The hydantoinase platform
Figure 1 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
|
The hydantoinase process was introduced in the 1970s for producing D-amino acids such as D-phenylglycine and p-OH-phenylglycine (7). Today, > 1000 tons of each of these amino acids are produced annually. They are used as side chains
for the -lactame antibiotics ampicillin and amoxicillin.
Figure 2 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
|
The D-hydantoinase process (see Figure 1) is an excellent example of a dynamic kinetic resolution process. As 5'-monosubstituted
hydantoins racemize spontaneously or enzyme-catalyzed under conditions used for biotransformation, a 100% yield of optically
pure D- or L-amino acid can be reached (8, 9). Another advantage of the hydantoinase route is that racemic 5'-monosubstituted
hydantoins can be easily synthesized from cheap starting materials through the reactions shown in Figure 2. In addition, if
the decarbamoylation step is done enzymatically, carbamoylases waste and by-product formation is extremely low (CO2 and NH4 are the only by-products), which is also advantageous in the product-isolation step. All these features make the hydantoinase
route very attractive for the industrial production of optically pure artificial amino acids (10). To date, low space-time
yields and high biocatalyst costs prevent the production of L-amino acids based on the hydantoinase process (11–13).
Therefore, Evonik expanded its hydantoinase platform for producing L-amino acids by focusing on strain development and process
optimization by biochemical engineering (14, 15). Despite significant progress in reducing the biocatalyst production cost,
increasing the activity of the biocatalyst and improving the space-time-yield process economics were still prohibitive for
commercialization of this process.
To address these problems, Evonik developed a new generation of an L-hydantoinase process based on a tailor-made recombinant
whole-cell biocatalyst. The biocatalyst costs have been reduced by designing recombinant Escherichia coli cells overexpressing a hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771. Despite this progress, the D-selectivity of the hydantoinase for D,L-methylthioethylhydantoin was significantly
limiting the space-time yield of the L-hydantoinase process (16, 17). As screening did not provide us with better hydantoinases,
the authors intended to invert the enantioselectivity of the hydantoinase by directed evolution (18). The productivity of
the process could be dramatically improved using the recombinant E. coli coexpressing the newly designed L-selective hydantoinase mutant with an L-carbamoylase and a hydantoin racemase. These improvements
have been confirmed at industrial scale and resulted in a process for producing various natural and nonnatural L-amino acids.
|
