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Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

  • Ruizhao Wang
  • Xiaoli Gu
  • Mingdong Yao
  • Caihui Pan
  • Hong Liu
  • Wenhai Xiao
  • Ying Wang
  • Yingjin Yuan
Research Article

Abstract

The conversion of β-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high β-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from Brevundimonas vesicularis DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtW were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both crtZ and crtW were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes.

Keywords

synthetic biology astaxanthin β-carotene hydroxylase β-carotene ketolase Saccharomyces cerevisiae 

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Notes

Acknowledgements

This work was supported by the International S&T Cooperation Program of China (2015DFA00960), the National Natural Science Foundation of China (Grant Nos. 31600052 and 21676192) and Innovative Talents and Platform Program of Tianjin (16PTSYJC00050).

Supplementary material

11705_2017_1628_MOESM1_ESM.pdf (388 kb)
Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

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Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Ruizhao Wang
    • 1
    • 2
  • Xiaoli Gu
    • 1
    • 2
  • Mingdong Yao
    • 1
    • 2
  • Caihui Pan
    • 1
    • 2
  • Hong Liu
    • 1
    • 2
  • Wenhai Xiao
    • 1
    • 2
  • Ying Wang
    • 1
    • 2
  • Yingjin Yuan
    • 1
    • 2
  1. 1.Key Laboratory of Systems Bioengineering (Ministry of Education)Tianjin UniversityTianjinChina
  2. 2.SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina

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