title: Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Aiming 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.
  
  
  CONCLUSIONS
  Using 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.
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. fotaaki@hotmail.co.uk.
authors.forename: Fotini A
authors.initials: FA
authors.lastname: Trikka
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. nikolchem@hotmail.com.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Nikolaidis
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. ath.anastasia@gmail.com.|Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion, 71003, Greece. ath.anastasia@gmail.com.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Athanasakoglou
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. a.andreadelli@gmail.com.
authors.forename: Aggeliki
authors.initials: A
authors.lastname: Andreadelli
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion, 71003, Greece. igneacodruta@yahoo.com.
authors.forename: Codruta
authors.initials: C
authors.lastname: Ignea
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. ntina_kotta@yahoo.com.
authors.forename: Konstantia
authors.initials: K
authors.lastname: Kotta
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. argiriou@certh.gr.
authors.forename: Anagnostis
authors.initials: A
authors.lastname: Argiriou
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion, 71003, Greece. s.kampranis@med.uoc.gr.
authors.forename: Sotirios C
authors.initials: SC
authors.lastname: Kampranis
authors.affiliation: Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece. antoniosmakris@yahoo.gr.
authors.forename: Antonios M
authors.initials: AM
authors.lastname: Makris
mesh_terms: D002338:Carotenoids / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D004875:Ergosterol / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*
chemical_list: D004224:Diterpenes; D013729:Terpenes; D002338:Carotenoids; C070760:sclareol; D004875:Ergosterol