Convincing microbial hosts to make new products

Environment and sustainability 1. nov 2021 3 min Professor and Group Leader Pablo Iván Nikel Written by Kristian Sjøgren

Researchers have found a way to get bacteria to produce compounds that they would not normally produce by making them dependent on the compounds to survive.

As cell factories, bacteria can be fantastic sources for making many types of biological molecules, and both researchers and industry have used bacteria to do this for many years.

However, bacteria do not normally make certain compounds: the ones they cannot use and that rarely occur in life forms. One example is fluorinated substances, which are used on a large scale in industry as pharmaceutical drugs and also to make fluoropolymers such as Teflon®.

Now, researchers have found a way to get bacteria to produce many of these compounds that they would not normally produce by making the bacteria dependent.

“We focus on how to get bacteria to produce many molecules that can only be produced today by traditional chemical processes that are often more expensive and generate various problematic byproducts. We therefore need new methods to make these products biologically, and we have worked on general ways to do this,” explains an author behind the new study, Pablo Iván Nikel, Senior Researcher and Group Leader at the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.

The research, published in Nature Communications, was a collaboration with the Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany and Wageningen University, Netherlands.

Inducing bacteria to produce the desired molecules

The challenge of persuading bacteria to produce compounds they do not use has always existed.

Researchers can easily insert the genes for producing a given organic molecule into bacteria, but if it does not have to use the organic molecules to survive, it rapidly blocks the process and these microbial hosts then produce very little (or none at all) in large industrial tanks.

The researchers took the manipulation of the bacteria’s genome a step further by making the bacteria depend on the organic molecules for their survival. Instead of blocking the biological process, the bacteria have to keep the process running for survival.

“Our study is a framework for how to make bacteria dependent on producing molecules they do not normally produce. We have implemented this strategy in my team for producing fluorinated compounds, but the principle applies to many biological molecules,” says Pablo Iván Nikel.

Making bacteria dependent

The background for the new method is that bacteria require 12 essential molecules to survive. The bacteria produce these molecules themselves and do not grow if they do not have access to these molecules.

The method starts by eliminating the activities that produce one or more of these 12 essential molecules.

“This very elegant approach, which has been used by other researchers before, was developed by the late Arren Bar-Even. The approach is a smart way of leveraging evolution to optimise cell factories,” explains Pablo Iván Nikel.

After the bacterial genome has been modified so essential molecules for growth are no longer produced, the pathway for production of the industrially useful molecule needs to be inserted in the bacterium. This synthetic pathway is designed such that it produces both the essential molecule and the compound of interest. Then, the bacteria are forced to use the synthetic pathway that provides access to the essential molecules, and they are thereby also forced to produce the industrially useful products.

“You could say that we have tricked the bacteria into becoming dependent on producing the compound we want them to make. Another perspective is that we can evolve the bacteria so that by getting better and better at producing a compound that benefits their growth, they also produce more and more of the compound that we want them to make,” says Pablo Iván Nikel.

Pablo Iván Nikel also explains that the method can be applied to produce specific molecules but also to assimilate substrates that microorganisms do not use.

“For example, you can make bacteria dependent on assimilating compounds that they would normally never absorb. They can thereby use a completely new substrate in the biological production of specific molecules,” he says.

Industrial quantum leap

According to Pablo Iván Nikel, this method has considerable perspectives within biotechnology.

One perspective in biotechnology is that it will make it easier to engineer bacteria that may be major producers of organic molecules that are industrially useful. Industry is interested in programming bacteria to produce specific molecules and how to get bacteria to produce the most. Traditionally, many variants of bacteria with the capacity to produce useful biological molecule have to be created and tested. The trick is to discover which of these bacteria are best at producing the molecule and then to develop them into a culture that can be used industrially.

In the past, this process involved considerable analysis of production rates (which involves costly and time-consuming analytical methods), but the new method makes identification much easier.

“Traditionally, many genetic variants are screened, but here we just have to find the bacteria that grow most rapidly. Since the production of the useful molecules is directly linked to the growth of the bacteria, the bacteria that grow most rapidly also produce the most of these molecules. Quantifying cell growth rather than the product will accelerate the development of new bacterial strains for industrial production enormously,” concludes Pablo Iván Nikel.

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