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

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.