Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum.

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L-1 ) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38% of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Lignin Precipitation and Fractionation from OrganoCat Pulping to Obtain Lignin with Different Sizes and Chemical Composition.

Fractionation of lignocellulose into its three main components, lignin, hemicelluloses, and cellulose, is a common approach in modern biorefinery concepts. Whereas the valorization of hemicelluloses and cellulose sugars has been widely discussed in literature, lignin utilization is still challenging. Due to its high heterogeneity and complexity, as well as impurities from pulping, it is a challenging feedstock. However, being the most abundant source of renewable aromatics, it remains a promising resource. This work describes a fractionation procedure that aims at stepwise precipitating beech wood (Fagus sp.) lignin obtained with OrganoCat technology from a 2-methyltetrahydrofuran solution, using n-hexane and n-pentane as antisolvents. By consecutive antisolvent precipitation and filtration, lignin is fractionated and then characterized to elucidate the structure of the different fractions. This way, more defined and purified lignin fractions can be obtained. Narrowing down the complexity of lignin and separately valorizing the fractions might further increase the economic viability of biorefineries.