Physiology of Pseudomonas putida and metabolic engineering of P. putida strain SEM10 for biological funneling of lignin-derived aromatics

Details

Supervisors

Stenuit, Benoît

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

enApproximately 40% of the chemical industry relies on aromatic compounds, most of which are currently synthesized from fossil-derived BTX (benzene, toluene, and xylene). Lignocellulosic biomass represents a sustainable alternative source of aromatics; however, while efficient valorization processes exist for the polysaccharide fraction, lignin remains largely underexploited due to its structural complexity and process-induced recalcitrance. This Master’s thesis investigated the biological funneling of lignin-derived monoaromatic compounds into value-added platform aromatics using a heterogeneous lignin oil obtained via reductive catalytic fractionation (RCF) of vine shoots. Dihydroferulic acid (DHCA), the second most abundant monoaromatic compound in this substrate, was identified as a key target for microbial conversion. More specifically, this study focused on the metabolic engineering of Pseudomonas putida SEM10 for the conversion of eugenol and DHCA into protocatechuic acid (PCA). The synthetic eugo8x gene, encoding a eugenol oxidase, was integrated into the SEM10 genome with the aim of enabling the catabolism of both substrates. The growth performance of the engineered strain SEM10::eugo8x was compared with that of P. putida SEM10 WT, KT2440, and SEM10.ΔpcaGH. When DHCA was supplied as the sole carbon source, SEM10::eugo8x, SEM10 WT, and strain KT2440 exhibited significant growth, suggesting the presence of a native, yet unidentified, DHCA catabolic pathway in P. putida. However, SEM10::eugo8x reached a lower final OD600 than SEM10 WT and KT2440, likely due to the increased metabolic burden associated with suicide plasmid integration. Prior to cultivation on lignin oil, methanol toxicity was assessed and showed that concentrations up to 3% (v/v) only moderately affected growth, mainly by increasing the lag phase and reducing the maximum specific growth rate. Cultivation on lignin oil resulted in growth inhibition and reduced biomass formation, which was attributed primarily to the toxicity of DHSA, along with other lignin-derived aromatic compounds. Interestingly, increasing the total aromatic concentration from 0.13 to 1.30 g L-1 led to a shorter lag phase and a higher specific growth rate, potentially due to enhanced DHCA availability promoting growth. An adaptive laboratory evolution experiment performed on SEM10 WT and SEM10::eugo8x did not result in strains capable of growing on eugenol as the sole carbon source. These findings suggest that the inserted eugo8x gene was either non-functional in this host strain or did not provide a selective advantage over the native DHCA catabolic pathway of SEM10. Moreover, mutations present in eugo8x compared to the wild-type eugo may have impaired its ability to degrade eugenol. Overall, this work demonstrates that Pseudomonas putida SEM10 is a robust and promising bacterial chassis for lignin-derived aromatic funneling, owing to its tolerance to stressful conditions and its intrinsic capacity to metabolize key lignin-derived compounds. At the same time, the results highlight the need to identify and evaluate additional enzymatic pathways to enable the efficient conversion of a broader range of lignin-derived aromatics.