditions were optimized to obtain 69% conversion when the initial water addition was 0.1 m L,the enzyme concentration was 80 mg/m L,and the reaction was carried out at a shaker speed of 200 r/min and 35℃ for 24 h.This study complements the current effective mutation breeding methods in Yarrowia lipolytica,further explores the biofunctional pathway of phenotypic enhancement of mutant strains,and provides theoretical basis and support for the later improvement of Yarrowia lipolytica lipase production;provides suitable lipase species for enzymatic synthesis of vitamin A palmitate,and at the same time,optimizes the esterification conditions,which is of great significance for the production of vitamin A palmitate in the application of modern industrial enzyme synthesis.