Quick abbreviations

1. Combining inhibitor tolerance and D-Xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellylose-based bioethanol production

2. Quick abbreviations • DW: Dry weight • YPD: Yeast extract peptone dextrose; media • HPLC: High performance liquid chromatography • PCR: Polymerase chain reaction.

3. D-xylose fermentation • What is D-xylose? • a sugar first isolated from wood • is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms and includes a formyl functional group. • It is the precursor to hemicellulose, one of the main constituents of biomass.

4. Goals • Lignocellulose-based bioethanol • High inhibitor tolerance in industrial Saccharomyces

5. Problems with fermentation • Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiaestrains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates.

6. -including the furan derivatives furfural and HMF, aliphatic acids, such as acetic acid, formic acid and levulinic acid, and various phenolic compounds, such as vanillic acid, vanillin, synringaldehyde, syringic acid, and 4-hydroxybenzoic acid • large amounts of several types of inhibitory compounds are released during the pretreatment process. • They inhibit microbial fermentation and growth, resulting in severely reduced ethanol yield and productivity. • pentose fermentation appears to be much more sensitive to the toxic inhibitors, the productivity of the yeast in high-density lignocellulose hydrolysatesis largely determined by the robustness of the pentose fermentation.

7. Ethanol Red • Strain derived from ethanol red known as GS1.11-26 was created through evolutionary engineering procedures (mutagenesis). • This strain seemed promising and had a high level of lignocellulosic fermentation but was unable to retain the same tolerance from Ethanol Red in high concentrations of ethanol and acetate which resulted in yield loss. • the strain would not be suitable for direct industrial application.

8. Game plan • development of three new xylose utilizing industrial yeast strains, derived from the GS1.11-26 strain, which lack its negative properties. • Two new strains are diploid and were obtained through meiotic recombination • diploid segregant from a strongly inhibitor-tolerant triploid strain and with a haploid segregantof Ethanol Red.

9. Diploid importance • Researches had an idea that diploid strain would be best. • Why? • the most stable genome size in S. cerevisiae appears to be the diploid state. • diploid segregantswere obtained with a much higher D-xylose utilization rate than the tetraploid parent.

10. 3-strains • Two of the strains (GSF3 35 and GSF767) have been derived through meiotic recombination of the efficient D-xylose utilizing strain GS1.11-26 (derived from Ethanol Red) with the most inhibitor tolerant strain obtained from a screening of more than 580 yeast strains.

11. Strain GSE16 has a purely Ethanol Red background, developed by backcrossing GS1.11-26 with a haploid derivative of Ethanol Red.

12. Comparison

13. Sugar consumption rates

14. Inhibitor tolerance

15. All three strains showed superior performance with respect to aerobic growth rate, glucose consumption rate and inhibitor tolerance compared to GS1.11-26.

16. Methods for strain development

17. Screening of strains for inhibitor tolerance in spruce hydrolysate medium • Initial screening of the yeast strain collection for inhibitor tolerance was performed on agar plates containing increasing concentrations of the whole slurry of spruce hydrolysate(40% to 70%). • most tolerant strain JT21653

18. Screening segregants for growth and fermentation in D-xylose medium • To select strains that were able to ferment D-xylose efficiently, small tube semi-anaerobic batch fermentationswere performed. • The rate of fermentation was estimated by following the weight loss due to CO2 release. • Strains were evaluated for fermentation performance in D-xylose-glucose and in spruce media • Strains must prove to grow well in high gravity fermentation (having good density compared to ethanol in mixture)

19. Determination of ploidyby flow cytometry • Laser based biophysical technology • Able to tell ploidy by suspending cells in a stream of fluid and passing them by an electronic detection apparatus.

20. Meiotic recombination • Strains of opposite mating type were crossed by mixing small amounts of cells from each strain on a YPD plate (Yeast Peptone). • A few colonies were analyzed by PCR and flow cytometry to identify diploids.

21. Cont. Spores were isolated by tetrad dissection using an MSM micromanipulator (Singer instruments, Somerset, UK).

22. Determination of mating type • Mating type was determined by PCR and pheromone assay. PCR was performed using a primer annealing to the MAT locus and a MATa or MATα specific primer. - mating type was further validated by a pheromone assay.

23. Growth rate assay • -check growth rates and compare

24. Analysis of cell mass and metabolite concentrations • - filtrate was used for HPLC analysis. Ethanol from the very-high gravity fermentations was measured by near infrared spectroscopy (Alcolyzer, Anton Paar).

25. Conclusion • three superior industrial S. cerevisiae strains that combine efficient D-xylose utilization with high inhibitor tolerance. • Since the background strain Ethanol Red has a proven record of successful industrial application, the three new superior strains have strong potential for direct application in industrial bioethanol production.

26. References • Demeke et al.: Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production. Biotechnology for Biofuels 2013 6:120. • Mielenz JR: Ethanol production from biomass: technology and commercialization status. CurrOpinMicrobiol 2001, 4:324–329. • Sims RE, Mabee W, Saddler JN, Taylor M: An overview of second generation biofuel technologies. BioresourTechnol 2010, 101:1570–1580. • St Charles J, Hamilton ML, Petes TD: Meiotic chromosome segregation in triploid strains of Saccharomyces cerevisiae. Genetics 2010, 186:537–550. • Ding MZ, Li BZ, Cheng JS, Yuan YJ: Metabolome analysis of differential responses of diploid and haploid yeast to ethanol stress. OMICS 2010,14:553–561.