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Summary  Co‐production of two or more desirable compounds from low‐cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co‐production strategy are still scarce. In this study, the ability of genome‐edited strain  Pseudomonas putida  EM42 to simultaneously valorize  d ‐xylose and  d ‐cellobiose – two important lignocellulosic carbohydrates – by converting them into the platform chemical  d ‐xylonate and medium‐chain‐length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with  P. putida  resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with a high yield. Xylose oxidation was subsequently coupled to the growth of  P. putida  with cytoplasmic β‐glucosidase BglC from  Thermobifida fusca  on  d ‐cellobiose. This disaccharide turned out to be a better co‐substrate for xylose‐to‐xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in  P. putida . Co‐production of extracellular xylose‐born xylonate and intracellular cellobiose‐born medium‐chain‐length polyhydroxyalkanoates was established in proof‐of‐concept experiments with  P. putida  grown on the disaccharide. This study highlights the potential of  P. putida  EM42 as a microbial platform for the production of xylonate, identifies cellobiose as a new substrate for mcl‐PHA production, and proposes a fresh strategy for the simultaneous valorization of xylose and cellobiose.

microbial biotechnology

Biotransformation of  d ‐xylose to  d ‐xylonate coupled to medium‐chain‐length polyhydroxyalkanoate production in cellobiose‐grown  Pseudomonas putida  EM42