Astaxanthin Overproduction Enhanced by Metabolomics-Guided Rational Metabolic Engineering in <i>Synechococcus</i> sp. PCC 7002

Astaxanthin, a natural red pigment with antioxidant and other physiological activities, is widely used in the feed, pharmaceutical, and cosmetic industries. <i>Haematococcus pluvialis</i> is a well-known microbial producer of astaxanthin; however, its slow growth and requirement for a complex two-stage cultivation under high-light conditions limit large-scale application due to increased contamination risk. As an alternative, <i>Synechococcus</i> sp. PCC 7002 offers rapid growth and robustness, but metabolic engineering strategies to enhance astaxanthin production in this strain remain underexplored. In this study, we applied a metabolomics-guided approach to identify novel metabolic bottlenecks and engineering targets. A base strain expressing β-carotene hydroxylase (<i>crtZ</i>) and ketolase (<i>crtW</i>) from <i>Brevundimonas</i> sp. SD212 produced 6.2 mg/g of DCW astaxanthin. Metabolome analysis revealed the accumulation of sedoheptulose-7-phosphate (S7P) and 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP), suggesting that the reactions catalyzed by transketolase (TKT) and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate synthase (IspG) are rate-limiting. Overexpression of <i>tkt</i> or <i>ispG</i> reduced the levels of their respective substrates, confirming relief of these bottlenecks. Notably, the TKT-overexpressing strain achieved an astaxanthin content of 10.3 mg/g-DCW, while the IspG-overexpressing strain showed no significant improvement. Further optimization of culture conditions─such as medium composition, light intensity, and temperature─led to an astaxanthin productivity of 7.5 mg/L/day. These results demonstrate the effectiveness of a metabolomics-driven design-build-test-learn (DBTL) approach for enhancing astaxanthin production in <i>Synechococcus</i> sp. PCC 7002.