Efficient bioproduction of 3-(4-thiazolyl)-l-alanine, an unnatural amino acid of pharmaceutical importance, by an enzymatic relay process

As an l -histidine analog, 3-(4-thiazolyl)- l -alanine (TAla) has been attracting increasing biotechnological interest because it can serve as a key building block in the development of many novel therapeutic peptides such as rovatirelin. Although several chemical and enzymatic methods have been reported for TAla production, the use of toxic agents and limitation imposed by a maximum theoretical conversion rate of 50% need to be discreetly considered before large-scale production. Here an enzymatic relay system overcoming these potential drawbacks was developed for efficient TAla production. Five aminoacylases (ACYs) were screened to synthesize TAla from racemic N -acetyl-3-(4-thiazolyl)- dl -alanine via l -enantioselective kinetic resolution. Four ACYs displayed the target activity, and the enzyme DdACY1, which is a novel ACY from Dictyostelium discoideum , was found to be promising due to its high activity and solubility. Using the E. coli whole cells expressing DdACY1 as biocatalysts, the optimal temperature and pH were determined to be 60-65°C and 8.5, respectively. The TAla yield reached approximately 45% under the optimized conditions. By coupling the Deinococcus radiodurans N -acyl-amino acid racemase DrNAAAR with DdACY1 in the same system, the maximum theoretical conversion rate of the reaction was overstepped and a 69.3% yield was achieved. Optimizing the dosage of DrNAAAR and adding 1 mmol/L Zn 2+ or Mg 2+ further improved the catalytic efficiency. In the scaled-up reaction containing 70 mmol/L racemic substrate, 77.1% of the substrate was converted into TAla in 24 h. After product isolation and purification, 0.454 g solid powder of TAla with high chemical (95.2%) and optical (99.9% ee ) purities was obtained. This study established an overall process for TAla bioproduction by coupling a novel ACY and a previously reported NAAAR via dynamic kinetic resolution. 3-(4-Thiazolyl)- l -alanine (TAla) is an l -histidine analog that can serve as a key building block in the development of many novel therapeutic peptides. Herein, a complete process, including enzymatic relay reactions and product isolation/purification, was established for efficient production of TAla with high chemical and optical purities.