Research On The Secondary Metabolites Of Marine Aspergillus Terreus C23-3 Under Chemical Induction

Background:Since marine fungi live in special habitats with high salt,high pressure,low oxygen,and oligonutrition,they form unique metabolic mechanisms and can produce a large quantity of secondary metabolites with novel structures as well as rich activities.Among them,there are many active ingredients that have strong antioxidant or acetylcholinesterase inhibitory or other activities to prevent and treat neurodegenerative diseases,providing a rich source of anti-AD(Alzheimer's disease)drug lead compounds.Purpose:the aim of this study is to screen chemically regulated cultural conditions that can obtain more abundant secondary metabolites,explore new methods of secondary metabolism regulation,expand the structural diversity and activity of important products such as aryl butyrolactones,and provide a basis for the discovery of anti-AD active drug lead compounds.Methods:A marine-derived Aspergillus terreus strain C23-3 featuring of producing butyrolactone I was used as the starting strain to conduct chemically regulated fermentation culture with different strategies,including diversified small molecule chemical induction strategy and simulated mutational biosynthesis strategy:(1)Diversified small molecule chemical induction strategyBased on two basic mediums,seawater potato sucrose liquid medium and brown rice medium,a series of concentration gradients of diversified precursor small molecules(3-amino-L-tyrosine,3-hydroxy-L-tyrosine,3-nitro-L-tyrosine,3,4-dihydroxyphenylpyruvate,farnesol,farnesylpyrophosphate(FPP)),oxidative stress agents(sodium bisulfite menadione(MSB),hydrogen peroxide(H₂O₂))and L-selenomethionine were used as chemical inducers to study the secondary metabolites of the strain under different conditions.The cultures were investigated on the colony morphology,thin layer chromatography characteristics(ultraviolet,fluorescence,chemical color development and bioautographic characteristics),HPLC fingerprints and molecular networking based on LC-MS/MS.(2)Simulated mutational biosynthesis strategyUsing seawater potato sucrose liquid medium as the basic medium,firstly the biosynthesis dynamics of butyrolactone I,a characteristic metabolite of strain C23-3,was studied and found out the time-point of the first rise and the peak time in butyrolactone I production;secondly,a gradient series of concentrations of diverse precursor small molecules(3-hydroxy-L-tyrosine,3,4-dihydroxyphenylpyruvate,farnesol)and 4-hydroxyphenylpyruvate synthase inhibitors,including prephenate dehydrogenase inhibitor(1,3-adamantane diacetic acid,N-(p-toluenesulfonyl)-L-phenylalanine)and Tyrosine aminotransferase inhibitor(phenelzine sulfate)were used as chemical inducers in the way of alone or combinational addition.The colony morphology,HPLC fingerprints of the strain metabolites and production of butyrolactone I under different culture conditions were investigated.Results:(1)Diversified small molecule chemical inductionThe micro-fermentation results showed that nine inducers could stimulate the changes of the types and yields of metabolites of strain C23-3 to varying degrees.Among them,four inducers including farnesol(1?mol/L),FPP(5?mol/L),MSB(10?mol/L)and 3-amino-L-tyrosine(1 mmol/L)displayed good effects,which could stimulate the strain to produce diversified butyrolactones and territrems.The regular fermentation results showed that H₂O₂ produced oxidative stress on the strain C23-3 under the brown rice medium,and induced the strain to produce higher yield of territrems;the TLC fingerprints of the metabolites of the strains under the other eight induction conditions showed remarkable differences,but their HPLC-DAD fingerprints did not change significantly.The large-scale fermentation showed that the metabolites of the strain under the condition of MSB(10?mol/L)induction may contain a series of butyrolactones.(2)Simulated mutational biosynthesisThe experimental result of dynamics of butyrolactone I(a characteristic metabolite of strain C23-3)showed that the yield of butyrolactone I,would change with the culture time,starting rapid synthesis on day 2 after inoculation and reaching peak on day 5.Tyrosine aminotransferase inhibitor-phenelzine sulfate had a significant inhibitory effect on the synthesis of butyrolactone I;pre-phenate dehydrogenase inhibitors-1,3-adamantane diacetic acid or N-(p-toluenesulfonyl)-L-phenylalanine alone had no significant inhibitory effect on the synthesis of butyrolactone I;however,there was a dose-dependent inhibitory effect when they were applied in combination with phenelzine sulfate,and the effect was stronger than that of phenelzine sulfate alone.The small molecule precursor analog 3,4-dihydroxyphenylpyruvate showed a significant inhibitory effect on the production of the main product butyrolactone I at a dose of 10 mmol/L.When 3,4-dihydroxyphenylpyruvate and two enzyme inhibitors(N-(p-toluenesulfonyl)-L-phenylalanine+phenelzine sulfate)worked together,they could greatly inhibit the production of butyrolactone I.Conclusion:The induction of diversified small molecules had a regulatory effect on the secondary metabolism of marine fungi,and could activate the expression of some silent gene clusters;co-action of 4-hydroxyphenylpyruvate synthase inhibitors and 3,4-dihydroxyphenylpyruvate could inhibit the production of butyrolactone I,the main product of the strain,and verify its biosynthetic pathway,which laid a preliminary foundation for further research on simulated mutational biosynthesis.These were useful explorations for the discovery of anti-AD active compounds.