Distribution Of Biosynthetic Pathway And Metabolic Regulation Of 5-Aminolevulinic Acid In Anoxygenic Phototrophic Bacteria

5-aminolevulinic acid?ALA?,a non-proteinogenic amino acid,is the precursor for the synthesis of heme,?bacteria?chlorophyll and VB₁₂,and widely applied in agriculture and medical fields.ALA biosynthetic pathway includes C4 and C5pathway.Anoxygenic phototrophic bacteria?APB?is a type of metabolic diversity group that relies on bacterial chlorophyll?BChl?as a core component for photosynthesis.ALA is an essential precursor for BChl synthesis.Therefore,ALA production by APB is also receiving attention and favor.However,the understanding of the distribution of C4 and C5 pathways in APB is limited and imperfect,the experimental evidence is lacking,and there are certain divergencies.In response to these problems,this study systematically analyzed the distribution characteristics and laws of C4 and C5 pathways in APB at the genomic level through bioinformatics analysis.Further,the ALA synthesis pathways of?proteobacteria purple bacteria?M.gracile YL28?were verified by experiments.Transform the ALA key enzyme gene of C5 pathway and the magnesium chelatase gene?bchHID?and the magnesium protoporphyrin methyltransferase gene?bchM?of the bacterialchlorophyll synthesis pathway derived from the APB into E.coli BL21 and Rhodopseudomonas palustris CGA009 successively to explore the regulation mechanism of ALA.The main results are as follows:?1?Genome analysis and validation of ALA synthesis pathway in APB.The genomic data of 117 strains APB were obtained,and the distribution characteristics of ALA synthase genes?gtr,gsa and hemA?in the genome were systematically analyzed.A new key enzyme gene of the C4 pathway was discovered,and a new understanding that the C4 and C5 pathways coexist in the green bacteria,?-and?-proteobacteria purple bacteria was proposed.Further,the similar sequences of GTR,GSA and ALAS of M.gracile YL28 were determination after cloning expression and nickel column purification method,verifying the function of the new ALA synthase gene,indicating that the two pathways?C4 and C5?coexist in the YL28 strain.?2?Regulation of C5 pathway gene on ALA synthesis.Four recombinant E.coli strains were obtained by cloning the C5 pathway gene derived from M.gracile YL28.The addition of glutamic acid or glucose to the culture system?LB?up-regulated the accumulation of ALA.Compared with the other three single-gene expression strains,the accumulation of ALA in the gtr-gsa co-expressing strain was significantly high.The key enzyme genes of C5 pathway derived from YL28 was introduced into R.palustris CGA009 to obtain 3 recombinant strains,then we found it promote the accumulation of ALA in recombinant R.palustris.?3?Regulation of BChl synthesis pathway gene on ALA accumulation.The magnesium chelate synthase gene?bchHID??6590 bp?and the protoporphyrin methyltransferase gene?bchM?derived from YL28 BChl synthesis pathway were cloned into E.coli to obtain two recombinant strains,and found the accumulation of ALA in them were decreased.It may be due to its enzymatic reaction product promoting the catabolism of ALA,and the BChl synthesis intermediate magnesium protoporphyrin monomethyl ester?MPE?was detected in the bchHID-bchM co-expression strain.In summary,this study proposed a new understanding for the distribution of ALA synthesis pathway in purple bacteria and green bacteria.It was first proposed that C4and C5 ALA synthesis pathway coexist in green bacteria,?-and?-proteobacteria purple bacteria,and found a new ALA synthetic gene for the C4 pathway.Preliminary studies on metabolic regulation of ALA lay a foundation for further study on it.