Metabolic engineering of Acinetobacter baylyi ADP1 for efficient utilization of pentose sugars and production of glutamic acid.

Efficient utilization of pentose sugars is critical for advancing sustainable biomanufacturing using lignocellulose. However, many host strains capable of consuming glucose and lignin-derived monomers are unable to utilize pentose sugars. Here, we engineered Acinetobacter baylyi ADP1 for the utilization of D-xylose and L-arabinose. We first modelled different pentose utilization pathways using flux balance analysis to choose the most optimal pathway. A marker-free approach combining transformation and selection facilitated the integration of the pentose catabolic gene clusters of the selected Weimberg pathway into the A. baylyi genome, generating strains capable of efficiently utilizing both D-xylose and L-arabinose as sole carbon sources without any additional engineering or adaptation. For D-xylose, the cells achieved the highest growth rate (μ=0.73 h^-1) reported to date for non-native hosts engineered for pentose utilization. For L-arabinose, a growth rate of μ=0.40 h^-1 was achieved, which also surpassed the growth rate on a native substrate of A. baylyi, glucose (μ=0.37 h^-1). Importantly, pentose utilization occurred simultaneously with glucose utilization. We then applied metabolic flux analysis using ¹³C labeled xylose to reveal D-xylose metabolism in the engineered strain. To demonstrate the potential for bioproduction, L-glutamate was selected as a target compound. Deletion of sucAB and gabT, and the overexpression of gdhA enabled L-glutamate production. With the engineered strain, a carbon yield of 34% during co-utilization with succinate and 70% via whole-cell catalysis using resting cells was achieved. Notably, L-glutamate production directly from industrially relevant hemicellulose hydrolysate was demonstrated. This study establishes a robust platform for pentose utilization and bioproduction in A. baylyi ADP1 and highlights the potential for metabolic optimization.