Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by l-amino acid deaminase

Abstract

L-Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from L-amino acids. In a previous study, a whole cell biocatalyst with the L-amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from L-glutamic acid, and the α-KG titer reached 12.79 g L⁻¹ in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L⁻¹ when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L⁻¹ when monosodium glutamate was continuously fed at a constant rate of 6 g L⁻¹ h⁻¹ (from 4 to 23 h) with an initial concentration of 50 g L⁻¹. Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min⁻¹ to 241.8 μM min⁻¹) and substrate affinity (the K_m for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.