Engineered, Evolved, and Re-Engineered Yeast for Producing Cellulosic Biofuels

While there are anticipated benefits from using cellulosic biomass as a feedstock to produce biofuels and chemicals, commercial production of cellulosic biofuel and chemicals has been hampered by technical difficulties, such as inefficient fermentation of cellulosic sugars, and the toxicity of acetic acid which are abundant in plant cell wall hydrolysates. As an effort to overcome the problems, we constructed a highly engineered Saccharomyces cerevisiae strain capable of converting mixed carbon components (cellobiose, xylose, and acetate) into ethanol simultaneously.

As S. cerevisiae cannot ferment xylose, we first constructed a rapid and efficient xylose fermenting S. cerevisiae (SR8) through a rational design that allows strong and balanced expression levels of the xylose metabolic genes (XYL1, XYL2, and XYL3), and evolution of the engineered strain for rapid xylose fermentation (1). Through systems-level characterization of the evolved strain, we learned that deletion of PHO13 is a beneficial genetic perturbation eliciting up-regulation of the pentose phosphate pathway (2). Second, we introduced a cellobiose metabolic pathway into the efficient xylose fermenting strain (SR8) for co-fermentation of cellobiose and xylose (3). Again, a rational design for integrating genes (cdt-1 and gh1-1) coding for cellobiose transporter and intracellular β-glucosidase, and evolution of the engineered strain were combined to isolate an engineered strain (EJ4) co-fermenting cellobiose and xylose rapidly. After measuring the copy numbers of cdt-1 and gh1-1 in the evolved strain, we learned that cellobiose fermentation can be improved by amplification of cdt-1 and gh1-1. Finally, we introduced an acetate reduction pathway, which can enhance xylose fermentation through redox coupling (4), into the xylose and cellobiose co-fermenting strain (EJ4) for efficient co-utilization of cellobiose, xylose, and acetate. The resulting strain was able to produce ethanol from a mixture of cellobiose, xylose and acetate with a substantially higher yield and productivity than the control strains, demonstrating the synergistic effects from integration of multiple metabolic pathways (5).

The Design, Build, Test and Learn (DBTL) cycle has been formulated as a standard practice of synthetic biology to construct optimal microbial strains with desired traits. Our strain improvement efforts exemplifies the DBTL cycle.

References

1. Kim, S. R., Skerker, J. M., Kang, W., Lesmana, A., Wei, N., Arkin, A. P., and Jin, Y. S. (2013) PloS ONE 8, e57048

2. Kim, S.R., Xu, H., Lesmana, A., Kuzmanovic, U., Au, M., Florencia, C., Oh, E.J., Zhang, G.C., Kim, K. H., Jin, Y.S. (2015). Applied and Enviromental Microbiology 81,1601-1609

3. Ha, S. J., Galazka, J. M., Kim, S. R., Choi, J. H., Yang, X., Seo, J. H., Glass, N. L., Cate, J. H., and Jin, Y. S. (2011). Proceedings of the National Academy of Sciences of the United States of America 108, 504-509

4. Wei, N., Quarterman, J., Kim, S. R., Cate, J. H., and Jin, Y. S. (2013). Nature Communications 4: 2805

5. Wei, N., Oh, E.J., Million, G., Cate, J.H., Jin, Y.S (2015). ACS Synthetic Biology 4, 707-13