Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production

Trikka et al. Microbial Cell Factories Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production Fotini A Trikka 0 Alexandros Nikolaidis 0 Anastasia Athanasakoglou 0 Aggeliki Andreadelli 0 Codruta Ignea Konstantia Kotta 0 Anagnostis Argiriou 0 Sotirios C Kampranis Antonios M Makris 0 0 Institute of Applied Biosciences/CERTH , P.O. Box 60361, Thermi, 57001 Thessaloniki , Greece 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. Conclusion: 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. Terpenoids; Saccharomyces cerevisiae; Sclareol; Carotenoid; Ergosterol biosynthesis - Background Terpenoids (isoprenoids) are an important class of secondary metabolites contributing more than 70,000 compounds to the rich chemical diversity of natural product structures (The Dictionary of Natural Products Online: http://dnp.chemnetbase.com/intro) [1]. Many terpenoids possess pharmaceutical properties and are currently used in clinical practice. Among them are taxol, a diterpene from yew, which has successfully been established as a major antineoplastic agent, and artemisinin, a sesquiterpene lactone, which is an effective antimalarial agent [2-7]. Recently, attention has focused on microbially produced terpenes as biofuels [8-12]. In addition, several terpenes have attracted the interest of the flavour and fragrance industry. Such examples include (+)-nootkatone, an oxidized sesquiterpene extracted from grapefruit [13], santalols, the main components of sandalwood essential oil [14], and sclareol (Figure 1), an industrially important diterpene precursor of a sustainable alternative to ambergris [15,16]. Most of commercially produced sclareol is derived by extraction from cultivated Salvia sclarea. The sclareol biosynthetic pathway has Figure 1 Pathway describing sclareol and carotenoid biosynthesis in yeast. Erg20p catalyzes the formation of C15 farnesyl pyrophosphate molecules (FPP) for isoprenoid and sterol biosynthesis. A variant (F96C) enzyme was previously engineered to catalyze geranylgeranyl pyrophosphate synthesis (GGPP). Fusion of ERG20 (F96C) to the Cistus creticus 8-hydroxy copalyl diphosphate synthase (CcCLS) generates 8-OH-CPP, which in turn is converted to sclareol either through spontaneous hydrolysis in the acidified culture medium or enzymatically by the Salvia sclarea sclareol synthase (SsSCLS). In carotenoid biosynthesis the inserted carotenogenic pathway cassette of X. dendrorhous expresses the the GGPP synthase (encoded by the crtE gene), the bifunctional enzyme phytoene synthase/lycopene cyclase (encoded by the crtYB gene) and phytoene desaturase (encoded by the crtI gene). recently been elucidated and reconstructed in E.coli and S. cerevisiae [15,16]. Terpenoids are biosynthesized from two C5 precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [17]. In yeast and mammals, IPP originates from acetyl-CoA through the intermediate mevalonic acid (MVA). IPP then gives rise to the higher order building blocks, geranyl pyrophosphate (GPP; C10), farnesyl pyrophosphate (FPP; C15) and geranylgeranyl pyrophosphate (GGPP; C20) through the action of prenyltransferases [17]. In yeast, most of the pathway output in the form of FPP is utilized for the biosynthesis of sterols. The terpene hydrocarbon scaffolds are generated by the action of mono-, sesqui-, and diterpene synthases that catalyze multistep reactions using GPP, FPP or GGPP as substrates, respectively. Although 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 number of terpenoids produced in heterologous systems is continuously growing as more pathways become elucidated and new genes are cloned and characterized. In parallel to the gene discovery effort to identify and characterize enzymes producing chemicals of value, there has been a continuous effort to generate high producing yeast strains. Approaches to improve terpenoid production in yeast have mostly focused on existing knowledge of the sterol biosynthetic pathway with considerable success [4,19,20] (reviewed in [21]). Some key interventions in this direction include a) the deregulation of HMG-CoA reductase (HMGR) by truncation of the regulatory transmembrane domain [22] or point mutations (K6R) in HMG2 which render the enzyme resistant to ubiquitination [23,24] and b) the suppression of the squalene synthase gene (ERG9), which controls the major isoprenoid substrate draining route, that of ergosterol synthesis [4,19,24,25]. However, the magnitude and complexity of genetic interactions identified in yeast cells [26], suggest that the output of a biosynthetic pathway may also be affected by a large number of seemingly unrelated factors. Taking advantage of genetic interaction data, a set of heterozygous gene deletions that increased endogenous hmgp levels and consequently improved sesquiterpene biosynthesis was identified [27]. Quick and inexpensive selection m (...truncated)