Improvement of a CrtO-type of β-carotene ketolase for canthaxanthin production in Methylomonas sp.

Two types of non-homologous β-carotene ketolases (CrtW and CrtO) were previously described. We report improvement of a CrtO-type of β-carotene ketolase for canthaxanthin production in a methylotrophic bacterium, Methylomonas sp. 16a, which could use the C1 substrate (methane or methanol) as sole carbon and energy source. The crtO gene from Rhodococcus erythropolis was improved for canthaxanthin production in an E. coli strain engineered to produce high titer carotenoids by error-prone PCR mutagenesis followed by in vitro recombination. The best mutants from protein engineering could produce ∼90% of total carotenoids as canthaxanthin in the high titer E. coli strain compared to ∼20% canthaxanthin produced by the starting gene. Canthaxanthin production in Methylomonas was also significantly improved to ∼50% of total carotenoids by the mutant genes. Further improvement of canthaxanthin production to ∼93% in Methylomonas was achieved by increased expression of the best mutant gene. Some mutations were found in many of the improved genes, suggesting that these sites, and possibly the regions around these sites, were important for improving the crtOʹs activity for canthaxanthin production.