Combinatorial iterative method for metabolic engineering of Yarrowia lipolytica: application for betanin biosynthesis.
Metab Eng
; 2024 Sep 09.
Article
en En
| MEDLINE
| ID: mdl-39260817
ABSTRACT
Combinatorial library-based metabolic engineering approaches allow lower cost and faster strain development. We developed a genetic toolbox EXPRESSYALI for combinatorial engineering of the oleaginous yeast Yarrowia lipolytica. The toolbox enables consecutive rounds of engineering, where up to three combinatorially assembled gene expression cassettes can be integrated into each yeast clone per round. The cassettes are integrated into distinct intergenic sites or an open reading frame of a target gene if a simultaneous gene knockout is desired. We demonstrate the application of the toolbox by optimizing the Y. lipolytica to produce the red beet color betanin via six consecutive rounds of genome editing and screening. The library size varied between 24-360. Library screening was facilitated by automated color-based colony picking. In the first round, betanin pathway genes were integrated, resulting in betanin titer of around 20 mg/L. Through the following five consecutive rounds, additional biosynthetic genes were integrated, and the precursor supply was optimized, resulting in a titer of 70 mg/L. Three beta-glucosidases were deleted to prevent betanin deglycosylation, which led to a betanin titer of 130 mg/L in a small scale and a titer of 1.4 g/L in fed-batch bioreactors. The EXPRESSYALI toolbox can facilitate metabolic engineering efforts in Y. lipolytica (available via AddGene Cat. Nr. 212682-212704, Addgene kit ID # 1000000245).
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1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Metab Eng
Asunto de la revista:
ENGENHARIA BIOMEDICA
/
METABOLISMO
Año:
2024
Tipo del documento:
Article
País de afiliación:
Dinamarca
Pais de publicación:
Bélgica