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. 2014 Jan 23;9(1):e86739.
doi: 10.1371/journal.pone.0086739. eCollection 2014.

Reconstruction of the carotenoid biosynthetic pathway of Cronobacter sakazakii BAA894 in Escherichia coli

Wei Zhang  1 , Xiaoqing Hu  2 , Liqin Wang  3 , Xiaoyuan Wang  2
Affiliations

Reconstruction of the carotenoid biosynthetic pathway of Cronobacter sakazakii BAA894 in Escherichia coli

Wei Zhang et al. PLoS One. .

Abstract

Cronobacter sakazakii could form yellow-pigmented colonies. However, the chemical structure and the biosynthetic pathway of the yellow pigments have not been identified. In this study, the yellow pigments of C. sakazakii BAA894 were purified and analyzed. The major components of the yellow pigments were confirmed as zeaxanthin-monoglycoside and zeaxanthin-diglycoside. A gene cluster containing seven genes responsible for the yellow pigmentation in C. sakazakii BAA894 was identified. The seven genes of C. sakazakii BAA894 or parts of them were reconstructed in a heterologous host Escherichia coli DH5α. The pigments formed in these E. coli strains were isolated and analyzed by thin layer chromatography, UV-visible spectroscopy, high performance liquid chromatography, and electron spray ionization-mass spectrometry. These redesigned E. coli strains could produce different carotenoids. E. coli strain expressing all the seven genes could produce zeaxanthin-monoglycoside and zeaxanthin-diglycoside; E. coli strains expressing parts of the seven genes could produce lycopene, β-carotene, cryptoxanthin or zeaxanthin. This study identified the gene cluster responsible for the yellow pigmentation in C. sakazakii BAA894.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Organization of carotenoid biosynthesis genes in C. sakazakii.
Direction of transcription is indicated by arrows. ORF numbers of the corresponding genes, and identities of the corresponding proteins in C. sakazakii strains ES5 and BAA894 are listed. Only the gene idi was annotated in the genome of C. sakazakii BAA894.
Figure 2
Figure 2. Analysis of yellow pigments produced by C. sakazakii BAA894.
A. TLC analysis of yellow pigments CS1 and CS2 produced by C. sakazakii BAA894. B. UV-visible spectra of purified CS1 and CS2. C. ESI/MS analysis of the purified CS1. E. ESI/MS analysis of the purified CS2.
Figure 3
Figure 3. Reconstruction of the carotenoid biosynthesis pathway of C. sakazakii in E. coli.
A. Seven genes crtE, idi, crtX, crtY, crtI, crtB and crtZ were amplified from the genome of C. sakazakii BAA894 and cloned into pWSK29 in various combinations. B. Different colors shown by cells of E. coli DH5α/pWSK29, DH5α/pWSK29-i, DH5α/pWSK29-Ei, DH5α/pWSK29-EiB, DH5α/pWSK29-EiIB, DH5α/pWSK29-EIB, DH5α/pWSK29-EiYIB, DH5α/pWSK29-EiZYIB, and DH5α/pWSK29-EiZYIBX.
Figure 4
Figure 4. Zeaxanthin-monoglycoside and zeaxanthin-diglycoside could be synthesized in E. coli when seven C. sakazakii genes were expressed.
A. TLC analysis of carotenoids EC1 and EC2 produced by DH5α/pWSK29-EiZYIBX. B. UV-visible spectra of purified EC1 and EC2. C. ESI/MS analysis of purified EC1. D. ESI/MS analysis of purified EC2. E. HPLC analysis of the hexose hydrolyzed from EC1.
Figure 5
Figure 5. C. sakazakii gene crtZ is responsible for the biosynthesis of cryptoxanthin and zeaxanthin from β-carotene.
A. TLC analysis of carotenoids EC3 and EC4 produced by DH5α/pWSK29-EiZYIB. B. UV-visible spectra of purified EC3 and EC4. C. ESI/MS of purified EC3. D. ESI/MS of purified EC4.
Figure 6
Figure 6. Lycopene and β-carotene could be synthesized in E. coli when C. sakazakii genes crtE, idi, crtI and crtB were expressed with or without crtY, respectively.
A. TLC analysis of carotenoids EC1 and EC2 produced by DH5α/pWSK29-EiIB and DH5α/pWSK29-EiYIB, respectively. B. UV-visible spectra of purified EC1 and EC2. C. HPLC spectra of purified EC1 and EC2.
Figure 7
Figure 7. The proposed pathway of carotenoid biosynthesis in C. sakazakii BAA894.
The names used in this article for each molecule were listed on the right.
See this image and copyright information in PMC

References

    1. Cazzonelli CI, Pogson BJ (2010) Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci 15: 266–274. - PubMed
    1. Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea F (2006) Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 46: 185–196. - PubMed
    1. Vachali P, Bhosale P, Bernstein PS (2012) Microbial carotenoids. Method Mol Biol 898: 41–59. - PubMed
    1. Miura Y, Kondo K, Saito T, Shimada H, Fraser PD, et al. (1998) Production of the carotenoids lycopene, β-carotene, and astaxanthin in the food yeast Candida utilis . Appl Environ Microbiol 64: 1226–1229. - PMC - PubMed
    1. Sedkova N, Tao L, Rouvière PE, Cheng Q (2005) Diversity of carotenoid synthesis gene clusters from environmental Enterobacteriaceae strains. Appl Environ Microbiol 71: 8141–8146. - PMC - PubMed

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