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A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
- Source :
- ACS Synthetic Biology
- Publication Year :
- 2021
- Publisher :
- American Chemical Society, 2021.
-
Abstract
- Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.
- Subjects :
- 0106 biological sciences
Yarrowia lipolytica
Indoles
Golden Gate Cloning
Biomedical Engineering
Yarrowia
Golden-Gate cloning
01 natural sciences
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Calcium Carbonate
Fungal Proteins
03 medical and health sciences
chemistry.chemical_compound
violacein
Biosynthesis
010608 biotechnology
fermentation optimization
Cloning, Molecular
Promoter Regions, Genetic
Gene
030304 developmental biology
Gene Library
Cloning
0303 health sciences
Natural product
biology
library construction
General Medicine
Hydrogen-Ion Concentration
biology.organism_classification
Yeast
chemistry
Biochemistry
Metabolic Engineering
Batch Cell Culture Techniques
promoter engineering
Transformation efficiency
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 21615063
- Volume :
- 10
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- ACS Synthetic Biology
- Accession number :
- edsair.doi.dedup.....f7759b1262c37bded9424543eaf49593