When bacteria have something against bacteria
Genetically engineered soil bacteria produce alternative to antibiotics

Ulm University

A research team involving the Ulm Institute of Microbiology and Biotechnology has been successful in producing antimicrobial agents in pure form with the help of genetically modified soil bacteria (Corynebacterium glutamicum). The bacteriocins produced by this method could prove to be useful as an alternative to antibiotics for fighting against bacterial pathogens. A further use for the antibacterial peptides could be in food preservation.

Bacteria strains like to be on their own. In order to keep unwelcome food competitors away, they produce antimicrobial substances to prevent other bacteria strains from spreading out in their environment. These so-called bacteriocins are already being used today in the food industry as a food preservative. Bacteriocins also have enormous potential in the field of medicine. In light of increasing antibiotic resistance, they are considered promising alternatives for treating infections caused by human pathogenic bacteria.

“Clinical application of such bacteriocins requires novel, large-scale methods that would make it possible to substantially improve the efficiency of production and the purity of the substance”, explains Professor Christian Riedel from the Institute of Microbiology and Biotechnology at Ulm University. In a study for the journal “Metabolic Engineering”, the microbiologist demonstrated how this is possible from a biotechnological perspective.

Up to now, bacteriocins have been produced exclusively with natural bacteria in complex fermentation processes employing complex and expensive growth media. At best, this produces semi-purified preparations or raw ferments. For medical purposes, such as a substitute for antibiotics, the bacteriocins from these “natural” fermentation procedures need to be purified in a highly complicated procedure, which is costly and therefore not very attractive. Now Christian Riedel and his team from Ulm, along with other researchers from Germany, Norway, Denmark and Austria, have succeeded in genetically modifying the bacterium Corynebacterium glutamicum in such a way that a highly effective antimicrobial peptide (Pediocin PA-1) is produced in its pure form. The bacterium used as the production host is a non-pathogenic soil bacterium that has long been known as a natural producer of amino acids – for example the flavour enhancer glutamate – and which today plays an important role as a biotechnological platform organism.

The genetically modified bacteria produce a bacteriocin against listeria

The team of researchers has equipped the bacterium with synthetic, precisely functionalised genes that manage the production of the bacteriocin. Pediocin PA-1 is especially effective against Listeria monocytogenes, bacteria that are widely spread in the environment. If they are ingested, however - for instance via contaminated food such as raw cheese - they can cause dangerous, and sometimes even fatal, listeriosis in human beings.

The scientists needed to overcome many challenges in order to successfully complete their project, which was published in the journal Metabolic Engineering. The biggest challenge was figuring out how to coax the bacteria to produce antimicrobial substances that could potentially be toxic for the producers. Why is it that the Pediocin PA-1 synthesised by C. glutamicum does not have any harmful effects on the soil bacteria? Riedel’s research team made use of one of the microbe’s unique biological traits. “Cornybacterium glutamicum does not have any receptors that bacteriocins can dock onto. It is therefore resistant to their antibacterial effect. Lucky for us!” explains Dr Oliver Goldbeck. The postdoctoral student is a research associate at the Institute of Microbiology and Biotechnology and first author of the study. The team was also able to scale up the synthetic bacteriocin production from laboratory scale to industrial large-scale pilot production.

Artificial genes make the bacteria less demanding - they consume nutrients from waste wood

A third aim of the project was to make the underlying fermenting technology more affordable and more efficient in terms of resource use. “Instead of using expensive growth media, we use waste products from the wood industry as a substrate for our production,” explains Riedel. In order to achieve this, the cooperation partners in Professor Christoph Wittmann ‘s (University of Saarland) team made further genetic modifications to the bacterial production host. “This makes it possible for our bacteria to process sugars and organic acids from the waste wood in order to ultimately produce the antimicrobial peptides,” the researcher explains. The scientists have thus not only been successful in getting “useful” bacteria to work for them in order to produce agents against “harmful” bacteria. They have also succeeded in making their beneficial organisms less demanding, meaning that feeding them will be less costly and more environmentally friendly in the future.

This project is a part of the international research network called “iFermenter”, which is coordinated by the Norwegian University of Science and Technology and is receiving funding from the EU within the framework of Horizon2020 in the amount of around 5.25 million euros. The focus of the joint project is on developing an intelligent biotechnological process that makes it possible to synthesise antimicrobial proteins from waste products in the wood industry. The objective is to feed sugar-containing waste material into a value-added process and thus simultaneously reduce the cost and increase the resource efficiency of biotechnological production processes. Ulm University is involved in “iFermenter” with sub-projects amounting to 452,000 euros.

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum
OliverGoldbeck, Dominique N.Desef, Kirill V.Ovchinnikov, Fernando Perez-Garcia, Jens Christmann, Peter Sinner, Peter Crauwels, Dominik Weixler, Peng Cao, Judith Becker, Michael Kohlstedt, Julian Kager, Bernhard J. Eikmanns, Gerd M. Seibold, Christoph Herwig, Christoph Wittmann, Nadav S.Bar, Dzung B.Diep und Christian U. Riedel in: Metabolic Engineering. 2021 Sep 4; 68:34-45.
doi: 10.1016/j.ymben.2021.09.002. Online ahead of print.


Group Bacteriocin-Study
The researchers involved in this project: Oliver Goldbeck, Christian U Riedel, Dominique N Desef and Bernhard J Eikmanns (from left). Missing: Dominik Weixler (Photo: Lisanne Wolters / Uni Ulm)
Fluorescent sensor bacteria
Fluorescent sensor bacteria help the researchers to detect antimicrobial peptides such as bacteriocins. The fluorescent microscopic images show individual bacteria (A) and cultures on agar plates (B and C). (A) Upon contact with the bacteriocin, the sensor bacteria glow blue-green around the edges. (Image: Christian Riedel / Uni Ulm)
Fluorescent sensor bacteria
(B) Around the white colonies of a bacteriocin producer, the inhibiting areolas are easily visible among the fluorescent sensor bacteria. (C) Dependent on the degree of dilution (increasing from right to left) and the bacteriocin concentration (increasing from bottom to top), the growth of the sensor bacteria is inhibited (Image: Christian Riedel / Uni Ulm)
Explanatory graphic on the recombinant production of bacteriocins
Explanatory graphic on the recombinant production of bacteriocins with Corynebacterium glutamicum (Image: Christian Riedel Uni Ulm)