Abstract

The present research deals with implementation of the bridge-induced chromosome translocation (BIT) technology and yeast artificial chromosome (YAC) recombineering for strain improvement of bioethanol producing transgenic yeast. We aimed to construct a YAC that carries cellulose degradation genes on it and then to apply the BIT technology. BIT technology allowed us to gain two advantages; one of them was to stabilize YAC into the yeast genome and the other one was to have increased a gene expression level consequent to the translocation event. Selection strategies were implemented to obtain novel genetic regulation that would achieved the final phenotype originally desired with the high cellulose degradation and high ethanol producing features. In conclusion, in our study, we utilized two novel technologies (Yeast Artificial Chromosome (YAC) recombineering and Bridge- Induced Translocation (BIT) technology to introduce new, multi-factorial genetic traits into a yeast strain, a process that would otherwise take several time-consuming and labor-intensive rounds of genetic engineering. This work describes the successful recombinant translocant yeast that is able to efficiently utilize cellulosic material as a carbon source with highly stable recombinant translocant chromosome and has high level of cellulases capacity.

Keywords: Bridge-Induced Translocation (BIT); Yeast Artificial Chromosome (YAC); Saccharomyces cerevisiae

References

1. Wyman Charles E. “Ethanol from Lignocellulosic Biomass: Technology, Economics, and Opportunities”. Bioresource Technology1 (1994): 3-15.
2. Lee Jeewon. “Biological Conversion of Lignocellulosic Biomass to Ethanol”. Journal of Biotechnology1 (1997): 1-24.
3. Lange Jean-paul and Shell Global Solutions. “Lignocellulose Conversion: An Introduction to Chemistry”. (2007) 39-48.
4. Fan Li-Hai., et al. “Self-Surface Assembly of Cellulosomes with Two Miniscaffoldins on Saccharomyces Cerevisiae for Cellulosic Ethanol Production”. Proceedings of the National Academy of Sciences of the United States of America (2012): 1-6.
5. Nielsen Jens., et al. “Metabolic Engineering of Yeast for Production of Fuels and Chemicals”. Current Opinion in Biotechnology3 (2013): 398-404.
6. Chang Jui-Jen., et al. “PGASO: A Synthetic Biology Tool for Engineering a Cellulolytic Yeast”. Biotechnology for Biofuels1 (2012): 53.
7. Kricka William., et al. “Metabolic Engineering of Yeasts by Heterologous Enzyme Production for Degradation of Cellulose and Hemicellulose from Biomass: A Perspective”. Frontiers in Microbiology 5 (2014): 174.
8. Mba Medie., et al. “Genome Analyses Highlight the Different Biological Roles of Cellulases”. Nature Reviews. Microbiology3 (2012): 227-234.
9. Elkins James G., et al. “Engineered Microbial Systems for Enhanced Conversion of Lignocellulosic Biomass”. Current Opinion in Biotechnology5 (2010): 657-662.
10. Tosato Valentina., et al. “Non-Reciprocal Chromosomal Bridge-Induced Translocation (BIT) by Targeted DNA Integration in Yeast”. Chromosoma1 (2005): 15-27.
11. Nikitin Dmitri., et al. “Cellular and Molecular Effects of Nonreciprocal Chromosome Translocations in Saccharomyces Cerevisiae”. Proceedings of the National Academy of Sciences of the United States of America28 (20088): 9703-9708.
12. Beatrice Rossi., et al. “Different aneuploidies arise from the same bridge-induced chromosomal translocation event in Saccharomyces cerevisiae”. Genetics3 (2010): 775.
13. Arnak Remigiusz., et al. “Yeast Artificial Chromosomes”. (2012): 1-10.
14. Hoffma CS and F Winston. “A Ten-Minute DNA Preparation from Yeast Efficiently Releases Autonomous Plasmids for Transformation of Escherichia Coli”. Gene 57 (1987): 267-272.
15. Wach A., et al. “New Heterologous Modules for Classical or PCR-Based Gene Disruptions in Saccharomyces Cerevisiae”. Yeast 10 (1994): 1793-1808.
16. Sambrook J., et al. Molecular Cloning; a Laboratory Manual. (CSH). CSH (Ed) Molecular Cloning: A Laboratory Manual (1989): 9.
17. Ito H., et al. “Transformation of Intact Yeast Cells Treated with Alkali Cations”. Journal of Bacteriology1 (1983): 163-168.
18. Kaiser C., et al. “Preparation of Chromo- Some-Sized Yeast DNA Molecules in Solid Agarose”. CSH (Ed) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press (1994): 179-180.
19. Larsson S., et al. “Effect of Overexpression of Saccharomyces Cerevisiae Pad1p on the Resistance to Phenylacrylic Acids and Lignocellulose Hydrolysates under Aerobic and Oxygen-Limited Conditions”. Applied Microbiology and Biotechnology1 (2001): 167-174.
20. Lynd Lee R., et al. “Microbial Cellulose Utilization: Fundamentals and Biotechnology”. Microbiology and Molecular Biology Reviews: MMBR3 (2002): 506-577.
21. Romanos Michael A., et al. “Foreign Gene Expression in Yeast: A Review”. Yeast6 (1992): 423-488.

Citation

Citation: Burcin Altun., et al. “Strain Improvement of Saccharomyces cerevisiae by Bridge-induced Chromosome Translocation (BIT) and YAC Recombineering Technology". Acta Scientific Microbiology 3.9 (2020): 73-82.

Copyright

Copyright: © 2020 Burcin Altun., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.