Biofuels Production programs
Building Pathways for Biodiesel Production in E. coli and S. cerevisiae
We have focused on the construction of synthetic pathways for production of biodiesel in E. coli and S. cerevisiae. In S. cerevisiae, we have worked toward developing approaches to engineering the production of fuels made from acetyl-CoA, using a C4 product as a model system.
Goal 1 of our program is to construct pathways for iterative chain elongation of hydrocarbons that is orthogonal to canonical fatty acid biosynthesis that does not require the use of ATP. We have developed a thiolase-dependent strategy that does not require malonyl-CoA or ATP. When combined with an octanoyl-CoA specific thioesterase, our synthetic pathway produced 850 mg/L octanoic acid in a non-engineered E. coli strain. The productivity has increased 8.5-fold in 2014 (100 mg/L). We have also started to explore the production of tailoring these acids to form the corresponding alcohols. Goal 2 is to develop a framework for understanding heterologous gene expression in S. cerevisiae. We plan to focus on elucidating the molecular mechanisms that lead to high translational efficiency as well as the pathways by which poorly expressed transcripts are derailed. Our strategy is to quantify the behavior of highly-expressed native yeast transcripts as compared to non-native transcripts and to begin identifying factors in both the coding and non-coding regions of the transcript that affect the efficiency of various steps in mRNA processing, transport, and translation.
Our group has focused on the construction of synthetic pathways for the production of hydrocarbons in the biodiesel range in S. cerevisiae and E. coli. Towards these efforts, we have initiated a program aimed at the discovery of non-canonical enzymes for the production of these targets using a variety of different approaches. Using bioinformatics coupled to biochemical studies, we have identified new gene clusters encoding enzymes capable of carrying out ATP-independent chain extension reactions with acetyl-CoA and used these clusters as a basis for engineering pathways in E. coli. In addition, we are carrying out the biochemical and structural characterization of new enzymes from branched-chain acid fermenting hosts that are capable of utilizing acyl-CoA substrates beyond acetyl-CoA. In a third line of research, we are studying anaerobic fatty acid biosynthesis in the protist, Euglena gracilis, in an effort identify enzymes that enable production under these conditions. In another distinct line of research, we are using a model pathway constructed in E. coli as a starting point for optimizing synthetic pathways for longer chain products derived from acetyl-CoA using S. cerevisiae as a host. In this area, we have been addressing two key roadblocks in this goal, which are the low heterologous gene expression and low acetyl-CoA availability in yeast.
In the course of these studies, we have improved production to 300 mg/L, which is approximately a 30-fold improvement over literature values. Characterization of transcript and enzyme levels appears to implicate low heterologous protein production compared to E. coli as the main limitation in product titers. We are continuing to work towards improving pathway expression as well as engineering improved acetyl-CoA generation pathways for S. cerevisiae.
In our work in new pathway construction, we have worked toward identifying new fatty acid pathways that would be interesting targets for engineering the production of biodiesels. We have utilized both bioinformatics and physiological studies for these efforts. Using bioinformatics approaches, we have found interesting classes of enzymes that can be used for carbon chain extension reactions. These enzymes have been cloned, heterologously expressed, and purified. We have biochemically characterized ~10 homologs from which we have identified a few candidates that may be useful for constructing biodiesel pathways. With regard to physiology, we have been studying the protist, Euglena gracilis, because of its reported five systems for fatty acid biosynthesis including a wax fermentation pathway. We have used RNAseq experiments to begin characterizing and identifying the key components for these pathways. Several of these candidates have also been cloned, heterologously expressed, and purified for further biochemical characterization.
Published in 2013
Production of Advanced Biofuels in Engineered E. coli, M. Wen, B. B. Bond-Watts, M. C. Chang, Current Opinion in Chemical Biology, doi: 10.1016/j.cbpa.2013.03.034, May 6, 2013.
Exploring Bacterial Lignin Degradation, Margaret E. Brown, Michelle C.Y. Chang, Current Opinion in Chemical Biology, doi: 10.1016/j.cbpa.2013.11.015, December 2013.