Engineering mixed sugar metabolic channels in Pseudomonas putida to produce vanillic acid.

Hydrolysis of lignocellulosic biomass produces the mixed sugars of glucose (60-70 %), xylose (20-30 %), and arabinose (2-20 %), etc. Using mixed sugars instead of pure glucose for microbial biosynthesis will reduce the cost of carbon source and maximize utilization of biomass. However, carbon catabolite repression and poor adaptation of metabolic pathways are obstacles in the synergistic utilization of mixed sugars, thus resulting in low carbon utilization efficiency. Here, we engineered the mixed sugar metabolic channels in Pseudomonas putida to achieve their synergistic utilization for producing vanillic acid, a valuable aromatic compound with broad applications in the food, pharmaceuticals, cosmetics, and chemical industries. Expressing O-methyltransferase (OMT) and deleting vanillate-O-demethylase (vanAB) realized vanillic acid accumulation in P. putida from glucose. Introducing the xylose isomerase pathway enabled the strain to produce vanillic acid from xylose. Deleting glucose dehydrogenase (gcd) and transcriptional regulator (hexR), together expressing two critical pentose phosphate pathway enzymes (transketolase and transaldolase) effectively balanced glucose-xylose metabolic channels for vanillic acid production. Further assembling the arabinose oxidation pathway established the efficient metabolism of three sugars. The final engineered strain (VA12) produced 2.75 g/L vanillic acid in fed-batch fermentation with 20 g/L glucose, 10 g/L xylose and 10 g/L arabinose. This study effectively reduces carbon catabolite repression in the synergistic utilization of mixed sugars, and represents the first case utilizing glucose-xylose-arabinose for vanillic acid production, illustrating the capability of transforming lignocellulose hydrolyzate into valuable chemical products.