Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids

Abstract

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.

FIG. 1.

Effect of pH on systems 1 (black) and 2 (white) to produce d-methionine from d,l-MTEH. The buffers used were 200 mM potassium phosphate buffer from pH 6.5 to 8 (circles), 100 mM borate-HCl buffer from pH 8 to 9 (inverted triangles), and 100 mM borate-NaOH buffer at pH 9.5 and 10.5 (right-side-up triangles). Reactions were performed in triplicate, and error bars represent the standard errors of the means.

FIG. 2.

Effect of temperature on systems 1 (•) and 2 (▴) to produce d-methionine from d,l-MTEH. Activity was measured at different temperatures by using 0.25 g of recombinant cells/ml in 100 mM borate-HCl buffer (pH 8) and 15 mM d,l-MTEH. Reactions were performed in triplicate, and error bars represent the standard errors of the means.

FIG. 3.

Comparison of reaction profiles of d-methionine (d-met) production from 15 mM d,l-MTEH without d-carbamoyl methionine (d-car-met) accumulation by using pSER42 (A) and pAMG3 (B) in E. coli strain BL21. Reactions and measurements were carried out in triplicate as described in Materials and Methods.

FIG. 4.

Initial reaction rates for the production of different optically pure d-amino acids from 5-monosubstituted hydantoins using systems 1 and 2. The reactions were performed as described in Materials and Methods at pH 8 and 55°C. TRP, d-tryptophan; TYR, d-tyrosine; pHPG, d-p-hydroxyphenyl glycine; VAL, d-valine; PG, d-phenylglycine; ABA, d-aminobutyric acid; PA, d-phenylalanine; NVA, d-norvaline; NLEU, d-norleucine; MET, d-methionine; LEU, d-leucine; d-aa, d-amino acid. Reactions were performed in triplicate, and error bars represent the standard errors of the means.

FIG. 5.

Profile of d-methionine (d-met) production from 300 mM d,l-MTEH without d-carbamoyl methionine (d-car-met) accumulation by using pSER42 in E. coli strain BL21 induced with 0.1 mM IPTG (A) and uninduced (B) in a large-scale reaction with an end volume of 300 ml. Reactions and measurements were carried out in triplicate as described in Materials and Methods. Error bars represent the standard errors of the means.