Projects

We are studying the production of fatty acid derived oleochemicals in the model bacterium Escherichia coli, photosynthetic cyanobacteria, and the oleaginous yeast Yarrowia lipolytica. Our work has combined traditional metabolic engineering, synthetic biology, systems biology, physiological characterization, functional genomics, and metabolic modeling approaches. Our goal is to understand the barriers to producing fatty acid derivatives at titers that approach the theoretical yield from renewable resources such as biomass sugars or electrochemically generated C2 compounds.

We target the production of fatty acid derived products for two reasons. First, fatty acids are found in nearly all forms of life. Therefore, if a superior organism capable of breaking down biomass, consuming alternative carbon sources, or tolerating final products is isolated, then we can transplant our findings into these organisms. Second, fatty acids can be converted to a wide array of commercial compounds including oils that could be used as lubricants, polyesters that can be used as structural materials, fatty alcohols that are useful as surfactants, esters that are today’s biodiesel, alkanes that are found in today’s petrodiesel, and olefins that could be used as fuels or chemical building blocks. In addition, fatty acid derivatives could be chemically refined alongside petroleum and integrate into today’s chemical industry.

Please see one of our reviews on the topic for further information.

• Yan Q, Pfleger BF. Revisiting metabolic engineering strategies for microbial synthesis of oleochemicals Metabolic Engineering. (2020) DOI: 1016/j.ymben.2019.04.009
• Mehrer CR, Hernandez-Loazada NJ, Lai RY, Pfleger BF. Production of Fatty Acids and Derivatives by Metabolic Engineering of Bacteria. Handbook of Hydrocarbon and Lipid Microbiology Series. Consequences of Microbial Interactions with Hydrocarbons, Oils and Lipids: Production of Fuels and Chemicals. Springer Nature, Cham, Switzerland. S.Y. Lee (ed.), (2018). DOI: 1007/978-3-319-31421-1_385-1
• Pfleger BF, Gossing M, Nielsen J. Metabolic engineering strategies for microbial synthesis of oleochemicals. Metabolic Engineering. May;29:1-11 (2015). DOI: 1016/j.ymben.2015.01.009
• Lennen RM, Pfleger BF. Microbial production of fatty acid derived fuels and chemicals. Current Opinion in Biotechnology Dec;24(6):1044-53. (2013). DOI: 1016/j.copbio.2013.02.028
• Lennen RM, Pfleger BF. Engineering Escherichia coli to synthesize free fatty acids. Trends in Biotechnology Dec;30(12):659-67 (2012). DOI: 1016/j.tibtech.2012.09.006

Current work is exploring alternative substrates, overcoming oleochemical toxicity, and reducing the maintenance demand required during oleochemical production.

Cyanobacteria are one of the most important organisms on earth. As the original photosynthetic organism, they are likely responsible for the Great Oxidation Event, allowing for the majority of life as we know it to evolve.  Cyanobacteria are highly adaptable and can be found in almost every environment, from the tropics to the Antarctic, lakes and oceans to deserts. Some cyanobacteria, including the strain used in the Pfleger lab, Synechococcus sp. strain PCC 7002, are readily transformable and capable of rapid growth.

The goal of our research in the Pfleger lab is to create fuels and chemicals directly through photosynthesis without the need for agricultural land or potable water. To accomplish this we are studying the physiology of these bacteria, creating new genetic tools to control gene expression with the goal of applying what we have learned to produce desirable molecules.

Natural products are a common chemical scaffold for building drugs capable of fighting infections and other diseases. As resistance to common antibiotics and other drugs increases, society is in dire need of additional weapons to fight infectious disease and cancer. We are interested in developing tools to produce natural products in industrially relevant quantities. We are therefore conducting studies aimed at engineering enzymes to produce new natural products, at engineering cells as a chassis for microbial synthesis, and at developing a deeper understanding of the factors that regulate flux to these complex molecules.

The manipulation of gene expression is essential for virtually every biochemical engineering project. As such, we are interested in developing tools to enable precise control over both native and heterologous gene expression while pursuing basic research to better understand the factors controlling gene product abundance. The Pfleger Lab is interested in studying gene expression as it relates to the elements that are important to consider when designing synthetic genetic systems as well as how microorganisms orchestrate optimal expression of native genes. The regulatory processes responsible for adjusting the amount of a gene product under a given set of environmental conditions can occur at the levels of transcription, translation, and/or gene product stability (i.e. mRNA/protein stability). Specifically, we are interested in a novel class of trans-regulatory elements called transcription activator-like effectors (TALEs). These proteins were originally discovered as virulence effectors native to plant pathogenic bacteria and are of significant importance to engineering gene expression, as they may be reprogrammed to bind any desired sequence of DNA. We have shown that TALEs can be used to efficiently repress prokaryotic genes of interest in a targeted fashion² and have developed a TALE induction system.  The unique specificity of these proteins presents an unparalleled opportunity to add layers of synthetic genetic regulation over top of endogenous regulatory mechanisms, preserving the function of native systems while simultaneously achieving engineering objectives.

For precise and predictable control of protein expression it would be ideal to simultaneously regulate multiple processes. One powerful, yet relatively unexplored mechanism is regulating gene expression at the level of mRNA stability. This is a complicated task due to the numerous enzymes that participate in mRNA degradation each with different affinities for certain elements of each transcript.⁴ By examining the specificities of ribonucleases (RNases) from a systems-level perspective in both the model organism Escherichia coli and the cyanobacterium Synechococcus PCC 7002 we hope to formulate the theoretical foundations for engineering transcript stability.

• Copeland MF, Politz MC, Johnson CB, Markley AL, Pfleger BF. A transcription activator-like effector induction system mediated by proteolytic degradation. Nature Chemical Biology. Apr;12(4):254-60 (2016). DOI: 1038/nchembio.2021
• Copeland MF, Politz MC, Pfleger BF. Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes. Current Opinion in Biotechnology. Mar 12;29C:46-54 (2014). DOI: 1016/j.copbio.2014.02.010
• Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger BF. CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus strain PCC 7002. Metabolic Engineering. Jul 29. pii: S1096-7176(16)30062-3 (2016) DOI: 10.1016/j.ymben.2016.07.007
• Gordon GC, Cameron JC, Pfleger BF. RNA-sequencing identifies new RNase III cleavage sites in Escherichia coli and reveals increased regulation of mRNA. mBio. Mar 28;8(2). pii: e00128-17. (2017) DOI:1128/mBio.00128-17
• Gordon GC, Cameron JC, Gupta STP, Engstrom MD, Reed JL, Pfleger BF. Genome-wide analysis of RNA decay in the cyanobacterium Synechococcus sp. strain PCC7002. mSystems. (2020) 5(4) DOI: 1128/mSystems.00224-20

The rhizosphere is the soil environment immediately adjacent to plant roots. It is home to a diverse community of microorganisms that can impact crop traits such as growth rate, drought tolerance, disease avoidance, and nutrient demand. The microbial community and the plant depend on each other and communicate through poorly understood chemical mechanisms. These interactions and the resulting benefits to crop yield are attractive opportunities for applying synthetic biology. We have recently entered this space by developing a synthetic biology toolbox for a promising diazotroph (nitrogen fixing bacterium) and initiated collaborations with experts on the UW-Madison campus.

• Chakraborty S, Venkataraman M, Infante V, Pfleger BF, Ané JM. Scripting a new dialogue between diazotrophs and crops. Trends in Microbiology. Accepted. E-pub ahead of print (2023) DOI: 1016/j.tim.2023.08.007
• Venkataraman M, Ynigez-Gutierrez A, Fernandes PI, Gray VI, Ané JM, Pfleger BF. A synthetic biology toolbox for manipulating bacteria in the rhizosphere. E-pub ahead of print. (2023) DOI: 1021/acssynbio.3c00414