Characterization of bacterial beta-carotene 3,3'-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli

Abstract

beta-Carotene hydroxylase (CrtZ) is one of rate-limiting enzymes for astaxanthin production. A complementation analysis was conducted using Escherichia coli transformants to compare the catalytic efficiency of bacterial CrtZ from Brevundimonas sp. SD212, Paracoccus sp. PC1 (formerly known as Alcaligenes sp. PC-1), Paracoccus sp. N81106 (Agrobacterium aurantiacum), Pantoea ananatis (Erwinia uredovora 20D3), marine bacterium P99-3, and P450 monooxygenase (CYP175A1) from Thermus thermophilus HB27. Each crtZ or CYP175A1 gene was expressed in E. coli transformants synthesizing canthaxanthin and beta-carotene due to the respective presence of plasmids pAC-Cantha and pACCAR16DeltacrtX. The carotenoids that accumulated in the resulting recombinant E. coli cells were examined by chromatographic and spectroscopic analyses. E. coli carrying Brevundimonas sp. SD212 crtZ showed the highest astaxanthin production efficiency among the transformants examined, while there was no significant difference in the catalytic efficiency for conversion from beta-carotene to zeaxanthin. Recombinant E. coli expressing the CYP175A1 gene, in addition to the genes for canthaxanthin synthesis, surprisingly accumulated adonirubin (phoenicoxanthin) as the main product, although the other recombinant E. coli did not accumulate any adonirubin. The present results suggest that the Brevundimonas sp. SD212 crtZ and T. thermophilus HB27 CYP175A1 genes could, respectively, be used for the efficient production of astaxanthin and adonirubin in heterologous hosts.