Abstract
BackgroundTerpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system.ResultsAiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation.ConclusionUsing an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0246-0) contains supplementary material, which is available to authorized users.
Highlights
Terpenoids have numerous applications in flavors, fragrances, drugs and biofuels
Identification of a working subset of gene deletions that enhance carotenoid production A heterozygous gene deletion in S. cerevisiae typically results in a 50% decrease in the corresponding protein levels [34]
To identify heterozygous gene deletions in yeast that support increased carotenoid, and diterpene, production, we set out to develop a system to screen the collection of yeast viable deletion strains using a carotenogenic screen
Summary
Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. Efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system. S. cerevisiae does not produce terpenoids, expression of plant derived terpene synthases in yeast cells revealed that it was possible for the enzymes to utilize the endogenous substrates (GPP, FPP, GGPP) and produce a range of terpenoid compounds [4,18]. The magnitude and complexity of genetic interactions identified in yeast cells [26], suggest that the output of a biosynthetic pathway may be affected by a large number of seemingly unrelated factors. To inactivate DOS2 in the rox haploid strain AM228, we Strain
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