Abstract
Astaxanthin, a natural red pigment with antioxidant and other physiological activities, is widely used in the feed, pharmaceutical, and cosmetic industries. Haematococcus pluvialis 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, Synechococcus 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 (crtZ) and ketolase (crtW) from Brevundimonas 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 tkt or ispG 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 Synechococcus sp. PCC 7002.