Structural Analysis And Biosynthetic Pathway Study Of Carotenoids In C.sakazakii

Cronobacter sakazakii can produce carotenoids which are helpful for its tolerance todesiccation and acidic environment, and this may be linked with the virulence of the pathogen.Now, little was known about the compositions and biosynthesis pathway of carotenoids in C.sakazakii. Here, we focused on the carotenoids produced by C. sakazakii BAA-894, aimed atrevealing the compositions and biosynthesis pathway of carotenoids in C. sakazakii. The mainconclusions are shown below:(1) Components of carotenoids produced by C. sakazakii BAA-894were confirmed inthis study. Carotenoids produced by C. sakazakii BAA-894were extracted, separated andpurified. Analysis with thin layer chromatography, ultraviolet-visible spectrum, ionchromatography and liquid chromatography-mass spectrometry showed that the carotenoidsmainly consist of zeaxanthin-monoglycoside and zeaxanthin-diglycoside.(2) The gene cluster crtE-idi-XYIBZ responsible for carotenoid production in C.sakazakii BAA-894was confirmed in this study. Based on sequence alignment, a gene clustercrtE-idi-XYIBZ containing seven ORFs homologous to carotenoids biosynthesis genes crtE,idi, crtX, crtY, crtI, crtB and crtZ was found in the genome of C. sakazakii BAA-894.Escherichia coli DH5α/pWSK29-EiZYIBX was constructed, and this strain could also producecarotenoids. Analysis showed that DH5α/pWSK29-EiZYIBX could producezeaxanthin-monoglycoside and zeaxanthin-diglycoside. This suggests that the gene clustercrtE-idi-XYIBZ is responsible for carotenoids production in C. sakazakii.(3) The functions of key genes in cluster crtE-idi-XYIBZ were confirmed in this study.Key genes in crtE-idi-XYIBZ were expressed in E. coli DH5α in different combination toconstruct six recombinant strains DH5α/pWSK29-i, DH5α/pWSK29-Ei, DH5α/pWSK29-EiB,DH5α/pWSK29-EiIB, DH5α/pWSK29-EiYIB and DH5α/pWSK29-EiZYIB. Analysis showedthat lycopene could be produced by DH5α/pWSK29-EiIB; β-carotene could be produced byDH5α/pWSK29-EiYIB; cryptoxanthin and zeaxanthin could be produced byDH5α/pWSK29-EiZYIB. This further elucidates the function of key genes in the clustercrtE-idi-XYIBZ of C. sakazakii.(4) The facilitation of idi for carotenoid production in C. sakazakii BAA-894wasconfirmed in this study. Compared with the common carotenoid biosynthesis gene cluster, thegene cluster of C. sakazakii holds one more gene idi. In order to study the function of idi incarotenoids production in C. sakazakii, recombinant strains C. sakazakii BAA-894/pWSK29-iand E. coli DH5α were constructed. The carotenoids production of BAA-894/pWSK29-i was80%higher than that of BAA-894/pWSK29, and the carotenoids production of E. coliDH5α/pWSK29-EiIB was60%higher than that of DH5α/pWSK29-EIB, indicating that the idiin C. sakazakii could facilitate the carotenoids production.