The Biosynthetic Route Of Mycosporine Amino Acids In Nostoc Flagelliforme

Nowadays,more and more UV radiation arrive earth surface.Increasing UV radiation which have a great impact on the entire biosphere not only induce human skin cancer,but also cause serious harm to photosynthetic organisms in aquatic and terrestrial ecosystems.Mycosporine-like amino acids(MAAs)is a kind of colorless,low molecular weight and water-soluble substance which have a strong ability to absorb ultraviolet radiation.We have discovered the synthetic gene cluster of MAAs(mysA-mysB-mysD-mysCl-mysC2)in Nostoc flagelliforme through excavating of the genomic data of Nostoc flagelliforme.The gene cluster of MAAs is different from others reported before and may be involved in the synthesis of a new ultraviolet absorption substance which is mycosporine-2-(4-deoxygadusolyl-omithine)(M-2DO).However,the synthesis pathway of M-2D0 is not clear.In this study,the heterologous expression system of the gene cluster of MAAs in Nostoc flagelliforme was constructed using Anabaena sp.PCC 7120 and Escherichia coli BL21,and the products were detected by liquid chromatography-mass spectrometry(LC-MS)and nuclear magnetic resonance(NMR)in order to reveal the complete synthesis pathway of M-2DO.At present,the following major research results have been obtained.1.The MAAs biosynthetic genes mysA-mysB?mysA-mysB-mysD?mysA-mysB-mysD-mysC2,mysA-mysB-mysC1 and mysA-mysB-mysD-mysCl were transferred to Anabaena PCC 7120.The synthesis product of each gene was studied.As a result,the products of all Anabaena PCC 7120 were 4-DG.We failed determine the synthesis pathway of MAAs through the expression of Anabaena PCC 7120.2.Then,the heterologous expression system of the gene cluster of MAAs in Nostoc flagelliforme was constructed using Escherichia coli BL21.Its synthetic substances were used through LC-MS for separation and identification of molecular weight and identify the role in sequence of MAAs gene clusters.High purity MAAs was obtainted by HPLC and the molecular structure was confirmed by 1H NMR.Ultimately,we get the synthesis route of MAAs:MysA and MysB participate in the synthesis of 4-deoxygadusol(4-DG),the precursor of MAAs.MysC1 might be responsible for attaching ornithine(or lysine)and 4-deoxygadusol at the C1 site to synthesize 4-deoxygadusolyl-ornithine(or 4-deoxygadusolyl-lysine).MysC2 connected 4-DG to 4-deoxygadusolyl-ornithine dehydrates and condenses at the ornithine side chain to form mycosporine-4-deoxygadusolyl-omithine.Finally,under the MysD acted,4-deoxygadusolyl-ornithine was added to the carbonyl of the ring to form the final product M-2DO.3.The mysA-mysB-mysCI-PET28a Escherichia coli BL21 was treated with ornithine at different concentrations.It revealed that lysine involved in the synthesis of MAAs was gradually replaced by ornithine with the increase of ornithine concentration through high-performance liquid chromatographic analysis.It indicate that there is a lack of enough ornithine in Escherichia coli BL21.The detection of the expression product in Anabaena sp.PCC 7120 didn't reveal the involvement of lysine,suggesting that there is no dihydrolase that can turn arginine to ornithine and ammonia in Escherichia coli BL21.