Biofuels Production projects

Improving The Efficiency Of Xylose Consumption In S. Cerevisiae By Taking Both A Systems And Synthetic Biology Approach Towards Improving Overall Pathway Flux

In order to tap xylose, the major constituent of hemicellulose and an important step in sustainable biofuels, this project team is working to improve the efficiency of xylose catabolism in Saccharomyces cerevisiae.  Enzyme expression is being optimized by systematically screening a library of promoters driving all enzymes in the pathway predicted to influence pathway flux, and bottlenecks at the protein level are being addressed by designing synthetic scaffolds to co-localize enzymes for increased enzymatic efficiency and to limit intermediate loss.

project Highlights

2014 Highlights

Hemicellulose and pectin represent two polysaccharides that can be inexpensively and efficiently hydrolyzed enzymatically. The predominant monosaccharide of hemicellulose, xylose, can be utilized by S. cerevisiae provided a few heterologous and pentose pathway enzymes are expressed. We have taken measures to optimize expression, but a couple of the enzymes still appear to require further optimization. Additionally, we are integrating libraries into the chromosome. Pectin is a fairly abundant polysaccharide that is currently treated as a waste stream. Galacturonic acid is the predominant sugar in pectin. Many bacteria and filamentous fungi are capable of utilizing galacturonic acid. We are trying to import both of these pathways into S. cerevisiae, although we have experienced difficulty in achieving activity of one type of enzyme from both pathways, the dehydratase. Ongoing efforts will determine what factors are present in E. coli, but are lacking from S. cerevisiae, that allows for dehydratase function in S. cerevisiae.

2013 Highlights

In response to the need for better expression optimization tools for studying and optimizing metabolic pathways, such as xylose utilization, our lab has characterized a set of constitutive promoters and developed a rapid genotyping assay in Saccharomyces cerevisiae. As a challenging model of an anabolic pathway, we aimed to control product formation in the highly branched violacein biosynthesis pathway. Most anabolic pathways, including biofuel pathways, do not easily lend themselves to high-throughput screens or selections so determining the optimal expression combinations of even modest-sized pathways are extremely time-demanding and/or impractical.  We demonstrated a modeling approach that could be trained on a small, random percentage of the total library and used to predict the overall production landscape.  This allowed us to preferentially push flux to any of the four major products of the branched violacein pathway. The long-term goal is to optimize xylose utilization in Saccharomyces cerevisiae (baker’s yeast). By assembling promoter libraries driving each gene in the fungal xylose utilization pathway imported from the native xylose utilizing yeast Schefferomyces stipitis, including extra copies of the pentose phosphate pathway (PPP) and glycolytic enzyme PYK, expression optimization was conducted by employing selection for growth on xylose as the sole carbon source. An often-cited limitation to xylose fermentation with these fungal enzymes is the cofactor imbalance between the first two enzymes -- xylose reductase and xylitol dehydrogenase. One solution to this problem has been to engineer proteins with switched cofactor preference. Benefits from using these engineered enzymes have been mixed and it is unclear the extent to which altered cofactor preference vs. altered enzyme kinetics is affecting strain performance. Using combinatorial expression engineering, we seek to better understand the role of the pentose phosphate pathway in xylose utilization, understand the impact of oxygenation on optimal expression patterns for xylose-utilizing strains by comparing aerobic and anaerobic enrichments, and decipher the impact of switched cofactor preference during xylose fermentations.

2012 Highlights

Efficient xylose utilization in S. cerevisiae requires the introduction of a few enzymes to convert xylose into a metabolite the cell can recognize as well as optimizing the expression levels of each pathway enzyme. Although S. cerevisiae has several favorable characteristics as a production host, it has not evolved for xylose utilization. Thus, not only must expression level of the enzymes that must be introduced be optimized, but also the expression levels of endogenous enzymes contributing to this catabolic pathway that now assumes the increased flux of a central metabolic pathway. In optimizing these enzyme expressions, we have taken a combinatorial approach to balance functional activities and reduce intermediate accumulation, potential off-pathway flux, and metabolic loads. In 2012, the xylose utilization pathway including the pentose phosphate pathway were constructed where each enzyme expression level was varied over approximately three orders of magnitude and the dependency of pathway flux on expression levels of each component enzyme in both aerobic and anaerobic conditions was explored.

2011 Highlights

Dueber's group successfully constructed a combinatorial library for expression of the five-gene violacein biosynthetic pathway and optimization of titers and yields of violacein and/or off-pathway products.  They determined the upstream promoters driving each library gene and designed a strategy for the multimerization of scaffolded assemblies into large complexes in order to effectively concentrate pathway enzymes and intermediates.

2010 Highlights

Dueber’s team characterized a set of 12 constitutively active yeast promoters of various strengths and adapted the Gibson method for constructing multi-gene pathways with these promoter libraries. They also tagged the xylose catabolic pathway enzymes without loss of activity and put the scaffold under various strength promoters.

 

Publications

Published in 2013

Identification and Characterization of a Galacturonic Acid Transporter from Neurospora crassa and its Application for Saccharomyces cerevisiae Fermentation Processes, J. P. Benz, R. J. Protzko, J. M. Andrich, S. Bauer, J. E. Dueber, C . R. Somerville, Biotechnology for Biofuels, V. 7(1), pp. 20, doi:10.1186/1754-6834-7-20, Feburary 6, 2014. 

 

Employing a Combinatorial Expression Approach to Characterize Xylose Utilization in Saccharomyces cerevisiae, L. N. Latimer, M. E. Lee, D. Medina-Cleghorn, R. A. Kohnz, D. K. Nomura, J. E. Dueber, Metabolic Engineering, doi:10.1016/j.ymben.2014.06.002, 2014. 

Published in 2013

Expression-Level Optimization of a Multi-Enzyme Pathway in the Absence of a High-Throughput Assay, M. E. Lee, A. Aswani, A. S. Han, C. J. Tomlin, J. E. Dueber, Nucleic Acids Research, 41(22), pp. 10668-10678, doi: 10.1093/nar/gkt809, September 17, 2013.

Published in 2012

Engineering Robust Control of Two-Component System Phosphotransfer Using Modular Scaffolds, Weston R. Whitaker, Stephanie A. Davis, Adam P. Arkin, and John E. Dueber, Proceedings of the National Academy of Sciences, doi: : 10.1073/pnas.1209230109, October 15, 2012 online.

Published in 2011

Spatial Organization of Enzymes for Metabolic Engineering, Hanson Lee, William DeLoache, John Dueber, Metabolic Engineering, doi: 10.1016/j.ymben.2011.09.003, September 17, 2011.

Metabolic Pathway Flux Enhancement by Synthetic Protein Scaffolding, Weston Whitaker, John Dueber, Methods in Enzymology (Chapter 19), 497, pp. 447-468, doi: 10.1016/B978-0-12-385075-1.00019-6, May 19, 2011.

Xylitol Does Not Inhibit Fermentation by Engineered Saccharomyces cerevisiae Expressing xylA as Severely As It Inhibits Xylose Isomerase Reaction In Vitro, Suk-Jin Ha, Soo Rin Kim, Jin-Ho Choi, Myeong Soo Park, Yong-Su Jin, Applied Microbiology and Biotechnology, doi: 10.1007/s00253-011-3345-9, June 8, 2011.

 


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