High-level biosynthesis of enantiopure germacrene D in yeast.
Appl Microbiol Biotechnol
; 108(1): 50, 2024 Dec.
Article
en En
| MEDLINE
| ID: mdl-38183482
ABSTRACT
Germacrene D, a sesquiterpenoid compound found mainly in plant essential oils at a low level as (+) and/or (-) enantiomeric forms, is an ingredient for the fragrance industry, but a process for the sustainable supply of enantiopure germacrene D is not yet established. Here, we demonstrate metabolic engineering in yeast (Saccharomyces cerevisiae) achieving biosynthesis of enantiopure germacrene D at a high titer. To boost farnesyl pyrophosphate (FPP) flux for high-level germacrene D biosynthesis, a background yeast chassis (CENses5C) was developed by genomic integration of the expression cassettes for eight ergosterol pathway enzymes that sequentially converted acetyl-CoA to FPP and by replacing squalene synthase promoter with a copper-repressible promoter, which restricted FPP flux to the competing pathway. Galactose-induced expression of codon-optimized plant germacrene D synthases led to 13-30 fold higher titers of (+) or (-)-germacrene D in CENses5C than the parent strain CEN.PK2.1C. Furthermore, genomic integration of germacrene D synthases in GAL80, LPP1 and rDNA loci generated CENses8(+D) and CENses8(-D) strains, which produced 41.36 µg/ml and 728.87 µg/ml of (+) and (-)-germacrene D, respectively, without galactose supplementation. Moreover, coupling of mitochondrial citrate pool to the cytosolic acetyl-CoA, by expressing a codon-optimized ATP-citrate lyase of oleaginous yeast, resulted in 137.71 µg/ml and 815.81 µg/ml of (+) or (-)-germacrene D in CENses8(+D)* and CENses8(-D)* strains, which were 67-120 fold higher titers than in CEN.PK2.1C. In fed-batch fermentation, CENses8(+D)* and CENses8(-D)* produced 290.28 µg/ml and 2519.46 µg/ml (+) and (-)-germacrene D, respectively, the highest titers in shake-flask fermentation achieved so far. KEY POINTS ⢠Engineered S. cerevisiae produced enantiopure (+) and (-)-germacrene D at high titers ⢠Engineered strain produced up to 120-fold higher germacrene D than the parental strain ⢠Highest titers of enantiopure (+) and (-)-germacrene D achieved so far in shake-flask.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
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Galactosa
Idioma:
En
Revista:
Appl Microbiol Biotechnol
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Appl. microbiol. biotechnol
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Applied microbiology and biotechnology
Año:
2024
Tipo del documento:
Article
País de afiliación:
India