Metabolic Engineering Of Aspergillus Flavipes For Aspochalasin D Production And Fermentation Optimization

Cytochalasins is a kind of polyketide-amino acid hybrid natural products with the isoindolone core and macrocyclic structure,and widely distributed in fungi.The typical biological activity of cytochalasins is binding to actin to affect a series of cellular processes.Aspochalasin D（AD）belongs to a class of cytochalasins with the L-leucine as precursor.AD has a series of unique biological activities,including anti-fouling,anti-cancer,anti-becterial and inhibiting serotonin release.AD has important potential application prospect in agriculture and pharmaceutical.However,The AD production by the original strains is low.Aspergillus flavipes 3.17641 with AD production capacity was used as the research strain.Fermentation optimization and metabolic engineering were employed to optimize the fermentation conditions and metabolic of AD synthesis,which significantly improved the AD production of the strain.This study not only provides a basis of the large-scale production and application of AD,but also provides a reference for enhancing the production of similar natural products in other fungi.The main results are as follows:（1）The strain 3.17641 was verified as A.flavipes and its fermentation product was identified as AD.The A.flavipes 3.17641 was used as wild type（WT）.The strain 3.17641was confirmed as A.flavipes by internal transcribed spacer（ITS）sequence molecular identification.The strain was fermented in liquid medium,and the ethyl acetate extract was separated by column chromatography and thin layer chromatography.The isolated product was characterized by nuclear magnetic resonance,and the main product of the strain was identified as AD.（2）Improve AD production by optimizing fermentation conditions of A.flavipes 3.17641.Single factor experiment was used to optimize the fermentation conditions,including temperature,culture time,initial p H,carbon source and nitrogen source.L-leucine is an important amino acid precursor for the synthesis of AD,the effect of L-leucine on the production of AD was tested.Based on single factor experiments,response surface experiment was used to furthe optimize the medium components,and the selected factors were glucose,soy peptone and L-leucine.The optimized fermentation conditions were determined as follows:temperature 29°C,culture time 7 days,initial p H 8.0,glucose 25.2g·L^-1,soy peptone 3.0 g·L^-1,L-leucine 1.4 g·L^-1.Under optimized fermentation conditions,the AD production of A.flavipes 3.17641 increased from 44.0 mg·L^-1 to 199.2 mg·L^-1,which is 4.5 folds higher than that from the initial condition.（3）Efficient production of AD by metabolic engineering of A.flavipes 3.17641.The resistance screening test of A.flavipes 3.17641 was carried out,and 200μg·m L^-1 genomycin and 100μg·m L^-1 bleomycin were selected as the resistance screening markers for strain modification experiments.The aspo gene cluster responsible for the synthesis of AD was verified by knockout of the core gene aspo E.Based on the biosynthetic pathway analysis of AD,the flavin-dependent oxidase encoded by aspo A catalyzed the double bond isomerization of AD and precursor TMC-196 to produce Aspochalasin P and Aspochalasin Q,respectively,which reduced the production of AD.The mutantΔaspo A was obtained by deletion of aspo A in A.flavipes 3.17641.The expression of aspo A in the mutantΔaspo A was abolished,while the expression levels of aspo B,aspo C and aspo E-H were not significantly different from those of WT.The AD production of the mutantΔaspo A reached 325.3 mg·L^-1,which was 1.6folds higher than that of WT.In order to further enhance the AD production of A.flavipes3.17641,the mutantΔaspo A::OEaspo G was obtained by overexpressing the transcription factor aspo G after the deletion of aspo A.Compared with WT,the expression level of aspo G in the mutantΔaspo A::OEaspo G was significantly increased by 29.8 folds,and the relative expression levels of aspo B and aspo F were increased by 19.1 folds and 14.4 folds,and the relative expression levels of aspo C,aspo E and aspo H were slightly increased by 3.5 folds,5.2folds and 2.5 folds,respectively.The production of the mutantΔaspo A::OEaspo G was 812.1mg·L^-1 under optimized fermentation conditions,which was 4.1 folds and 2.5 folds higher than that of WT and the mutantΔaspo A,respectively.