The Study On Cascade Biocatalytic Synthesis Of Phenylpropionic Acids

Phenylpropionic acids,such as phenylalanines,phenylpyruvic acids,phenyllactic acids,and phenylacrylic acids,are important chemicals which have been widely used in the synthesis of pharmaceuticals,cosmetics,and fine chemicals.Herein,we report an artificially designed amino-group-transformation biocatalytic process,which uses simple phenols,pyruvate,and ammonia to synthesize（S）-α-tyrosine derivatives,4-hydroxyphenylpyruvic acid derivatives,（R）-α-tyrosine derivatives,and（R）-β-tyrosine derivatives.The main results were described as follows:1.Designing an amino-group-transformation biocatalytic cascade.Based on synthesis route analysis of phenylpropionic acids,we designed an amino-group-transformation biocatalytic cascade,which is presented as introduction and transformation of amino group to synthesize phenylpropionic acids.This biocatalytic cascade has two parts:an amino-group-introduction module（AI）and three amino-group-transformation modules（AT）.AI can convert phenols,pyruvate,and ammonia to（S）-α-tyrosine derivatives by amino introduction.Phenols,pyruvate,and ammonia can be converted to 4-hydroxyphenylpyruvic acid derivatives by amino introduction and oxidative deamination,（R）-α-tyrosine derivatives by amino introduction and stereoinversion,and（R）-β-tyrosine derivatives by amino introduction and shift,via cascade AI with AT1,AT2,and AT3,respectively.2.Construction of amino-group-introduction and amino-group-transformation modules.The enzymes with better properties were screened by database mining and literature mining to construct basic modules.The variant M379V of the recombinant tyrosine phenol lyase from Citrobacter freundii was selected to construct AI.The L-amino acid deaminase from Proteus vulgaris was selected to construct AT1.The L-amino acid deaminase from Proteus vulgaris,variant CgDAPDH^BC621（R196M/T170I/H245N/Q151L/D155G）of D-amino acid dehydrogenase from Corynebacterium glutamicum,and glucose dehydrogenase from Bacillus megaterium were selected to construct AT2.The variant C107S of phenylalanine aminomutase from Taxus chinensis was selected to construct AT3.3.By assembling different modules to synthesize phenylpropionic acids.Phenols 1a-d was tranformed into（S）-α-tyrosine derivatives（S）-α-3a-d with E.coli 01 in AI by conversion optimization,with the conversion of 94-99%,and high ee（>98%）.Four recombinant E.coli strains E.coli 10-13,forα-keto acid synthesis,were constructed by assembling AI and AT1.The best strain E.coli 10 was used to transform 1a-d into the corresponding4-hydroxyphenylpyruvic acid derivatives 4a-d,and the conversion was 95-99%.By assembling AI and AT2,six recombinant E.coli strains E.coli 14-19,for（R）-α-amino acid synthesis,were constructed.The best strain E.coli 19 was used to transform 1a-d into the corresponding（R）-α-tyrosine derivatives（R）-α-3a-d,with the conversion of 91-96%,and high ee（>98%）.The variant TcPAM^C107S,L104A obtained by protein engineering showed higher enzyme activity to L-tyrosine derivatives in AT3.By assembling AI and AT3,four recombinant E.coli strains E.coli 20-23,for（R）-β-amino acid synthesis,were constructed.But the conversion of the best strain E.coli 23 was not ideal.A duplicate TcPAM^C107S,L104A107S,L104A gene was cloned into pRSF-TcPAM^C107S,L104A-CfTPL^M379V and transformed into E.coli BL21（DE3）to obtain a new strain E.coli 24.This strain was used to transform 1a-d into the corresponding（R）-β-amino acid（R）-β-5a-d,with the conversion of 68-86%,and high ee（>98%）.The synthesis of five products was scaled up to 100 mL and purified by chromatography,extraction,and crystallization.Then,the structure and molecular weight of the purified products were analyzed and identified by NMR and HRMS,which further proved the industrialization potential and accuracy of this cascade system.