Peak performance through a molecular diet

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To reach their full potential, the bacterial strains are genetically altered in the laboratory. This enables ab increase in the bacteria’s production rates of proteins and enzymes or a minimisation of unwanted by-products. Source: Stern-Enzym GmbH

14.03.2012  - 

Trimming bacteria for use in biotechnological production is a tricky business. Sometimes the microbes produce the desired enzyme, and sometimes they don’t. The reasons for this often recalcitrant behaviour usually remains a mystery to the researchers. In the cooperative project P27, part of the work of the Biocatalysis2021 Cluster funded by the Federal Ministry for Education and Research (BMBF), researchers are working on optimising the production performance of a commercially extremely interesting bacterium. In the spotlight here is Bacillus subtilis, the grass bacillus. The bacterium is currently being prepped for the production of proteins and enzymes that are greatly desirable for the food and pharmaceutical industries.

The researchers in the Cluster Biocatalysis2021– one of five networks in the BMBF funding initiative BioIndustrie 2021 – are occupied with projects relating to industrial biotechnology. These include the P27 ¬project, which also has the title ‘New Bacillus expression systems.’ The BMBF is funding the project with a total of 760,000 euros over a period of three years.

The domestication of the grass bacillus

A major hurdle in the production of enzymes and proteins today is the provision of suitable expression systems such as bacteria or yeasts. Only the best production strains can produce error-free amino acid chains and fold them into the proper three-dimensional structure, thereby producing the desired protein on a large scale.

“Our consortium wants to convert the well characterised wild-type bacterium Bacillus subtilis 6051a into a modern production strain. We want throw overboard the unnecessary genetic ballast of the bacterium – this only prevents the bacteria from carrying out its work efficiently,” says Lutz Popper, coordinator of the P27 project and head of R&D at SternEnzym GmbH & Co. KG based in Ahrensburg. Good quality production strains of the grass bacillus do exist, but these are the property of large companies and corporate groups. Moreover, the freely available laboratory strains are not suitable for large-scale production. This is now set to change thanks to the work of the P27 consortium. The Ernst Moritz Arndt University in Greifswald is responsible for the molecular biological aspects of the project. The internationally active company Miltenyi Biotech GmbH in Teterow is providing the expression systems and is also responsible for the fermentation technology. The third component is SternEnzym. There, scientists are testing the produced enzymes for their functionality in the area of food production.

High-throughput screening helps to speed up the development process.Source: Stern-Enzym GmbH

The obstacles and successes of scientific work

Two researchers from the team headed by Thomas Schweder, Professor of Pharmaceutical Biotechnology at the University of Greifswald, are in charge of genetic optimisation. Their task is to remove all unnecessary genetic sequences from the grass bacillus, and to create a form of minimal bacterium that is designed to do only one thing: synthesise desirable enzymes for industrial application. This microorganism is not even able to move itself forward. The ability has been unlearned; it is deemed to cost too much energy and is not needed for the production of proteins. “Of course, removing the genetic material was not quite as easy as it sounds,” recalls the coordinator. “But after a few setbacks we were ultimately able to prove the feasibility of our concept.” The optimised design of the production strain has already produced a variety of proteins such as a xylanase and an amylase. The consortium has now set itself the goal of upscaling its protein production. “We are currently working with smaller fermenters of up to ten litres, but we should be seeing production on a larger scale by the end of the year,” says the 52-year-old food technologist Popper.
 

In the meantime, the production of a brewers’ yeast-derived enzyme called sulfhydryl oxidase is causing the scientists to scratch their heads. “The yeast is infinitely lazy when it comes to the production of this enzyme,” discloses Popper. Because it is known as a very safe production organism due to the absence of undesirable metabolic products, the P27 grass bacillus appears well suited to its task. In reality, however, it is somewhat stubborn in this system: The protein is indeed produced, but it is not active.

The possible causes are being investigated, and Lutz Popper is confident that the P27 team ¬will get to the bottom of this issue.The long and rocky road to commercial applicationThe P27 project partners have succeeded in designing a microbe that is stable and which can uniformly convert raw materials into the desired substance, all whilst remaining positively tight-fisted with its energy reserves. The commercial utilisation of some of the synthesised proteins is being pursued by project partners SternEnzym GmbH and Miltenyi, and a patent application is in the works. Moreover, it is planned to use this optimised strain to produce other enzymes of all possible stripes.

Author: Andrea van Bergen