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One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica

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Abstract

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire β-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21 %, and the highest production of β-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.

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Acknowledgments

This work was supported by the National Basic Research Program of China (973: 2012CB721105 and 2014CB745101), National High-Tech Research and Development Program of China (863: 2012AA02A704) and the Knowledge Innovation Program of the Chinese Academy of Sciences (Y31M5A211). We thank Prof. Catherine Madzak for offering transformation protocols of Y. lipolytica.

Supporting information

Supplementary Table 1—Strains and plasmids used in this work

Supplementary Table 2—Primers used in this work

Supplementary Table 3—Procedure of elution for HPLC analysis

Supplementary Figure 1—Construction of engineering β-carotene biosynthesis pathway from X. dendrorhous in Y. lipolytica, namely XD

Supplementary Figure 2—A histogram representing the copy number of each cassette in mutant strain CIBTS 1176 by realtime qPCR

Author information

Authors and Affiliations

  1. College of Bioengineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Shuliang Gao & Daijie Chen

  2. School of Pharmacology, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China

    Linna Han

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

    Li Zhu & Mei Ge

  4. CAS 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

    Sheng Yang & Yu Jiang

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

    Sheng Yang & Yu Jiang

  6. Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China

    Sheng Yang

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

    Daijie Chen

  8. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai, 200237, China

    Sheng Yang & Daijie Chen

Authors
  1. Shuliang Gao
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  2. Linna Han
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  3. Li Zhu
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  4. Mei Ge
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  5. Sheng Yang
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  6. Yu Jiang
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  7. Daijie Chen
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Corresponding authors

Correspondence to Sheng Yang, Yu Jiang or Daijie Chen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supporting information

Supplementary Table 1—Strains and plasmids used in this work.

Supplementary Table 2—Primers used in this work.

Supplementary Table 3—Procedure of elution for HPLC analysis.

Supplementary Fig. 1—Construction of engineering β-carotene biosynthesis pathway from X. dendrorhous in Y. lipolytica, namely XD.

Supplementary Fig. 2—A histogram representing the copy number of each cassette in mutant strain CIBTS 1176 by realtime Qpcr.

Supplementary material 1 (DOCX 381 kb)

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Gao, S., Han, L., Zhu, L. et al. One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica . Biotechnol Lett 36, 2523–2528 (2014). https://doi.org/10.1007/s10529-014-1634-y

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  • DOI: https://doi.org/10.1007/s10529-014-1634-y

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