JBEI team develops new synthetic system and approach for dynamic regulation of metabolic pathways to improve microbial production of fuels and chemicals production
Researchers with the US Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have developed a new synthetic system and approach that can significantly boost the engineered microbial production of renewable fuels or chemicals.
This new technique—dubbed a dynamic sensor-regulator system (DSRS)—uses a transcription factor that senses key intermediates and dynamically regulates the expression of genes involved in end-product production. In one demonstration, the DSRS substantially improved the stability of biodiesel-producing strains of E. coli, increasing the titer to 1.5 g/liter and the yield threefold to 28% of the theoretical maximum. The DSRS should also be applicable to the microbial production of other chemical products, both fatty acid-based and beyond.
The DSRS is an amazing and powerful new tool, the first example of a synthetic system that can dynamically regulate a metabolic pathway for improving production of fatty acid-based fuels and chemicals while the microbes are in the bioreactor.—Jay Keasling, CEO of JBEI, and leader of this research
Keasling, who also serves as the Associate Laboratory Director for Biosciences at Lawrence Berkeley National Laboratory (Berkeley Lab) is the corresponding author of a paper describing this research in Nature Biotechnology.
High productivities, titers and yields are essential for the microbial production of chemicals to be economically viable, particularly for low-value bulk chemicals and biofuels. However, product titers and yields are often limited by metabolic imbalances. Expression of pathway genes at too low a level creates bottlenecks in biosynthetic pathways, whereas expression at too high a level diverts cellular resources to the production of unnecessary RNAs, proteins or intermediate metabolites that might otherwise be devoted to the desired chemical. Furthermore, heterologous enzymes or pathway intermediates are sometimes toxic to the host. Overproduction of toxic enzymes or intermediates leads to growth retardation or adaptive responses that reduce yield and productivity, such as genetic modifications that remove or inactivate the pathway genes.
Several strategies have been developed to regulate gene expression, including engineering the strengths of promoters, intergenic regions and ribosome binding sites. These methods provide static control of the gene expression level. If a control system is tuned for a particular condition in the bioreactor and the conditions change, the control system will not be able to respond and suboptimal product synthesis will result. Ideally, the desired metabolic pathway would be dynamically regulated in response to the physiological state of the cell.—Zhang et al.
The challenge with building such a dynamic regulatory system, the authors note, is having a sensor that can measure key intermediates in the synthesis cascade and having cognate regulators that can control gene expression to improve production of the desired chemicals.
The team sought to develop a control system that would make the process of improving yields easier by dynamically balancing the enzymes responsible for producing the product. The researchers selected a biosynthetic strain of E. coli engineered to convert glucose into biodiesel in the form of fatty acid ethyl ester (FAEE) with a yield of 9.4% of the theoretical maximum, as the target.
The first step in engineering the DSRS was to develop biosensors for a key intermediate, which, in the FAEE biosynthetic pathway, is fatty acyl-CoA and, to a lesser extent, free fatty acids. They then developed a set of promoters (segments of DNA) that boost the expression of specific genes in response to cellular acyl-CoA levels. These synthetic promoters only become fully activated when both fatty acids and the inducer reagent known as “IPTG” are present.
For a tightly regulated metabolic pathway to maximize product yields, it is essential that leaky gene expressions from promoters be eliminated. Since our hybrid promoters are repressed until induced by IPTG, and the induction levels can be tuned automatically by the FA/acyl-CoA level, they can be readily used to regulate production of biodiesel and other fatty acid-based chemicals.—Fuzhong Zhang
Introducing the DSRS into the biodiesel-producing strain of E.coli improved the stability of this strain and tripled the yield of fuel, reaching 28% of the theoretical maximum. With further refinements of the technique, yields should go even higher, the team says.
We have demonstrated that one can construct a DSRS by assimilating two different natural ligand-responsive transcription factors and engineering regulators with different responses. Additionally, we demonstrated how one system could be practically used to regulate production of a desired chemical. Given these two examples and the numerous other naturally occurring sensors available, it should be possible to regulate nearly any biosynthetic pathway for which a natural sensor for the product or an intermediate exists or for which a sensor can be easily evolved. Furthermore, quorum sensors or carbon source sensors could be used to initialize the upstream genes to create a fully internally controlled pathway.
With the development of advanced techniques for biosensor design (such as computer-aided design of proteins or model-driven RNA device engineering), it should one day be possible to dynamically regulate any metabolic pathway, regardless of whether a natural sensor is available or not, and increase yields, titers and productivities to make microbial production of commodity chemicals and fuels economically viable.—Zhang et al.
This research was supported in part by the DOE Office of Science, and in part by the National Science Foundation through the Synthetic Biology Engineering Research Center (SynBERC).
JBEI is one of three Bioenergy Research Centers established by the DOE’s Office of Science in 2007. It is a scientific partnership led by Berkeley Lab and includes the Sandia National Laboratories, the University of California campuses of Berkeley and Davis, the Carnegie Institution for Science, and the Lawrence Livermore National Laboratory.
Fuzhong Zhang, James M Carothers, & Jay D Keasling (2012) Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature Biotechnology. doi: 10.1038/nbt.2149