Improving astaxanthin production in Escherichia coli by co-utilizing CrtZ enzymes with different substrate preference.

BACKGROUND: The bifunctional enzyme ß-carotene hydroxylase (CrtZ) catalyzes the hydroxylation of carotenoid ß-ionone rings at the 3, 3' position regardless of the presence of keto group at 4, 4' position, which is an important step in the synthesis of astaxanthin. The level and substrate preference of CrtZ may have great effect on the amount of astaxanthin and the accumulation of intermediates. RESULTS: In this study, the substrate preference of PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from Pantoea Agglomerans were certified and were combined utilization for increase astaxanthin production. Firstly, PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from P. Agglomerans were expressed in platform strains CAR032 (ß-carotene producing strain) and Can004 (canthaxanthin producing strain) separately to identify their substrate preference for carotenoids with keto groups at 4,4' position or not. The results showed that PCcrtZ led to a lower zeaxanthin yield in CAR032 compared to that of PAcrtZ. On the contrary, higher astaxanthin production was obtained in Can004 by PCcrtZ than that of PAcrtZ. This demonstrated that PCCrtZ has higher canthaxanthin to astaxanthin conversion ability than PACrtZ, while PACrtZ prefer using ß-carotene as substrate. Finally, Ast010, which has two copies of PAcrtZ and one copy of PCcrtZ produced 1.82 g/L of astaxanthin after 70 h of fed-batch fermentation. CONCLUSIONS: Combined utilization of crtZ genes, which have ß-carotene and canthaxanthin substrate preference respectively, can greatly enhance the production of astaxanthin and increase the ratio of astaxanthin among total carotenoids.

Coordinated Expression of Astaxanthin Biosynthesis Genes for Improved Astaxanthin Production in Escherichia coli.

Astaxanthin has great potential commercial value in the feed, cosmetics, and nutraceutical industries due to its strong antioxidant capacity. In this study, the Escherichia coli strain CAR026 with completely balanced metabolic flow was selected as the starting strain for the production of astaxanthin. The expression of ß-carotene ketolase (CrtW) and ß-carotene hydroxylase (CrtZ), which catalyze the conversion of ß-carotene to astaxanthin, was coordinated, and a bottleneck was eliminated by increasing the copy number of crtY in CAR026. The resulting strain Ast007 produced 21.36 mg/L and 4.6 mg/g DCW of astaxanthin in shake flasks. In addition, the molecular chaperone genes groES-groEL were regulated to further improve the astaxanthin yield. The best strain Gro-46 produced 26 mg/L astaxanthin with a yield of 6.17 mg/g DCW in shake flasks and 1.18 g/L astaxanthin after 60 h of fermentation under fed-batch conditions. To the best of our knowledge, this is the highest astaxanthin obtained using engineered E. coli to date.