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Many of today’s pharmaceuticals, biofuels, and other chemicals are produced by microbial fermentation. But the churning metabolism of bacteria in a bioreactor raises the cells’ temperature, which in turn causes heat stress that hobbles production efficiency. Now, researchers have devised a gene network that boosts the efficiency of Escherichia coli, a workhorse of industrial fermentation, at high temperatures (ACS Synth. Biol. 2016; DOI: 10.1021/acssynbio.5b00158).
Three years ago, biochemical engineer Chun Li and his group at the Beijing Institute of Technology set out to make E. coli cells more thermotolerant. Their goal was to reduce the amount of electricity used and waste water generated in keeping the temperature inside bioreactors at 37 °C, the microbe’s optimal growth temperature. Industry experts told Li that lowering the mercury by even a few degrees would save considerable cost. “Our aim was to make a gene circuit that allows bacteria or yeast to grow well at high temperatures,” Li says.
Previously, researchers have tried to engineer thermotolerance by introducing so-called heat-shock proteins (HSPs), taken from microorganisms that grow well at higher temperatures, into E. coli to protect the cells from heat stress. But overexpressing HSPs alone can negatively affect cell health by shutting down aspects of cell metabolism.
Li and colleagues therefore created a more nuanced way to expand the temperature range at which E. coli could thrive. They strung together the genes for two HSPs that conferred heat resistance: one at 40-43 °C and one at 43-46 °C. They then linked each gene to DNA encoding a special RNA sequence called an RNA thermometer, which allowed the genes’ expression only at a specific temperature.
As microbes grow vigorously, their cell density increases and becomes a big source of heat stress. So the researchers also designed a mechanism to limit bacterial density. They coupled the temperature-sensing arm of their system to a set of bacterial genes that is activated when cell density exceeds a threshold—a cellular phenomenon known as quorum-sensing—and linked those, in turn, to genes that caused programmed cell death. High temperature would thus trigger a mechanism that decreased cell density to a level that kept the heat stress burden in check.
The E. coli strain engineered with this system, which the researchers call an intelligent microbial heat regulating engine, or IMHeRE, grew better than a control strain of unmodified E. coli at 43 °C, and held its cell density steady. The experimental strain also more quickly re-adapted to growth at 37 °C.
Finally, the researchers tested the engineered microbe’s ability to produce an industrially relevant fermentation product. To their surprise, at 40 °C, E. coli engineered with IMHeRE showed a five-fold increase in the production of lysine, an amino acid added to animal feed to boost growth, compared to control E. coli at any temperature. With the circuit, “high temperature is no longer a stress but can accelerate production,” the researchers write.
Li says that pilot testing under industrial conditions, conducted after the current study was submitted, showed the system works well in a 150-L fermenter. Next, his team is tackling another challenge: Low pH boosts productivity of many fermentation cultures but is a cell stressor. Therefore, they are developing a system that increases cell tolerance of acidity.
“There’s a clear hope in the field that we can start working towards smart organisms that can respond to—and even anticipate—changes they experience in culture,” says James M. Carothers, a chemical engineer at the University of Washington. “What’s interesting about this approach is that they tied a bunch of things together into an integrated system.” But he notes that E. coli is well-adapted to produce lysine; other products might not show the same yield improvements with this system.
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