NMR spectroscopy can aid in the battle against microorganisms by predicting their next move

Microorganisms frequently mutate into new strains, where some of them may give rise to novel diseases. Scientists play a crucial role in identifying and combating these diseases by developing new vaccines. Nuclear Magnetic Resonance (NMR) spectroscopy is an invaluable tool in understanding the process of how these microorganisms mutate, and with new methods, we can start predicting how the microorganisms will develop not only presently but also in the future. Jens Øllgaard Duus’ research is dedicated to understanding these microorganisms using various NMR methods.

Jens Øllgaard Duus is professor at the Technical University of Denmark (DTU) and a former center leader at the Carlsberg NMR center, where he used NMR to optimize the production of beer. Currently, he applies the techniques acquired from Carlsberg to understand how mutations affect diseases and convert food residues into biofuel.

After pursuing his education as an engineer at DTU, Jens Øllgaard Duus wrote his PhD under the supervision of Professor Klaus Bock. When Klaus moved to the Carlsberg Laboratory, Jens moved with him, and together they helped establish the Danish NMR center with Flemming Poulsen and his group.

Initially equipped with an old 500 MHz NMR machine, their efforts soon led to the acquisition of the first 600 MHz machine in Scandinavia and applied for a grant for a Danish NMR center at Carlsberg. As he was finishing his PhD, Jens helped design the center and installed the first 800 MHz instrument in Denmark. Over time, he advanced to become a group leader and then a professor at the Carlsberg Laboratory. In his last years at Carlsberg, he was the head of the Carlsberg laboratory.

Ten years ago, he left Carlsberg to serve as professor at DTU. Together with Charlotte Gotfredsen, he received a grant from the Villum Foundation to establish a new NMR center at DTU.

Jens’ research group at DTU is relatively small, mainly due to his focus on department and education management. His group has never been much larger than a couple of PhDs and postdocs.
The core of their research has always been the structure and function of carbohydrates, where they try to understand carbohydrates biological function in relation to bacteria and how they can help understand biological processes and develop vaccines.

NMR for diagnosis of pathogenic bacteria

Jens has worked with SSI Diagnostica, a spin-off company from the Statens Serum Institute (SSI), focusing on the study of pathogenic bacteria such as pneumococci. These bacteria are responsible for causing lung inflammation, commonly referred to as pneumonia. New strains of pneumococci emerge constantly, and when this happens, the immune defense often stops recognizing the pathogenic bacteria due to modifications in their surface.

The immune system probes the surface polysaccharides of the bacteria when trying to assess whether the bacteria are pathogenic. If it does not recognize these polysaccharides as pathogenic, the immune system does not elicit an immune response and the bacteria survive.
When bacterial mutations change these polysaccharides, they are therefore made unrecognizable to the immune system. Jens isolates these surface polysaccharides, uses NMR to determine their primary structure, and characterizes the carbohydrates that they consist of and their conformations.
These carbohydrates serve as diagnostic tools and as antigens in many vaccines as for example pneumonia, particularly for older populations.

“This battle between humans and microorganisms can almost be considered eternal. We must create new vaccines all the time since the microorganisms mutate constantly. Since we can determine the structure very precisely and sequence the genes of the bacteria, we can predict the target carbohydrates for the coming year. NMR is a vital tool for understanding and predicting this.” Jens explains.

Development of special NMR methods

Most of the time, Jens uses standard NMR pulse sequences for carbohydrate analysis. However, since different carbohydrates have a very similar structure, high-field NMR is needed to determine their structure accurately. Hence, Jens has been interested in acquiring 800 MHz or higher field instruments, like the one from Carlsberg.

A crucial aspect of carbohydrate structure is their stereochemistry. The slight difference in conformation allows enzymes to distinguish one carbohydrate from another. However, NMR data have a hard time discerning this unless they operate at very high frequencies.

Jens has also dedicated time working with the dissolution Dynamic Nuclear Polarization (d-DNP) technique, trying to understand enzymatic metabolism when microorganisms metabolize glucose. By using hyperpolarized glucose, he and other NMR scientists can observe the important pathways in glucose metabolism for each yeast strain and identify potential optimization bottlenecks.

During this investigation, they discovered a new method using deuterium-labeled glucose and d-DNP. By feeding the labeled glucose to yeast, they could observe, in real time, the yeast absorbing the glucose and converting it into ethanol and CO₂ through glycolysis. This allowed them to measure the formation, structures, and disappearance of molecules every few seconds.

Jens has also contributed to the development of new NMR tools. At Carlsberg, together with Ole W. Sørensen he developed a pulse sequence called H2BC, which detects two-bond couplings between protons and carbon. The more general HMBC method is widely used by organic chemists across the world. However, it lacks the ability to distinguish between protons and carbon two or more bond away. To address this limitation, they developed a new pulse sequence called H2BC, which can observe only the two-bond correlations. This has become a standard setting on most NMR instruments.

At DTU, they do not have engineers who build their machines. Most of the development happens in the software, not the hardware. They develop new computer systems for their instruments and then order the hardware from a contractor. Therefore, a strong understanding of mathematics and programming has become crucial in the field of NMR spectroscopy.

“NMR is a very interdisciplinary field. You have to be a chemist, biologist, physicist, mathematician, and programmer all at the same time.” He adds.

NMR spectroscopy uses for beer and bioethanol

When Jens was at Carlsberg, their research revolved around beer. They extensively studied various specialized enzymes involved in the breakdown and reconstruction of carbohydrates in barley.

“When barley sprouts, it naturally releases enzymes to break down the starch in the grain, making the carbohydrates available for the sprouting process. This same process is also utilized in beer production where sprouting is halted before all the starch is consumed but after the enzymes have been released. In an NMR tube, we can copy this enzymatic reaction by analyzing the carbohydrate spectra, adding the enzyme, and monitoring the enzymatic conversion in real-time.” he explains.

Today, they use the same method to study biomaterials One example is the conversion of biomass into glucose, which subsequently can be fermented into bioethanol. Since the use of food sources like grains or corn for bioethanol production is undesirable, they explore the potential of residual products like corn stalks using NMR spectroscopy and enzyme analysis.

Additionally, their goal is to achieve a comprehensive understanding of enzymatic functions. They use d-DNP to study how glycosides are converted by living organisms and examine how specific C-13 labeled carbohydrates are converted by enzymes.

“We still lack understanding of the initial step when a carbohydrate binds to an enzyme. But because d-DNP is so sensitive, we can detect new intermediates in the early stages of the enzymatic reaction. Although this may not be practically important per se, it plays a crucial role in understanding the basic science behind it.” Jens elaborates.

Collaboration with academia and the industry

Jens engages in collaboration with researchers from DTU who utilizes enzymes in different ways. Rather than breaking down molecules, they use enzymes to transfer carbohydrates to natural substances.

Most natural substances used in medicine or food often have poor physical-chemical properties, such as low solubility. To achieve improved solubility, enzymes can be utilized to add carbohydrates to natural substances in a single step, eliminating the requirement for chemical reagents or catalysts.

Jens Øllgaard Duus collaborates with DTU Biosustain, utilizing enzymes to glycolyze natural substances and determine the resulting products through NMR spectroscopy. However, he primarily works with the biological departments at DTU, such as Bioengineering and Food and Health. Although he no longer works in the fields of yeast and fermentation techniques like he did at Carlsberg he still uses many of the same techniques. Therefore, it makes sense for him to continue within the world of biology.

Jens also has good collaborations with companies. Many of them come to DTU to analyze their samples, and one of DTU’s strengths is working with and starting new companies.

“Right now, we are collaborating with a company called Foss, which manufactures analytical equipment for the dairy industry. We also collaborate with Topsoe using analytical chemistry methods to study biofuels. Companies are generally very open to doing collaborative student projects with us, and some of our students do their thesis or PhDs in collaboration with companies.” he adds

Jens also teaches several courses at DTU. Together with Charlotte, he teaches an introductory NMR course for graduate students. He also teaches a course in instrumental analytical chemistry.

“This course is important because much of the research done at DTU is qualitative, but companies require quantitative methods to measure the amount of a particular substance and its precise measurement.” Jens tells about the course.

In his course, students learn about methods such as mass spectrometry, biosensors, and electrochemistry that can achieve these objectives.

Strength of Danish NMR

“The strength of Danish NMR is that we have managed to collaborate for many years, even since the arrival of the first instruments in Denmark. For example, the first 270 MHz instrument at UCPH was a joint application across the country.” Jens says.

“The collaboration really took off after the creation of the Danish NMR center at Carlsberg. Many joint applications were submitted, and people managed to work together instead of competing. This collaborative spirit is still evident today, and very few groups compete within the NMR scene.” he explains.

“Another strength is diversity. UCPH and AAU focus on proteins, SDU traditionally focuses on DNA, AU excels in solid-state NMR, and here at DTU, we study small molecules and carbohydrates.” Jens further elaborates. “For example, if I have a student who wants to do a project in protein NMR, I will send them to UCPH, and I believe they would do the same. This way, we ensure that students work on projects with experts in their chosen field, rather than doing projects solely at their own university just to retain the students.” he adds.

“This openness and collaboration originated from Carlsberg, where we shared our NMR instruments with all universities. Carlsberg’s neutrality brought together all the universities, and I hope we can maintain this in the future.” Jens ends.

Written by: Jonatan Emil Svendsen