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Astaxanthin is a carotenoid of significant commercial value due to its superior antioxidant potential and wide applications in the aquaculture, food, cosmetic and pharmaceutical industries. A higher ratio of astaxanthin to the total carotenoids is required for efficient astaxanthin production. β-Carotene ketolase and hydroxylase play important roles in astaxanthin production. We first compared the conversion efficiency to astaxanthin in several β-carotene ketolases from Brevundimonas sp. SD212, Sphingomonas sp. DC18, Paracoccus sp. PC1, P. sp. N81106 and Chlamydomonas reinhardtii with the recombinant Escherichia coli cells that synthesize zeaxanthin due to the presence of the Pantoea ananatis crtEBIYZ. The B. sp. SD212 crtW and P. ananatis crtZ genes are the best combination for astaxanthin production. After balancing the activities of β-carotene ketolase and hydroxylase, an E. coli ASTA-1 that carries neither a plasmid nor an antibiotic marker was constructed to produce astaxanthin as the predominant carotenoid (96.6%) with a specific content of 7.4 ± 0.3 mg/g DCW without an addition of inducer.

Xylanases, which cleave the β-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.