Tomas Strucko

EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for bakers yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.

Metabolic engineering of yeast for fermentative production of flavonoids

Yeast Saccharomyces cerevisiae was engineered for de novo production of six different flavonoids (naringenin, liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin) directly from glucose, without supplementation of expensive intermediates. This required reconstruction of long biosynthetic pathways, comprising up to eight heterologous genes from plants. The obtained titers of kaempferol 26.57±2.66mgL-1 and quercetin 20.38±2.57mgL-1 exceed the previously reported titers in yeast. This is also the first report of de novo biosynthesis of resokaempferol and fisetin in yeast. The work demonstrates the potential of flavonoid-producing yeast cell factories.

CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside.

Laboratory evolution reveals regulatory and metabolic trade-offs of glycerol utilization in Saccharomyces cerevisiae

Most microbial species, including model eukaryote Saccharomyces cerevisiae, possess genetic capability to utilize many alternative nutrient sources. Yet, it remains an open question whether these manifest into assimilatory phenotypes. Despite possessing all necessary pathways, S. cerevisiae grows poorly or not at all when glycerol is the sole carbon source. Here we discover, through multiple evolved lineages, genetic determinants underlying glycerol catabolism and the associated fitness trade-offs. Most evolved lineages adapted through mutations in the HOG pathway, but showed hampered osmotolerance. In the other lineages, we find that only three mutations cause the improved phenotype. One of these contributes counter-intuitively by decoupling the TCA cycle from oxidative phosphorylation, and thereby hampers ethanol utilization. Transcriptomics, proteomics and metabolomics analysis of the re-engineered strains affirmed the causality of the three mutations at molecular level. Introduction of these mutations resulted in improved glycerol utilization also in industrial strains. Our findings not only have a direct relevance for improving glycerol-based bioprocesses, but also illustrate how a metabolic pathway can remain unexploited due to fitness trade-offs in other, ecologically important, traits.

Development of an efficient glycerol utilizing Saccharomyces cerevisiae platform strain via adaptive laboratory evolution

No Abstract is available for this article.

Applied systems biology - vanillin production in Saccharomyces cerevisiae

We have selected the mediterranean shrub Euphorbia characias as an experimental model to study the complexity of plant latex chemistry. Latex is a mixture with diversified composition, that includes alkaloids, terpenoid compounds, polymeric substances such as resins and gums, starch, oil and a large number of proteins and enzymatic activities. The aim of the present study is to contribute to the knowledge of this plant product evaluating the antioxidant properties of extracts of E. characias and searching for polymeric substances as natural rubber. We analyzed different extracts from the latex of E. characias and performed a new extraction method (involving the use of trichloacetic acid, TCA) that turned out to be easier, faster and higher reproducible if compared to common extraction methods involving organic solvents like methanol, ethanol, and petroleum ether/methanol. TCA extract of E. characias latex exhibits antioxidant activities determined as total content of free-radical scavenging, polyphenols and total flavonoids. GC-MS analysis confirms the presence of several compound identified as antioxidant molecules. E. characias latex contains a natural rubber. The optimum rubber extraction is achieved with acetic acid followed by cyclohexane/ethanol treatment. The rubber content was shown to be 14% (w/v) of the E. characias latex and the gel content is 2.5% of the rubber weight. E. characias natural rubber showed a molecular weight of 93000 and Mw/Mn of 2.9. On the basis of H NMR, C NMR and FT-IR spectroscopy, the structure of this rubber can be identified as cis-1,4-polyisoprene. This study was partially supported by a grant from Regione Autonoma della Sardegna, Progetti di ricerca di base CRP2-22.Session 1—Maternal Medicine 1. INCIDENCE OF SUPPLEMENTAL POLYDRUG USE IN IRISH PREGNANT WOMEN ON METHADONE MAINTENANCE PROGRAMME Akhter P, Coulter-Smith S, Lee J, Brennan M, Clarke T, Geary M; Dublin, Ireland. 3. MAXIMAL EXERCISE TESTING CAN BE SAFELY USED TO ASSESS CARDIOVASCULAR RESERVE IN PREGNANCY Barker D, Mason G, Schlosshan D, McLoughlin H, Blackburn M, Simpson N, Tan LB; Leeds, UK 4. PREGNANCY OUTCOME IN RENAL TRANSPLANT RECIPIENTS AT ADDENBROOKES HOSPITAL: A 10 YEAR REVIEW Burrell C, Lees C, Daniels I, Somoye G; Cambridge, UK 5. HYPERTENSION IN PREGNANCY: A SURVEY OF CLINICAL PRACTICE TO DOCUMENT CURRENT PRACTICE IN THE UK AND REFINE QUESTIONS FOR A RANDOMIZED CONTROLLED TRIAL (RCT) Churchill D, Duley L, Farrell B; Wolverhampton, UK 6. THE IMPACT OF ACQUIRED THROMBOPHILIA ON MATERNAL AND FETAL WELLBEING IN A LOW-RISK POPULATION Cooley SM, Donnelly J, Walsh T, Geary MP, Gillan J, MacMahon C; Dublin, Ireland 7. FREE MATERNAL DNA IS INCREASED BEFORE 20 WEEKS GESTATION IN WOMEN WHOSE PREGNANCIES ARE SUBSEQUENTLY COMPLICATED BY IUGR BUT NOT PREECLAMPSIA Crowley A, Fitzpatrick P, Sheils O, O’Leary J, O’Herlihy C, Byrne B; Dublin, Ireland 8. IMPACT OF NATIONAL GUIDELINES ON THE MANAGEMENT OF VENOUS THROMBOEMBOLISM IN PREGNANCY Doumouchtsis SK, Tartaglia MA, Thomas GMA, Wilson MP, Thomson AJ; Paisley, UK 9. AN AUDIT ON THE MANAGEMENT OF ANAEMIA IN PREGNANCY IN A LARGE DISTRICT GENERAL HOSPITAL Dua A, Schram CMH, Karunakaran B; Blackburn, UK 10. MECHANISMS OF ACTION OF TNF-a ON ENDOTHELIUM-DEPENDENT RELAXATION IN OMENTAL ARTERIES OBTAINED FROM WOMEN WITH HEALTHY PREGNANCIES Gillham JC, Taggart MJ, Baker PN; Manchester, UK 11. THE QUEENMOTHERS HOSPITAL REVIEW OF PRE-GESTATIONAL DIABETES MELLITUS Hale J, Nicoll AE, Munaza S, Macara LM, Small M, Capaldi B, Cameron AD; Glasgow, UK 12. HEPATITIS B VACCINATION IN AT RISK DRUG USING WOMEN IN PREGNANCY Hyde J, Blunsden V, Irish C; Bristol, UK 13. ALVEOLAR O2 AND CO2 TENSIONS DURING PREGNANCY, MEASURED WITH A NOVEL NON-INVASIVE TECHNIQUE Litos M, Hadjistavrou C, Antsaklis A, Xygakis A, Jordanoglou J; Athens, Greece 14. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) LEVELS IN DIABETIC PREGNANCY: RELATIONS TO NORMAL PREGNANCY AND GLYCAEMIC CONTROL Manderson JG, McClure N, Patterson CC, Hadden DR, Traub AI, McCance DR; Belfast, UK 15. HYPOTHYROIDISM IN PREGNANCY: INCREASE IN THYROXINE DOSE MAY BE TOO LATE USING OUR CURRENT PROTOCOL Masheshwari S, Singh A, Trinder J; Bristol, UK 16. PREGNANCY CARE IN OBESE WOMEN. AN OBSERVATIONAL STUDY IN A DISTRICT GENERAL HOSPITAL Nawar-Youssef MN, Iqbal F, Odukoya OA; Scunthorpe, UK 17. MANAGEMENT OF POST-PARTUM HYPERTENSION (PHT) Ogunnoiki O, Hirsi-Farah S, Gray G, Nelson-Piercy C; London, UK 18. STUDY ON SCREENING FOR GESTATIONAL DIABETES ParveenAS,PeploeD,Bell-ThomasS;Abergavenny,UK 19. DOES THE INTRODUCTION OF A SCREENING PROCESS FOR MENTAL HEALTH PROBLEMS IMPROVE DETECTION OF AT RISK WOMEN IN THE ANTENATAL PERIOD? Scholefield HJ, Hernon M, Topping J; Liverpool, UK 20. MATERNAL OBESITY AND ITS IMPACT ON PREGNANCY OUTCOME Singhal T, Vogiatzi M, Parmeshwaran S, Howarth ES; Leicester, UK 21. RETROSPECTIVE ANALYSIS OF THE OUTCOME OF HIV PREGNANCIES IN A LARGE DISTRICT GENERAL HOSPITAL Sivarajan S, Hooi A, Roy M, Thomas P, Modi M, Howard R, Sahoo S; London, UK 22. PLACENTAL ABCA1 PROTEIN EXPRESSION IN ANTI-PHOSPHOLIPID SYNDROME TetlowN, Albrecht C, Lakasing L, Soumian S, Patel P, Sullivan M, Nicolaides K, Williamson C; London, UK 23. VIRILIZATION IN PREGNANCY ASSOCIATED WITH OVARIAN LEIOMYOMAS Udayasankar V, Moselhi M, Fielding A; Swansea, UK Journal of Obstetrics and Gynaecology (April 2005) Vol. 25, Supplement 1, S27–S31