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Production of β-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica

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Abstract

Objectives

To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene β-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce β-carotene.

Results

To increase the integration efficiency of a 3.8 kb carS under the control of P GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg β-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica.

Conclusion

Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to β-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.

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Acknowledgements

We thank Professor Hal S. Alper for the generous gift of the pMCSCen1 plasmid. This work was financed by the Ministry of Science and Technology of China (973:2014CB745101).

Supporting information

Supplementary Table 1—Primers used.

Supplementary Fig. 1—Transmembrane prediction analysis of carB enzymes from (A) Blakeslea trispora and (B) Mucor circinelloides.

Supplementary Fig. 2—Transmembrane prediction analysis of (A) carRA enzyme from Blakeslea trispora and (B) carRP enzyme from Mucor circinelloides.

Supplementary Fig. 3—URA3 disruption of CIBTS1961 via the CRISPR-Cas9 system.

Supplementary Fig. 4—Disruption of URA3 in CIBTS1961, mediated by the CRISPR-Cas9 system.

Author information

Authors and Affiliations

  1. School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Shuliang Gao & Daijie Chen

  2. College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai, 200234, China

    Yangyang Tong

  3. Shanghai Laiyi Center for Biopharmaceutical R&D, 800 Dongchuan Road, Shanghai, 200240, China

    Li Zhu & Mei Ge

  4. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China

    Yu Jiang & Sheng Yang

  5. Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai, 201201, China

    Yu Jiang & Sheng Yang

  6. Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai, 200040, China

    Daijie Chen

  7. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), 200 North Zhongshan Road, Nanjing, 211816, China

    Sheng Yang

Authors
  1. Shuliang Gao
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  2. Yangyang Tong
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  3. Li Zhu
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  4. Mei Ge
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  5. Yu Jiang
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  6. Daijie Chen
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  7. Sheng Yang
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Corresponding author

Correspondence to Daijie Chen.

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Gao, S., Tong, Y., Zhu, L. et al. Production of β-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica . Biotechnol Lett 39, 921–927 (2017). https://doi.org/10.1007/s10529-017-2318-1

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  • DOI: https://doi.org/10.1007/s10529-017-2318-1

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