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The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction

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Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has emerged as the dominating tool for genome engineering, while also changes the speed and efficiency of metabolic engineering in conventional and non-conventional yeasts. Among these CRISPR-Cas systems, CRISPR-Cas9 technology has usually been applied for removing unfavorable target genes. Here, we used CRISPR-Cas9 technology to delete the gal80 gene in uracil-deficient strain and had successfully remolded the engineered Saccharomyces cerevisiae that can produce artemisinic acid without galactose induction. An L9(34) orthogonal test was adopted to investigate the effects of different factors on artemisinic acid production. Fermentation medium III with sucrose as carbon sources, 1% inoculum level, and 84-h culture time were identified as the optimal fermentation conditions. Under this condition, the maximum artemisinic acid production by engineered S. cerevisiae 1211-2 was 740 mg/L in shake-flask cultivation level. This study provided an effective approach to reform metabolic pathway of artemisinic acid-producing strain. The engineered S. cerevisiae 1211-2 may be applied to artemisinic acid production by industrial fermentation in the future.

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Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeat

PAM:

Protospacer adjacent motif

sgRNA:

Single-guide RNA

5-FOA:

5-Fluoroorotic acid

ura3 :

Orotidine-5′-phosphate decarboxylase gene

gal80 :

Galactose metabolism negative regulator

Gal80:

Gal4 inhibitor protein

SD-URA:

Synthetic dextrose minimal medium without uracil

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Acknowledgements

This research was supported by grants from the Drug Innovation Major Project (2017ZX09101002-003-003 and 2018ZX09711001-007-003), National Key Research and Development Program of China (2018YFA0902000), and National Natural Science Foundation of China (31870059).

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Authors and Affiliations

  1. NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tian Tan Xi Li No. 1, Beijing, 100050, China

    Limei Ai, Weiwei Guo & Liping Bai

  2. Zhejiang Key Laboratory of Antifungal Drugs, Zhejiang Hisun Pharmaceutical Company Limited, Taizhou, 318000, Zhejiang, China

    Wei Chen & Yun Teng

  3. CAMS Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China

    Liping Bai

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  1. Limei Ai
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Correspondence to Yun Teng or Liping Bai.

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Ai, L., Guo, W., Chen, W. et al. The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction. Curr Microbiol 76, 1313–1319 (2019). https://doi.org/10.1007/s00284-019-01752-2

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