**Background:** Astaxanthin is a red carotenoid antioxidant used in pharmaceuticals, cosmetics, nutraceuticals, and aquaculture. It is valued for preventing oxidative stress-related diseases such as cataracts, cancers, Parkinson's, and Alzheimer's. Commercial production relies on chemical synthesis (yielding mixed stereoisomers) or microalgae cultivation (Haematococcus pluvialis), which is slow and requires stressful conditions. Heterologous production in Escherichia coli offers advantages of rapid growth and simple extraction, but yields are typically lower. Most E. coli studies use bacterial ketolases/hydroxylases (CrtW/CrtZ). This work is the first to evaluate the plant-derived enzymes CBFD1 and HBFD1 from Adonis aestivalis combined with an overexpressed isoprenoid pathway (IUP) for astaxanthin production in E. coli.
**Methods:** E. coli MG1655(DE3) strains were engineered with three upstream isoprenoid pathways: (1) wild-type endogenous MEP, (2) chromosomally integrated trcMEP (overexpressing dxs-idi-ispDF), and (3) the artificial isopentenol utilization pathway (IUP) via plasmid pSEVA228-pro4IUP (expressing CK, IPK, IDI). Downstream carotenoid genes included crtE/ggpps, crtB, crtI, crtY from Pantoea agglomerans, and cbfd1/hbfd1 from A. aestivalis. Three plasmid configurations were tested: (i) pAC-BETAipi + pCBFD1 (strains ASTA 1-3), (ii) single-operon p5T7-Astaipi (ASTA 4-6), and (iii) two-operon system p5T7-lycipi-ispA + pAC-ASTA (ASTA 7-9). Cultures were grown in LB or M9 minimal media at 30°C, induced with 0.1 mM IPTG (plus 25 mM isoprenol for IUP strains). Total carotenoids were quantified by spectrophotometry at 475 nm, and composition was analyzed by HPLC with a C30 column.
**Key Results:** In LB media, the trcMEP strain (ASTA 2) achieved the highest total carotenoid titre of 6.05 ± 0.95 mg/L at 36 h, a 2.5-fold increase over wild-type. The IUP strain (ASTA 3) reached similar carotenoid content (2.87 ± 0.67 mg/g) but lower cell density. In M9 minimal media, the IUP strain (ASTA 3) dramatically outperformed others, producing 11.3 ± 0.55 mg/L total carotenoids—a 13-fold increase over wild-type and 11-fold over trcMEP in M9. The IUP strain continued producing until 48 h, while others ceased by 12 h. When using the single-operon p5T7-Astaipi (ASTA 4-6), the IUP strain (ASTA 6) showed a 3.6-fold decrease in titre compared to ASTA 3. The two-operon system (ASTA 7-9) improved performance for trcMEP and wild-type strains (2.8-fold and 4.8-fold increases, respectively), while IUP strain ASTA 9 maintained similar titre (3.65 ± 0.39 mg/L at 24 h). HPLC analysis revealed that astaxanthin purity increased with operon optimization: ASTA 3 produced 34.6 ± 3.0% astaxanthin (with 40.7 ± 4.3% β-carotene), ASTA 6 produced 56.4 ± 0.8% astaxanthin, and ASTA 9 achieved 73.5 ± 0.2% astaxanthin with no detectable canthaxanthin. The specific yield for ASTA 9 was 2.66 mg/g (with 73.5% purity) and for ASTA 6 was 3.91 mg/g (with 34.6% purity).
**Clinical Implications:** This study provides a novel metabolic engineering platform for producing biologically derived astaxanthin with high purity in E. coli, using plant enzymes (CBFD1/HBFD1) for the first time in combination with the IUP pathway. The finding that IUP performs best in minimal media (M9) is advantageous for industrial scale-up due to lower cost and greater reproducibility. The achieved astaxanthin purity of 73.5% (strain ASTA 9) is competitive with bacterial CrtW/CrtZ systems, though lower than the highest reported (>95%). The accumulation of intermediates (β-carotene, isozeaxanthin) indicates bottlenecks in the xanthophyll pathway, suggesting that further optimization of enzyme ratios, promoter strength, or fusion proteins could improve conversion. For nutraceutical and aquaculture applications, this work offers a scalable microbial alternative to chemical synthesis and microalgae cultivation, potentially enabling cost-effective production of single-isomer astaxanthin for preventing oxidative stress-related diseases.