Analysis Of Genes Involved In Zooxanthellae MAAs Biosynthesis And Identification Of DHQS-OMT Genes

Objective:The genes related to the biosynthetic pathways of zooxanthellae cytosolic amino acid MAAs were mined using stable isotope-labeled metabolic analysis and transcriptome data,and the DHQS-OMT gene was characterized and expressed heterologously to understand the mechanism of zooxanthellae resistance to ultraviolet rays and lay the groundwork for later industrial application.Methods:Using Na H¹³CO3-labeled zooxanthellae strain SYSC-14-11,a thorough scan of the extract in positive and negative ESI ionization mode,combined with the database and retention time using UPLC-Q-Exactive MS analysis technology,was executed to identify the 13C-labeled metabolites.Based on the transcriptome annotation results,the genes related to zooxanthellae biosynthesis were screened.The ORF of a fusion gene DHQS-OMT,which may be involved in the synthesis of MAAs precursors,was constructed into the prokaryotic expression vector BL21 to detect whether there were new products generated using prokaryotic expression,purification,renaturation and HPLC.Results:（1）A total of 10 kinds of ¹³C-labeled compounds were identified to be involved in MAAs biosynthesis by metabolite analysis.Among them,4 labeled compounds were confirmed to be the precursors or primary metabolites of MAAs biosynthesis pathway according to the literature.Further analysis also indicated that shikimic acid pathway was occurred in zooxanthellae.On the other hand,it was speculated that pentose phosphate pathway,from demethyl 4-deoxygadusol（DDG）to 4-deoxygadusol,might also be existed in the biosynthesis of MAAs based on the metabolites.（2）A total of 233 genes related to MAAs biosynthesis pathways were screened from the transcriptome,including 97 DHQS annotations,35 OMT annotations,27 ATP-grasp domain annotations and 74 NRPS annotations.Through the local Blast,9 unigenes were obtained by screening.Then,the homologous evolution analysis was carried out.Finally,a highly similar fusion gene that may be involved in the production of MAAs precursors was selected and named as the DHQS-OMT fusion gene.Further analysis suggested that DHQS-OMT fusion gene was clustered with invertebrate,which inferred that horizontal gene transfer via endosymbiosis might be occurred.Bioinformatics analysis of the gene shows that the length of ORF is 1998bp,which encoded 665 amino acids.The predicted molecular weight and isoelectric point is73.02 k Da and 5.29,respectively.And it is a hydrophilic protein with special functional domains such as DHQS and OMT,which were closely related with DHQS of Cyanobacterium,and OMT of fungi,respectively.（3）After synthesizing the ORF of DHQS-OMT gene,it was constructed into a prokaryotic expression vector.The molecular weight of obtained protein is between 60-75 k Da,which is in line with the expected molecular weight of this protein.The HPLC results of bacterial extracts showed that 2 isolated peaks were found at the retention time of 2.5 min and 5 min in the bacterial extract when compared with that without IPTG induction,which indicated that new products were formed.And the mass spectrometry identification is required in the future study.Conclusion:It was found that both shikimic acid pathway and pentose phosphate pathway were involved in the biosynthesis of MAAs in the zooxanthellae.And a fusion gene was screened out.It was expected that this study would provide the foundation for the research concerning the gene function identification and biosynthetic pathways of zooxanthellae MAAs.