Live, but don’t die or repeat

With various gene editing techniques now available, scientists have gotten pretty good at developing modified microbes able to produce a whole range of useful chemical compounds, including biofuels and industrial chemicals. What is proving trickier, however, is getting these modified microbes to produce the compounds at high enough yields for commercial production.

One problem is that the compounds, as well as some of the by-products generated during their production, can be toxic to the modified microbes at high enough concentrations, inhibiting their activity and limiting yields. Another problem is that the microbes often only produce the chemicals at a certain point during their lifecycle, usually when they’re not doing other important activities such as reproducing, which also obviously limits yields.

Up to now, scientists have tended to focus on the first of these problems, by trying to develop strains of the modified microbes that are better able to withstand the toxicity of the compounds. This is often done through adaptive evolution, by growing the microbes in a solution containing a high concentration of the toxic compounds of interest, picking out the best growing strains and then transferring them to a solution containing a slightly higher concentration of the compounds. Do this multiple times and the result should be a strain better able to withstand high concentrations.

In a recent example of this approach, a team of scientists in the US, led by Aiqin Shi at the University of Florida, Gainesville, exposed a strain of Escherichia coli able to produce ethanol by fermentation to the hydrolysate produced when plant biomass is broken down by a dilute acid solution. Although this dilute acid treatment is very effective at releasing cellulose from the biomass for subsequent enzymatic conversion to glucose, it tends to produce a hydrolysate containing a load of toxic compounds, such as organic acids, furan derivatives, and phenolic compounds, that inhibit the activity of the fermenting microbes.

As reported in a paper in Biotechnology and Bioengineering, Shi and his team exposed 500 generations of the E. coli strain to increasing concentrations of the dilute acid hydrolysate. This resulted in the creation of a new strain that could withstand the hydrolysate at concentrations of over 50%, whereas the initial strain was inhibited by concentrations of 40%.

The researchers conducted a genetic analysis of this new strain, which revealed that its increased robustness was mainly caused by the loss of just two genes. When they deleted either of these genes in the initial E. coli strain, they found that it became just as good at withstanding high concentrations of the hydrolysate. This suggests that the genes work in different ways, while also offering a faster way to enhance the robustness of fermenting microbes than adaptive evolution.

That still doesn’t solve the other problem, but this is what a team of Chinese scientists led by Liming Liu at Jiangnan University in Wuxi decided to focus on. Again, they were working with a strain of E. coli, but this strain had been modified to produce two related chemical products: the bioplastic poly(lactate-co-3-hydroxybutyrate) (PLH) and butyrate.

To get the strain to produce these two products for as long as possible, Liu and his team attempted to increase its lifespan while also reduce the time it spent reproducing. They did this by using a gene editing technique known as recombinase-based state machine to delete two genes and overexpress a third. One of the genes coded for a carbon storage regulator known to be important for replication, while the other two genes helped to control lifespan.

As the scientists report in a paper in Nature Catalysis, the new strain did live for longer and replicate less. This led it to build up more PLH in its cytoplasm, accounting for over 50% of the contents of the cell, and produce more butyrate, with a yield of almost 30g per liter. In addition, this longer-lasting strain also grew to be 13 times larger than the initial strain, making it more robust and providing even more room to accumulate PLH. 

The views represented here are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd. or of the SCI.