Evolutionary Rewiring of Sugar Transporter Gene manXYZ Enables Highly Coordinated Non-glucose Metabolisms in...

Achieving highly coordinated metabolism and efficient conversion of lignocellulose-derived non-glucose sugars remains a major challenge for biorefinery microbial cell factories. Herein, we report an evolutionary rewiring of an engineered lactic acid bacterium Pediococcus acidilactici G1 with enhanced co-utilization of glucose, xylose, arabinose, mannose, and galactose for l-lactic acid generation. Transcriptomic analysis revealed the extraordinary upregulation of a unique sugar transporter gene cluster, manXYZ, while genome re-sequencing identified two convergent mutations in the transcriptional regulators CcpA (T65I) and LevR (E159G). Back mutation, reverse metabolic engineering, and electrophoretic mobility shift assays (EMSA) were then employed to investigate the regulation of manXYZ expression by CcpA T65I and LevR E159G. Collectively, our findings demonstrate that recurrent mutations in transcriptional regulators CcpA and LevR drove the significant upregulation of the manXYZ sugar transporter cluster during adaptive evolution, highlighting the importance of sugar transport in improving non-glucose sugar metabolism. This study not only develops a promising strain for practical biorefinery applications but also provides a new sugar transporter solution, rather than regular intracellular metabolic pathway engineering, for highly efficient non-glucose sugars utilization in P. acidilactici.