Function And Regulation Of The Lipoxygenase Family In Neoporphyra Haitanensis

Oxylipins in many algae are found to be generated from both C18 and C20 polyunsaturated fatty acids(PUFAs)by lipid oxidation pathway,hold diversity in chemical structures,and play a key role in the chemical defense and signaling under different environmental stresses.Although lipoxygenase(LOX)is a key enzyme to initiate the lipid oxidation pathway,but it is still poorly reported in algae.Herein,the present work was focused on a comprehensively study on the LOX gene family in Neoporphyra haitanensis,including member composition,molecular characteristics,enzyme activity,the molecular mechanism of substrate transformation,as well as the defensive response under diverse intertidal environmental conditions.Furthermore,based on the multi-functional activity of NhLOX1,its potential as a novel biocatalyst for the one-step biosynthesis of aromatic compounds from fatty acids was analyzed.The main results are summarized as follows:(1)The NhLOXs family showed multiple origin characteristics in evolution.By blast search and conserved domain analysis,a total of six LOXs homologous genes were identified in N.haitanensis genome.Phylogenetic analysis showed that LOXs from different species were mainly clustered into two groups that were early divergent from a common ancient ancestor.Some LOXs from multicellular red algae,including NhLOX1 and 2,gathered closely to ones from some marine bacteria,fungi,green algae,higher plants and animals,while the remaining red algal LOXs clustered well with homologs from bacteria,cyanobacteria and brown algae in another group.In addition,gene expansion of LOXs obviously occurred among Bangiaceae species.A variety of transcript isoforms of NhLOX1 and 2 were obtained in the full length c DNA library,suggesting their diverse regulatory role at the level of post-transcriptional modification.(2)The NhLOXs family had the characteristics of broad substrate selectivity,non-specific oxidation site and multifunctional enzymatic activity.Recombinant proteins of NhLOXs were obtained by over-expression in E.coli.Combining with HPLC-MSMS and GC-MS detection,the enzymatic activity of NhLOXs in vitro was analyzed.Results showed that there were significant differences in substrate selection,preference and peroxidation sites among six NhLOX members.Both NhLOX1 and 2 showed multifunctional activity.Except for LOX activity,NhLOX1 exhibited the enzymatic activity similar to allene oxide synthase(AOS)and hydroperoxidase lyase(HPL),which simultaneously existed in the sole functional domain at the C-terminal part of proteins.NhLOX1 could catalyze the peroxidation of C18-PUFAs at C-9/13,C20-PUFAs at C-8/9/12 and docosahexaenoic acid(DHA)at C-10/11/14,respectively,resulting in diverse products such as mono-hydroperoxides(mono-ROOH),di-hydroperoxides(di-ROOH),ketols and C5-C9 volatile aldehydes/alcohols/ketones(VOCs).Differently,NhLOX2 held both LOX and hydroperoxidase-like(HPI)activity,but only used substrates that harbor vinyl bonds no less than 3.So,by NhLOX2 catalysis,γ-linolenic acid was transformed at C-6/9/13,C20-PUFAs at C-8/9/12 and DHA at C-4/7/10/14/20 to generate corresponding mono-ROOH,di-ROOH and hydroxylated products.Nevertheless,both NhLOX1 and 2showed substrate preference for C20-PUFAs,especially used eicosapentaenoic acid(EPA)as the most optimum substrate,suggesting their close relationship with fatty acids composition in N.haitanensis.However,assays in vitro indicated that NhLOX3-6 had no catalytic activity on C18-PUFAs and arachidonic acid(ARA),and could only convert a few of EPA and DHA into5-/8-hydroxy eicosapentaenoic acid(5-/8-HEPE)and 7-/10-hydroxy docosahexaenoic acid(7-/10-HDHE).In summary,NhLOXs-depending lipid oxidation seems to provide a sufficient enzymatic basis for the abundant oxylipins biosynthesis in N.haitanensis.(3)The selectivity of substrate oxidation site and AOS-like activity of NhLOX1 were controlled by specific amino acids at the active center.Results revealed that Phe-642,Phe-826,Gln-777 and Asn-575 in NhLOX1 were closely related to the activity of double-peroxiding and AOS-like to substrates by conjoint analysis using 3D modeling,enzyme-substrate molecular docking and gene site-directed mutation.These four amino acids may be the key sites playing as the spatial-localization factors for substrates in the active pocket of NhLOX1.In addition,Phe-826 or Asn-575 mutant proteins catalyzed ARA to generate more C8-/C9-VOCs than the wild NhLOX1 did,suggesting that the HPL activity was more active when losing these two potential locators.Based on these data,we proposed a new hypothetical model for the substrate catalytic mechanism of multifunctional LOXs,in which two steps of reaction happen when C18-and C20-PUFAs are transformed into di-ROOH or ketols.In the enzymatic reaction of di-ROOH biosynthesis,C18-and C20-PUFAs are likely to take different "head to tail" directions to insert into the active pocket of NhLOX1.Then,substrates may undergo double peroxidation under the localization of Phe-642 and Phe-826.However,when products are ketols,both C18-and C20-PUFAs are inserted into the active pocket by the carboxyl head.Meanwhile,the changes of enzymatic activity of different NhLOX1 isoform proteins suggested that the N-terminal SRPBCC domain and the connecting loops in the middle region may be involved in the stability of spatial conformation.Deletion of these sequences resulted in the change of the catalytic activity and the double-peroxidation capacity of NhLOX1 to different substrates,and even the loss of HPL activity.By comparing the catalysis characteristics of LOXs from Shewanella violacea and Chondrus crispus,results further indicated that the AOS-like activity in NhLOX1 was spcies-specific,while the catalysis like HPL has been obtained by some ancient ancestors from marine bacteria.Nonetheless,more details about the catalytic mechanism of algae LOXs are still needed to study by new X-ray crystal data.(4)The Nhloxs family exhibited different transcription patterns in the growth of N.haitanensis and in response to(a)biotic stresses.In silico prediction of cis-regulatory active elements revealed that there were abundant stress response elements in the promoter region of Nhloxs,involving in phytohormone responsiveness,light and abiotic stress regulation,and the binding sites of defense-related transcription factors.Furthermore,according to the full-length c DNA library and RNA-seq transcriptome,it was found that the high level of the transcription of Nhlox1 was main in the gametophyte,while Nhlox5 and 6 was expressed mainly in the filamentous conchocelis.Broadly,the transcription level of Nhloxs in the diploid stage was significantly lower than that in the haploid.In addition,the expression of Nhlox2 was significantly upregulated in the carpotetrasporangium stage and was at the highest level of transcription among all members,suggesting that this gene may play an important role in regulating sexual differentiation of N.haitanensis.By the real-time quantitative PCR detection,it was found that Nhlox1 and 5 were main the genes upregulated at the initial stage of oligoagar exposure to N.haitanensis gametophyte,and the transcription of Nhlox2 was more strongly raised at 20 min.However,under the eliciting of Flg22,the expression of Nhlox1 was rapidly upregulated and maintained at a high level,while Nhlox5 and 6 were significantly activated after 20 min.Compared with biotic elicitors,the expression patterns of Nhloxs were obviously different under abiotic stresses.Under the drought stress,most members were significantly upregulated,with an exception of Nhlox3,and the expression change of Nhlox2 was the most severe,followed by Nhlox1.When the gametophyte were faced the mechanical damage,all members of Nhloxs responded positively and significantly,and exhibited higher levels of transcription,especially the three members of Nhlox1-3.Additionally,the expression of Nhlox1,2 and 6 were up-regulated under heat shock,while the rest of three members were significantly inhibited to transcribe.Inversely,the transcriptome analysis suggested that the expression of Nhlox2 and 5 could be activated by low temperature,while other members were significantly declined in their transcription,showing the time-and temperature-dependence.(5)NhLOX1 holds the ability to generate C8-C9 VOCs by one-step catalysis reaction.When C18～C22 PUFAs were used as substrates of NhLOX1,the yields of C8–C9 VOCs were high,e.g.,2E-nonenal from ARA,and 2E,6Z-nonadienal from EPA.Hydrolyzed oils were also tested as substrates.The reactions mainly generated 2E,4E-pentadienal and 2E,4E-octadienal from hydrolyzed sunflower seed oil,corn oil,and fish oil,respectively.NhLOX1 showed good stability after storage at 4 °C for two weeks and broad tolerance to p H and temperature.These desirable properties of NhLOX1 make it a promising novel biocatalyst for the industrial production of volatile aroma compounds.In conclusion,this present work successfully identified six LOX members and their functions in N.haitanensis,found two novel multifunctional LOXs,and proposed a new hypothesis of fatty acid peroxidation mechanism for the multifunctional LOXs in algae.The key molecular evidences were obtained on the scientific questions of how the abundant and diverse oxylipins in algae are generated by the lipid oxidation pathway and what the regulatory patterns of the LOX family are adopted during the growth and stress responses under exposure to the complex intertidal environment conditions.These results will be valuable for the further study on the functional and molecular regulatory network of the eukaryotic algae LOX family,and provide basic and substantial data to support the role of lipid oxidation pathway in the environmental adaptation and defense system of intertidal algae.