Abstract
2,4 pyridine dicarboxylic acid (2,4 PDCA) is an analogue of terephthalate, and hence a target chemical in the field of bio-based plastics. Here, Pseudomonas putida KT2440 strains were engineered to efficiently drive the metabolism of lignin-derived monoaromatics towards 2,4 PDCA in a resting cells-based bioprocess that alleviates growth-coupled limitations and allows biocatalysts recycling. Native β-ketoadipate pathway was blocked by replacing protocatechuate 3,4-dioxygenase by the exogenous LigAB extradiol dioxygenase. Overexpression of pcaK encoding a transporter increased 8-fold 2,4 PDCA productivity from protocatechuate, reaching the highest value reported so far (0.58 g L-1h−1). Overexpression of the 4-hydroxybenzoate monooxygenase (pobA) speed up drastically the production of 2,4 PDCA from 4-hydroxybenzoate (0.056 g L-1h−1) or p-coumarate (0.012 g L-1h−1) achieving values 15-fold higher than those reported with Rhodococcus jostii biocatalysts. 2,4 PDCA was also bioproduced by using soda lignin as feedstock, paving the way for future polymeric lignin valorization approaches.
Highlights
Circular economy should aim for a more efficient fate of raw mate rials or feedstocks through recycling of the generated goods and through the reincorporation of industrial, domestic wastes and residuals into the production-consumption cycle
Re-routing the P. putida KT2440 β-ketoadipate pathway for 2,4 Pyridine dicarboxylic acids (PDCA) production in resting cells In P. putida KT2440, catabolism of a variety of lignin-derived monomers present in lignocelluloses mixtures, e.g., pcoumaric acid (pCA), coniferyl alcohol or ferulic acid (Linger et al, 2014; Salvachúa et al, 2015), is channeled to the central intermediate protocatechuic acid (PCA), which is further metabolized through the β-ketoadipate pathway to yield acetyl-CoA and fuel the Krebs cycle (Jimenez et al, 2002)
To re-route PCA towards 2,4 PDCA, a derivative P. putida KT2440 strain was generated by replacing the native ortho-cleavage 3,4 protocatechuate dioxygenase activity by a heterologous meta-cleavage 4,5 protocatechuate dioxygenase coded by the ligAB genes from Sphingobium sp
Summary
Circular economy should aim for a more efficient fate of raw mate rials or feedstocks through recycling of the generated goods and through the reincorporation of industrial, domestic wastes and residuals into the production-consumption cycle. While the carbohydrate part (cel lulose and hemicellulose) is used for paper, nylon or biofuel production, among other uses, the aromatic component (lignin) is extracted in amounts higher than 50 Mt per year worldwide (Bajwa et al, 2019) and is mostly considered a residue Part of it is used for macromolecular applications (production of glues, adsorbents or raw material for carbon nanofibers) (Chung and Washburn, 2013; Esposito and Antonietti, 2015; Liu et al, 2015; Norberg et al, 2013; Sen et al, 2015; Upton and Kasko, 2016) and most of it is combusted for heat and energy production within factory facilities (Liu et al, 2015). As demonstrated in several publications, lignin streams can be revalorized through green biotech nology using genetically modified bacteria for the production of com pounds of undisputable commercial necessity and interest such as muconic acid (Chen et al, 2021; Salvachúa et al, 2018), adipic acid (Vardon et al, 2015), pyridine dicarboxylic acids (Johnson et al, 2019; Spence et al, 2021) or vanillin (Sainsbury et al, 2013), among others
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