Biosynthesis Pathway Of Bacterioruberin In The Halophilic Archaea Halorubrum

Bacterioruberin and its derivatives are locted on the cell membranes of halophilic archaea,and belong to hydroxylated C50 non-cyclic carotenoids.Compared with the common C40 carotenoids,bacterioruberin also processes antimicrobial,antiviral,antihemolytic and antitumor and multiple biological functions.At present,bacterioruberin is mainly obtained by halophilic archaea fermentation,but the low yield makes it difficult to achieve large-scale production.The low yield of bacterioruberin is mainly due to the low intracellular content,and the study on biosynthesis pathway of bacterioruberin is helpful to enhance the intracellular content.In this paper,halophilic archaea Halorubrum sp.IT-4with high content of bacterioruberin was used as the experimental material.Via the morphological observation and physiological and biochemical identification,the basic characteristics of Halorubrum sp.IT-4 were clarified.Based on the whole genome map,the de novo synthesis pathway of Halorubrum bacterioruberin was preliminarily analyzed.Through the carotenoids content and transcriptome in different growth periods,mined the candidate genes related to bacterioruberin synthesis.which provide theoretical support for metabolic regulation and further improvement of bacterioruberin content.The study also lay a theoretical foundation for the development and utilization of new halophilic archaea carotenoids resources.The main research results were as follows:(1)Halorubrum sp.IT-4 cells is short rod-like.The size of short rod-like formwas0.5~1μm×1~1.5 μm.Halorubrum sp.IT-4 is Gram negative;the colony is round with diameter of 0.5~1 mm,smooth surface,circular,convex and with entire margins and red color.Catalase activity and oxidase is positive.(2)The chromosomal genome of Halorubrum sp.IT-4 contained 2,9962,65 bp,with GC content of 68.78 %.A total of 2956 coding genes were predicted.Based on the genome,it was speculated that Halorubrum sp.IT-4 synthesized isopentenyl diphosphate through the mevalonate pathway,then converted into lycopene,further synthesized bacterioruberin.The biosynthesis pathway of lycopene is clear based on a variety of research,but the pathway from lycopene to bacterioruberin has not been clarified.In this study,9 genes related to bacterioruberin biosynthesis were selected from the genome and conduct bioinformatics analysis,including cru F encoding carotenoid hydrase,crt D-1,crt D-2 and crt D-3 genes encoding carotenoid desaturase,lye J-1 and lye J-2 genes encoding lycopene elongase,β-carotene dioxygenase(brp)and bacteriorhodopsin(bop).(3)Carotenoids anlysis results showed that the total carotenoids and bacterioruberin content of Halorubrum sp.IT-4 cells increased gradually with the increase of culture time within 192 h,and the total carotenoids content was the highest at 192 h,which reached4.5×10-10±0.26×10-10 μg/cell,and the content decreased after 192 h.(4)Halorubrum sp.IT-4 cells at different growth stages(24 h,48 h,96 h and 192 h)were collected for comparative transcriptome analysis.The expression of selected 8 genes were analyzed by fluorescence quantitative PCR,and 75% genes were similar with transcriptome results.The transcriptome result showed that crt D-1,crt D-2 and crt D-3increased significantly at 96 h and 192 h,whilethe expression of cru F,lye J-1 and lye J-2genes decreased significantly with the increase of culture time.Besides,the expression of brp and bop,which indirectly affect the synthesis of bacterioruberin,were proportional to the culture time.These two genes are involved in the synthesis of bacteriorhodopsin in halophilic archaea,which is a competitive pathway with bacterioruberin,and could be used as candidate genes for metabolic regulation.