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 et technologies de l'environnement, à finalité spécialisée

Abstract

Given the depletion of non-renewable resources and growing environmental concerns regarding the petrochemical industry, there is an increasing need to discover sustainable bio-sourced alternatives. Lignin stands out as a particularly attractive opportunity for the production of aromatics and the synthesis of biobased products and chemicals. However, its extraction and fractionation from lignocellulosic biomass present challenges due to the degradation of lignin quality with current industrial processes. Emerging pretreatment techniques offer new opportunities for lignin valorization, particularly with subsequent biochemical processes using naturally occurring microorganisms and enzymes to convert lignin monomers into high-value-added products. This master’s thesis aimed first to optimize a minimal medium for Pseudomonas putida KT2440, a bacterium known for its ability to degrade aromatics and its suitability as a host for heterogeneous gene expression. Later, an improved streamlined strain P. putida SEM10 was utilized as a host for genetic manipulation. The second objective of this work was to quantify the bacterium's growth and assess its ability to thrive on aromatic compounds derived from lignin extracts. To achieve this, reductive catalytic fractionation (RCF) was considered for lignin extraction and depolymerization from vine shoots. Analysis of the lignin oil extracted via RCF enabled identification of its monomeric composition, crucial for subsequent bioconversions. Following this analysis, several candidate genes were identified from the literature, for integration into the genome, aiming to facilitate the metabolic funneling of these lignin-derived monomers into valuable metabolites. Genome sequencing of two aromatic-degrading strains was also conducted to identify other potential candidate genes. A protocol for genetic engineering was tested, initially validated by in silico gene insertion for aromatic funneling. Then, in vivo gene knockout was performed aiming at the accumulation of protocatechuate (PCA) from lignin monomers. PCA is an intermediate metabolite of interest since it is a valuable precursor for producing high-value phenolic compounds used in various industrial applications. This metabolic modification yielded promising results and underscores the potential of genetic engineering approaches to enhance lignin valorization processes, paving the way for future advancements in sustainable biobased industries.