Chemical-Enzymatic Synthesis Of 2-amino-1-(4-nitrophenyl)-1,3-propanediol

2-amino-1-（4-nitrophenyl）-1,3-propanediol,commonly known as chlormycoramine（ANP）,belongs to the group of amino alcohols with four optical isomers,of which（1R,2R）-ANP is the key active precursor for the production of chloramphenicol.Since ANP has two active centers and the O atoms and N atoms in the structure have good coordination ability,it has wide application value.In this study,a new pathway for the synthesis of（1R）-ANP using p-nitro-α-acetamino-β-hydroxyphenylacetone（p-NAH）as a substrate was constructed by combining chemical hydrolysis and biocatalysis.The details of the study are as follows:1.Preparation of AHNA and screening of carbonyl reductase.2-Amino-3-hydroxy-1-（4-nitrophenyl）-1-propanone（AHNA）was generated by chemical hydrolysis of p-NAH in 82%yield.Using AHNA as a substrate,159 carbonyl reductases extant in the laboratory were screened to obtain an enzyme,Lvchun,capable of catalyzing the generation of（1R）-ANP from AHNA.sequencing and multiple sequence alignment analysis revealed that Lvchun is a short-chain dehydrogenase derived from Novosphingobium aromaticivorans（GenBank:WP₀11906790.1）.2.Molecular modification of the short-chain dehydrogenase Lvchun.Sequence alignment analysis revealed that the Ser110 site of Lvchun is a potential site related to the stereoselectivity of the enzyme,and sentinel saturation mutations were performed on this site.The d.e.values of mut-S110E and mut-S110F in the obtained mutants were greater than 99%,but their activities were only 6.64%and 10.18%of the wild type,respectively;while the mut-S110L activity was 1.74 times higher than that of the wild type with a d.e.value of-5.07%.By homology modeling,molecular docking combined with alanine scanning,six amino acid sites,Gly108,Val112,Phe214,Lys222,Leu216 and His243 were selected for targeted saturation mutagenesis,and the mutant mut-V112Y was obtained with 3.47 times more activity than the wild type with a d.e.value of 4.10%.The d.e.value of mut-L216Q was greater than 99%,but the activity was only 27.94%of the wild type.Beneficial stacking mutations failed to obtain mutants with both increased enzyme activity and stereoselectivity.The main reason for the increased activity is that the amino acid substitution at the site shortens the distance between the substrate AHNA and C4 on the nicotinamide ring of NADH and the critical catalytic residues in the substrate binding pocket.3.The enzymatic properties of mut-V112Y and CbFDH were studied.The results showed that the optimum reaction temperature of mut-V112Y was 30℃,and it could still have more than 85%activity at this temperature for 24 h.The optimum reaction pH was 7.5,and it was more stable in a neutral-base environment.The optimum temperature of CbFDH was 40℃,and it could still maintain 80%residual activity for 24 h below 42℃;it was more stable in a neutral-base environment,and the optimum reaction pH was 8.0.4.Establishment and optimization of dual enzyme reaction system.In order to reduce the reaction cost,the co-expression and fusion expression strains of mut-V112Y and cbfdh genes were constructed,and the heterologous co-expression and fusion expression of mut-V112Y and CbFDH were realized in E.coli.It was verified that the catalytic efficiency of the dual enzyme co-expression catalytic system was higher than that of the dual enzyme fusion expression and dual bacteriophage coupled reaction system.It was also found that high concentrations of substrate had an inhibitory effect on enzyme activity.Using the dual enzyme co-expression recombinant strain as a whole-cell biocatalyst,the optimum pH of the reaction was 7.5 at 50 mM substrate concentration,the optimum temperature was 30℃,the optimum concentration of lyophilized bacterial powder was 25 g/L,the optimum concentration of coenzyme NAD+was 0.75 mM,and the optimum concentration of co-substrate sodium formate was 65 mM.Under the optimum reaction conditions,the product concentration reached 14.56 in 30 min,and the yield was 29.12%.In summary,this paper successfully constructed a biosynthetic pathway for the synthesis of（1R）-ANP using p-NAH as substrate by combining chemical hydrolysis with enzyme catalysis,and further improved the catalytic efficiency and reduced the production cost by combining molecular modification of enzyme,construction of recombinant strain and optimization of reaction conditions,which provides a reference for the industrial production of（1R）-ANP.