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Ntoanidou S.,Aristotle University of Thessaloniki | Kaloumenos N.,Hill International | Diamantidis G.,Aristotle University of Thessaloniki | Madesis P.,Institute of Applied Biosciences CERTH | Eleftherohorinos I.,Aristotle University of Thessaloniki
Pesticide Biochemistry and Physiology | Year: 2016

Applications of 64 times higher rates of azimsulfuron and halosulfuron-methyl failed to reduce by 50% growth of 10 putative resistant (R) Cyperus difformis (smallflower umbrella-sedge) populations collected fromrice fields located in Northern Greece. However, the growth rate of the susceptible (S) population was reduced by 50% by 1/4 of the recommended rate of both herbicides. The als gene sequencing identified point mutations at Pro-197 position, leading to amino acid substitution by Ala or Ser of the ALS enzyme. The in vitro activity of the ALS enzyme indicated that the I50 values (herbicide concentration required for 50% reduction of the ALS activity) ranged from 10.7 to 55.5 μM azimsulfuron and from 6.7 to 50.6 μM halosulfuron-methyl, whereas the respective values for the S population were 0.09 and 0.11 μM. These results strongly support that cross-resistance of 10 C. difformis populations was due to point mutations of the als gene, which resulted in a less-sensitive ALS enzyme. This study reports the presence of a 1240 bp intron in the C. difformis als gene with the Pro-197 pointmutations near the splice junction. © 2015 Elsevier B.V.

Trikka F.A.,Institute of Applied Biosciences CERTH | Nikolaidis A.,Institute of Applied Biosciences CERTH | Ignea C.,University of Crete | Tsaballa A.,Institute of Applied Biosciences CERTH | And 10 more authors.
BMC Genomics | Year: 2015

Background: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S.pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. Results: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. Conclusions: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites. © 2015 Trikka et al.

Trikka F.A.,Institute of Applied Biosciences CERTH | Nikolaidis A.,Institute of Applied Biosciences CERTH | Athanasakoglou A.,Institute of Applied Biosciences CERTH | Athanasakoglou A.,University of Crete | And 6 more authors.
Microbial Cell Factories | Year: 2015

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 750mg/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. © Trikka et al.; licensee BioMed Central.

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