Microbial Diversity And Secondary Metabolites Of Microalgae Prorocentrum Lima

The microalgae of Prorocentrum genus is a typical red tide dinoflagellate,which has an important impact on the marine ecological environment,and has always attracted people's attention.At the same time,its metabolites are diverse and have good biological activities.The representative metabolites,oakadaic acid?OA?and its derivative dinophysistoxin-1?DTX-1?,are the main components of diarrhea shellfish poisoning.It will directly affects people's health through seafoods and may lead to cancer.However,it has certain medicinal value.Therefore,the metabolites of Prorocentrum spp.have become a research hotspot of marine natural products.For further study on the relationship between the microalgae-producing toxin and its co-existing microorganisms,Protocentrum lima PL11 was analyzed by Illumina MiSeq high-throughput sequencing technology to analyze the species diversity and abundance of co-existing microorganisms in the sample.A total of 42065 sequences and68 OTUs were obtained by sequencing.The rarefaction curve indicated that the sequencing depth was sufficient.The Simpson value was 0.097,and the Shannon value was 2.807.The co-existing bacteria of simple belonged to 5 phylums,14 classes,26orders,38 families and 54 genera.Among them,there are 4 dominant phylums?>5%?,including Dinoflagellata?36.8%?,Ascomycota?26.3%?,Basidiomycota?10.5%?and?Bicosoecida,10.5%?.There are 3 dominant classes?>5%?,including Saccharomycetes?10.5%?,Dinophyceae?24.3%?,and the unknown class?21.4%?.There are 6 dominant genera?>5%?,including unknown genus of Nannocystaceae?21.8%?,Pyruvatibacte sp.?11.6%?,Phaeodactylibacter sp.?9.4%?,unknown genus of Hyphomonadaceae?8.5%?,unknown genus of OM190?8.1%?,and Roseovarius sp.?7.9%?.Nine strains were isolated from P.lima PL11 and classified into eight genera,among which were two Ochrobactrum strains,and each one of Microbacterium sp.,Algoriphagus sp.,Ponticoccus sp.,Hoeflea sp.,Labrenzia sp.,Sphingopyxis sp.,and Erythrobacter sp..The secondary metabolites of P.lima was studied.By combination use of thin layer chromatography,normal phase silica gel column,reversed phase silica gel column,MCI column,Sephadex LH-20 gel column,semi-preparative high performance liquid chromatography and recrystallization,17 monomer compounds was separated from the extract.15 structures?1–15?were identified by means of modern spectroscopy methods(LC-TOF MS,¹H-and ¹³C-NMR)and their physicochemical properties.Ten monomeric compounds?1–4,8,11,12,14,15?were isolated from the metabolites of P.lima PL11and PL-L;five monomeric compounds?5–7,9,13?isolated from the metabolites of P.lima PL01.Among them,OA?1?,OA methyl ester?2?,and prorocentrolide?3?were typical DSP and cyclic imine toxin metabolites from Prorocentrum spp.,respectively.Compounds 4-9,13 and 15 are reported for the first time from this genus of microalgae.Three compounds,?Z?-1,6,6-trimethyl-7-oxabicyclo[2,2,1]heax-2?9?-en-10-oic acid?4?,apo-9'-fucoxanthinon?5?and 3,5-Dihydroxy-6,7-megastigmadien-9-one?6?which contained unique multiple substituted cyclohexane ring and unsaturated function groups should have the carotenoid origin.Compounds 7–15 are benzoic acid,3-indolecarboxylic acid,3-indolylaldehyde,thymine,thymidine,cytosine,5-Hydroxy-3,4-di-methyl-5-pentyl-2?5h?-furanone,DHA ethyl and 5-aminopentanoic acid.The scallops and mussels were selected to study the metabolic transformation of toxins in shellfish.The extract of algal cells and the shellfish tissue were detected by LC-MS.The density of algae cells increased significantly after the experiment.The cyclic imine toxin,prorocentrolide,was detected in the extract of algae cells.No prorocentrolide was detected in mussels and scallops after exposure to P.lima of 10⁴ and10³ cells/L for 48 h.Another toxin,3-Hydroxy-2-methylenepropyl ester was detected in the scallop group S1 and S2.In addition to toxins,there are three main components with molecular weights of 678.89?v%is 24.95?,791.57?v%is 21.80?,and 904.67?v%is7.31?,respectively.The accumulation of the main components 1 and 2 in mussels was greater than that of scallops,and there was no significant difference between the different concentrations.P.lima is a typical toxin-producing red tide dinoflagellate.Make full studies of its secondary metabolites that contain well-active toxins and compounds with novel structures.Further exploration of its metabolic transformation in shellfish is of great significance to the food safety of Chinese seafood.