Strategic redesign of the central metabolism in Pseudomonas putida boosts NADPH generation and product formation

Abstract The biotechnological synthesis of chemicals presents a greener and more sustainable alternative to traditional petrochemical processes. However, achieving titers, rates, and yields (TRY) allowing for economic competitiveness remains a significant challenge. A promising strategy to address this bottleneck is the increase of intracellular NADPH levels, a crucial reducing cofactor essential for the biosynthesis of numerous compounds. Here, we have engineered Pseudomonas putida with enhanced NADPH levels by redirecting flux towards the Pentose Phosphate pathway, introducing a phosphoketolase shunt and overexpressing key NADPH-generating enzymes. This strategy resulted in strains with enhanced NADPH pools, increasing TRY levels of the NADPH-dependent biosynthesis of furan dicarboxylic acid (FDCA) and violacein. Furthermore, we address the growth deficiencies typically associated with cofactor level alterations by integrating gntZ, rpe, and xfpk into the genome of P. putida ΔgcdΔeddΔhexR. This genomic integration, coupled to optimal ribosome binding site (RBS) sequences selected from a library, ensures stable gene expression and optimal growth rates without the need for plasmid maintenance. Lastly, we assessed the FDCA production using 1-L bioreactors, demonstrating the superior capabilities of the engineered strains in batch and fed-batch conditions. This strategic redesign of the central metabolism has yielded an NADPH-enhanced P. putida strains with improved product formation and a robust defense against oxidative stress.