Efficient Coutilization of Methanol with Pentose Sugars by Engineered <i>Corynebacterium glutamicum</i> 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 13 C-methanol revealed that 70.78% of PCA carried at least one 13 C and 0.09% of PCA carried six 13 C. 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.