Comparative analysis of precursor supply and pathway reinforcement strategies for shikimic acid biosynthesis in Pseudomonas putida

Abstract The shikimate pathway is a central metabolic route for the biosynthesis of high-value aromatic compounds. Traditional metabolic engineering strategies have largely focused on overexpressing pathway-specific genes, enhancing precursor supply, or introducing feedback-resistant variants of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase to increase pathway flux. However, how these distinct strategies differentially affect flux distribution and the accumulation of key intermediates, specifically 3-dehydroshikimate (DHS) and shikimic acid (SA), remains largely unexplored in Pseudomonas putida ( P. putida ). Here, we systematically assess the effects of commonly applied engineering strategies on DHS and SA production using distinct SA pathway modules. The results show that overexpression of transketolase I ( tktA ), phosphoenolpyruvate synthase ( ppsA ), and SA pathway genes ( aroE , aroB , aroQ-II ), together with heterologous expression of feedback-resistant aroG , in a genome-edited P. putida EM42 strain harboring pykA / pykF deletions and a hexR knockout, yielded approximately 71 mg L⁻ 1 shikimic acid, corresponding to a > 284-fold increase relative to the wild-type EM42. Notably, the findings reveal that following the relief of precursor constraints, the achievable maximum titer is unaffected by the particular DAHP synthase isoform utilized. Furthermore, the standalone overexpression of ppsA and tktA yielded SA and DHS levels comparable to those achieved with the additional expression of the aforementioned pathway genes. However, despite this phenotypic convergence in titers, comparative transcriptomic analysis revealed that the genetic route taken to reach these levels is associated with markedly distinct transcriptional configurations of the host metabolic network. In the same genetic background, amplification of ppsA - tktA alone is associated with an increased transcriptional investment in the biosynthesis of essential cofactors and metabolic precursors, whereas overexpression of the SA pathway is accompanied by enrichment of protocatechuate catabolic genes, consistent with the intrinsic aromatic funneling capacity of P. putida under conditions of elevated shikimate pathway flux. These results demonstrate that evaluating engineering success solely based on final product yields obscures substantial differences in underlying metabolic states. Accordingly, we propose that tailored engineering strategies guided by key pathway intermediates are critical for the rational optimization of P. putida as an efficient cell factory.