Multi-enzyme cascade for improving β-hydroxy-α-amino acids production by engineering L-threonine transaldolase and combining acetaldehyde elimination system

Abstract L-threonine transaldolase (PsLTTA) could asymmetric synthesize β-hydroxy-α-amino acids (HAAs) with excellent stereoselectivity, while the poor yield limited its further application. Here we provided a combinatorial strategy to improve HAAs production, by directed evolution of PsLTTA towards enhanced activity and introducing an acetaldehyde elimination system to avoid acetaldehyde over-accumulation. A novel high throughput screening (HTS) method for evaluating PsLTTA activity was developed and applied for directed evolution of PsLTTA. Subsequently, we co-expressed alcohol dehydrogenase and formate dehydrogenase to construct an acetaldehyde elimination system to remove acetaldehyde inhibition. Moreover, the above positive strategies were integrated. As a result, the (2S,3R)-p-methylsulfonyl phenylserine yield reached 154.0 mM and with 94.6% de value, the highest productivity and stereoselectivity of (2S,3R)-HAAs reported by enzymatic synthesis so far. Taken together, our studies provided an efficient and green route for chiral synthesis of (2S,3R)-HAAs, which might contribute to the industrialization production of these useful building blocks.