Efficient Coutilization of Methanol with Pentose Sugars by Engineered Corynebacterium glutamicum for Protocatechuic Acid Production.

Protocatechuic acid (PCA) is a valuable phenolic compound, and its rising demand has facilitated sustainable PCA bioproduction. Compared to glucose, low-cost methanol and pentose sugars like xylose do not have food-competing uses. Thus, we decided to engineer Corynebacterium glutamicum to coutilize them for PCA production. First, we introduced the PCA biosynthesis pathway into a methanol-essential strain and deleted its degradation pathway to achieve PCA production from methanol. Subsequently, we repaired the rpe deletion and introduced evolved xylAB (gX), achieving the highest PCA yield of 0.449 mol/mol xylose. Additionally, isotope labeling with 13C-methanol revealed that 70.78% of PCA carried at least one 13C and 0.09% of PCA carried six 13C. Furthermore, the xylose-containing hydrolysate from orange peels supported PCA production to a high titer of 10.61 g/L. Overall, we demonstrate the first PCA production by a methanol-essential strain and the highest yield, providing options for more sustainable bioproduction from food-waste feedstocks.