Study On Fermentative Production Of Jasmonic Acid By Lasiodiplodia Iranensis

Jasmonic acid is a cyclopentane compound with fragrance.It has good application values in agriculture,flavor,fragrance and medicine industries.The content of jasmonic acid is low in plants and the extraction amount is limited;while the chemical synthesis of jasmonic acid is long and is always limited by a low yield and optical purity.As a result,people pay more attention to the production of jasmonic acid by fermentation.In this thesis,the screening of jasmonic acid–producing strains,the identification of the products,the optimization of the fermentation medium and the culture conditions and the inference of the jasmonic acid metabolic pathway were studied.The method of producing jasmonic acid by fermentation was preliminarily established,which laid the foundation for the large-scale production of jasmonic acid.Details as follows:?1?Screening and identification of jasmonic acid-producing strains.A total of 221 strains were screened from different screening material.Strain DWH-2 produced more jasmonic acid was obtained by TLC rapid detection method.GC-MS analysis was used for the qualitative analysis of the product.Jasmonic acid concentration was 340 mg·L^-1 by HPLC.The morphological and molecular biology of the strain was identified and the strain was identified as Lasiodiplodia iranensis,which was named Lasiodiplodia iranensis DWH-2.The strain has been preserved in China Center for Type Culture Collection with a number of CCTCC NO:M2017288.?2?Identification of product configuration.Ethyl acetate was used to extract the fermentation broth and the organic phase was evaporated.Purification of concentrated products was conducted by using semifinished liquid phase.Only one configuration was detected in the product by chiral column.The configuration was determined by circular dichroism analysis was?3R,7R-JA?.?3?Optimation of fermention medium and culture conditions.After controlling a single variable and orthogonal experiments,the medium composition was(g·L^-1):sucrose 50,NaNO₃4.5,KH₂PO₄ 2.0,KCl 0.4,MgSO₄·7H₂O 6,FeSO₄·7H₂O 0.9,corn syrup 1.5,soybean oil 10,trace elements 10 mL·L^-1.The trace element composition was as follows(g·L^-1):ZnSO₄·7H₂O0.03,MnSO₄·H₂O 0.0018,CuSO₄·5H₂O 0.0026,Na₂MoO₄·2H₂O 0.003.The optimal fermention condition in flasks wew under non-agitated condition,the seeds were inoculated for3 to 4 days,the medium initial pH was 5.5,the liquid volume was 100/500?m L/m L?,and the inoculation volume was 12%,the culture temperature is 28°C and the fermentation period is 7days.After optimization,the yield of jasmonic acid was up to 1.5 g·L^-1,which was 4⁵ times higher than before optimization.At the same time,experiments were also conducted in a 3 L fermentor.The yield of jasmonic acid was about 1.29 g·L^-1 by static culture,and the aeration volume was 0.1vvm,the fermentation period was about 12 days.?4?Analysis of intermediate metabolites and inference of metabolic pathway.The mycelial disruption solution was used as the enzyme source,the composition and content of the bacteria in different fermentation phases were determined by the external addition method.The existence of key metabolites such as?-linolenic acid,hydroperoxide and OPDA was confirmed.Based on the experimental results and literature analysis,?-linolenic acid was used as a starting material in this strain through a series of enzymatic reaction to produces hydroperoxide,OPDA,and ultimately produces jasmonic acid.