Evolving Nonphosphorylative Metabolism for Improving Production of 2-Oxoglutarate Derivatives

The bioconversion of lignocellulosic biomass into value-added products provides an alternative solution to environmental and economic challenges. Nonphosphorylative metabolism can convert pentoses and d-galacturonate into 2-oxoglutarate (2-KG) in a few steps, facilitating the production of 2-KG derivatives. However, the efficiency of the Weimberg pathway from Caulobacter crescentus, a type of nonphosphorylative metabolism, is constrained by the low activity of CcXylX, 2-keto-3-deoxy-d-xylonate dehydratase. To overcome this limitation, we engineered CcXylX through directed evolution. A resulting CcXylX mutant exhibited a 3-fold higher kcat value and notably enhanced the production of 2-KG derivatives from d-xylose, a major component of lignocellulosic hydrolysates, including a 32% increase in l-glutamate titer (8.3 g/L) and a 79% increase in l-proline titer (4.3 g/L) compared with the wild-type CcXylX. This research holds promise for advancing lignocellulosic biotechnology and provides insights into economically viable production of other 2-KG derivatives besides l-glutamate and l-proline.