Fig. 1: Pseudomonas putida EM42 PD310 used as a template strain in this study.

Schematic illustration of (a) upper sugar metabolism of P. putida EM42 PD310 and (b) pSEVA2213_xylABE plasmid construct. The PD310 strain¹⁸ capable of growth on D-xylose and its co-utilization with D-glucose bears low-copy-number plasmid pSEVA2213 with constitutive P_EM7 promoter and a synthetic operon that encodes exogenous xylose isomerase pathway (XylA xylose isomerase and XylB xylulokinase) and xylose/H⁺ symporter XylE from Escherichia coli BL21(DE3). The xylA and xylE genes are preceded by synthetic ribosome binding sites (RBS), while xylB was left with its native RBS. Note that the elements in the plasmid scheme are not drawn to scale. The PD310 strain was also deprived of the gcd gene, which encodes periplasmic glucose dehydrogenase (PP_1444) to prevent the transformation of xylose to the dead-end product xylonate (XLN). The exogenous pathway converts xylose to xylulose 5-phosphate (X5P), which enters the EDEMP cycle formed by the reactions of the pentose phosphate pathway (brown arrows), the Embden-Meyerhof-Parnas pathway (orange arrows), and the Entner-Doudoroff pathway (blue arrows). Abbreviations: GLN, gluconate; G6P, glucose 6-phosphate; G3P, glyceraldehyde 3-phosphate; Km, kanamycin; 2KG, 2-ketogluconate; 6PG, 6-phosphogluconate; RK2, a broad-host-range origin of replication; T0, transcriptional terminator.