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Current Microbiology

, Volume 76, Issue 11, pp 1313–1319 | Cite as

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

  • Limei Ai
  • Weiwei Guo
  • Wei Chen
  • Yun TengEmail author
  • Liping BaiEmail author
Article

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.

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

Notes

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).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

284_2019_1752_MOESM1_ESM.docx (296 kb)
Supplementary file1 (DOCX 296 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal BiotechnologyChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
  2. 2.Zhejiang Key Laboratory of Antifungal DrugsZhejiang Hisun Pharmaceutical Company LimitedTaizhouChina
  3. 3.CAMS Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal BiotechnologyChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina

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