Mechanism Research On The Metabolism Of Aromatic Amino Acids By Proteus Mirabilis And The Preliminary Application

L-aromatic amino acid metabolites derived from intestinal microbiota can link various factors with host pathophysiology and can be involved in pharmacologically interfered with when accumulated to a certain dose.Proteus mirabilis is opportunistic human pathogen in intestine,and its abundance increases with the occurrence of intestinal inflammation.In this study,P.mirabilis JN 458 was used as the original strain.L-aromatic amino acid metabolic pathways were confirmed,and the relevant genes involved in L-aromatic amino acid metabolism were analyzed in the genome of P.mirabilis.The related enzymes involved in the metabolism was verified by biochemical analysis.The regulation of genes related to L-aromatic amino acid metabolism was analyzed by RT-q PCR.A novel pathway for 2-phenylethanol synthesis was constructed using Escherichia coli.The main results were listed as the following points.（1）The metabolic pathways and potential genes of aromatic amino acids have been confirmed.17 L-aromatic amino acid metabolites in P.mirabilis JN 458 were determined by metabolite profiling.Based on the above metabolite profiling,the metabolic pathways have been confirmed.25 potential genes involved in L-aromatic amino acid metabolism in the genome of P.mirabilis were obtained.Two L-aromatic amino acid transaminase genes,eightα-hydroxy acid dehydrogenase genes,three aldehyde dehydrogenase genes,and threeω-transaminase genes were obtained.Besides,two L-amino acid deaminases,five alcohol dehydrogenases,one L-aromatic amino acid decarboxylase,and oneα-keto acid decarboxylase in P.mirabilis might be also involved in L-aromatic amino acid metabolism.（2）The functions of enzymes related to aromatic amino acid metabolism were verified.Two L-aromatic amino acid transaminases could catalyze L-aromatic amino acid into the corresponding aryl pyruvate.Besides,these enzymes could catalyze 2-phenylethylamine to phenylacetaldehyde,as well.L-aromatic amino acid transaminase（AAAT-1）usedα-ketoglutarate as a receptor of the amino group showed high catalytic activity for L-tyrosine and L-tryptophan,with k_cat/K_m of 6.61 s^-1·mmol^-1 and 2.79 s^-1·mmol^-1,respectively.Fiveα-hydroxy acid dehydrogenases（MDH,ALLD,GHRA,GHRB,and PHDGH）could catalyze aryl pyruvate to aryl lactate.MDH had a high catalytic activity for phenylpyruvate,and k_cat/K_m was 1.31 s^-1·mmol^-1.Three aldehyde dehydrogenases（SADH,ALDH,and BADH）could catalyze aryl acetaldehyde to aryl acetate.When NADP⁺was used as a coenzyme,ALDH showed high catalytic activity for phenylacetaldehyde,k_cat/K_m was 2.3 s^-1·mmol^-1.（3）The expression levels of genes related to aromatic amino acid metabolism were analyzed by RT-q PCR.11 related genes involved in 2-phenylethanol synthesis via three pathways were analyzed by RT-q PCR.As a consequence,11 related genes were significantly upregulated indicating that L-phenylalanine was transformed into 2-phenylethanol via the L-amino acid deaminase pathway,the Ehrlich pathway,and the L-aromatic amino acid decarboxylase pathway under aerobic conditions.9 related genes were significantly upregulated indicating that L-phenylalanine was transformed into 2-phenylethanol via the Ehrlich pathway under anaerobic conditions.In vitro biochemical analysis experiments,three other L-aromatic amino acid metabolic pathways might exsit in P.mirabilis JN 458.22 related genes involved in aromatic amino acid metabolism were analyzed by RT-q PCR.The results showed that L-aromatic amino acids were metabolized to aryl acetate,aryl lactate,aryl ethylamine,and aryl alcohol upregulated by 19genes under aerobic conditions;L-aromatic amino acids were metabolized to aryl acetate,aryl lactate,aryl ethylamine,and aryl alcohol upregulated by 18 genes.（4）A novel synthetic pathway of 2-phenylethanol was constructed in E.coli.The recombinant strain E-p AEAKa was constructed using plasmid p ACYCDuet-1 to express L-amino acid deaminase andα-keto acid decarboxylase,and p ETDuet-1 to express alcohol dehydrogenase（ADH-1）.The recombinant strain E-p AEAKa could transformed L-phenylalanine into 2-phenylethanol.The rate-limiting enzyme L-amino acid deaminase was modified based on the model of Pm LAAD constructed by homologous modeling.The mutant Pm LAAD V438G/K147V/K151E was constructed by three-point mutations.The relative enzyme activity of mutant Pm LAAD V438G/K147V/K151E is 3.53 times that of wild type Pm LAAD.The E-p REm KAG was conrtructed using plasmid p RSFDuet-1 to express Pm LAAD V438G/K147V/K151E andα-keto acid decarboxylase,and p ETDuet-1 to express alcohol dehydrogenase（ADH-4）and glucose dehydrogenase.The E-p REm KAG transformed10 g·L^-1 L-phenylalanine into 7.28±0.19 g·L^-1 2-phenylethanol for 8 h by adding an OD₆₀₀of13 wet cells at a temperature of 40°C and p H of 7.The conversion rate reached 98.46%.When the aeration rate was 0.4 vvm and glucose supplemented with an addition of 20 g·L^-1,E-p REm KAG whole cells transformed 10 g·L^-1 L-phenylalanine to 7.24±0.12 g·L^-1 2-phenylethanol,the conversion rate reached 97.92%.