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. 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.
We are studying the production of fatty acid derived oleochemicals in the model bacterium . 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.
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.
To understand the fundamental barriers to producing fatty acid derivatives, we have studied the biosynthesis of free fatty acids in the model bacterium . The general strategy for producing free fatty acids in E. coli is threefold:
1. Block the consumption of fatty acids and hydrocarbons by disrupting beta-oxidation, thereby providing a "SINK" for carbon flux.
2. Overexpress an acyl-ACP thioesterase to deplete the key regulatory signal, long-chain acyl-ACP, and provide a metabolic path to free fatty acids that by-passes phospholipid synthesis. This step provides a "PULL" of carbon flux into fatty acid biosynthesis.
3. Overexpress rate limiting steps or enzymes that catalyze reactions whose total activity is too low. This step provides a "PUSH" of carbon flux into fatty acid biosynthesis.
To further increase the yield of free fatty acids, we have applied the following approaches:
· Designed genetic modification (Lennen et al., 2010)
· Random mutagenesis and high throughput screening (Hoover et al.,, 2012)
· Balancing expression of key enzymes (Lennen et al., 2010)
· Functional genomics analysis of free fatty acid producing cells in comparison to engineered controls (Lennen et al., 2011)
· Metabolic and kinetic modeling of our cells (Youngquist et al., 2012)
· Exploring nutrient limitation conditions (Younquist et al., 2013)
We are currently expanding these approaches as we pursue strains that approach the theoretical yield on renewable resources. This work is supported by the National Science Foundation and The Dow Chemical Company.
Engineering Gene Expression for Biochemical Engineering
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.1 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.3 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.
3. Copeland, M.F., Politz, M.C., Johnson, C.B., Markley, A.L. & Pfleger, B.F. A transcription activator-like effector (TALE) induction system mediated by proteolysis. Nat Chem Biol advance online publication (2016).