Evolving Nonphosphorylative\nMetabolism for Improving\nProduction of 2‑Oxoglutarate Derivatives

The bioconversion of lignocellulosic biomass into value-added\nproducts\nprovides an alternative solution to environmental and economic challenges.\nNonphosphorylative metabolism can convert pentoses and d-galacturonate\ninto 2-oxoglutarate (2-KG) in a few steps, facilitating the production\nof 2-KG derivatives. However, the efficiency of the Weimberg pathway\nfrom Caulobacter crescentus, a type\nof nonphosphorylative metabolism, is constrained by the low activity\nof CcXylX, 2-keto-3-deoxy-d-xylonate dehydratase. To overcome\nthis limitation, we engineered CcXylX through directed evolution.\nA resulting CcXylX mutant exhibited a 3-fold higher <i>k</i><sub>cat</sub> value and notably enhanced the production of 2-KG\nderivatives from d-xylose, a major component of lignocellulosic\nhydrolysates, including a 32% increase in l-glutamate titer\n(8.3 g/L) and a 79% increase in l-proline titer (4.3 g/L)\ncompared with the wild-type CcXylX. This research holds promise for\nadvancing lignocellulosic biotechnology and provides insights into\neconomically viable production of other 2-KG derivatives besides l-glutamate and l-proline.