Fast-forward evolution: Nudging microorganisms to help make humanity sustainable

Creating balance is the key. In recent years, humanity has strived to restore the delicate equilibrium between its eternal pursuit of greater prosperity and well-being and a planet and climate struggling to keep pace. Surprisingly, the planet’s microorganisms emerge as allies in this battle for balance. Jack Pronk has dedicated his career to seeking assistance from yeasts, best known for their roles in bread rising and beer brewing. Nevertheless, with some careful nudging and balancing, these microorganisms can contribute far more significantly.

“Our dream is to establish a biobased economy using bacteria, yeasts, and fungi. An example is producing fuel ethanol from sugarcane or corn using yeast, which efficiently converts glucose into ethanol. However, this approach is controversial because it could compete with food production. To address this, we are exploring, and have already identified, methods to modify these organisms. This enables them to transform agricultural and forestry waste into biofuels and fine chemicals,” explains Jack Pronk, Professor and Head of the Department of Biotechnology at Delft University of Technology (TU Delft) in the Netherlands.

Survival under ice

Jack Pronk has always been captivated by nature, exploring it both at the macro level, such as being a dedicated fisherman, and seeking to understand the science behind it. Thus, studying biology at Leiden University was a natural choice for him. However, after just one year of BSc studies, he grew disillusioned with the textbooks filled with information on anatomy and the systematics of plants and animals.

“I was ready to quit. And even if I would continue, I saw myself probably becoming a full-time teacher. Fortunately, my parents persuaded me to give it another year. Then, in the second year, we had extended practicals in laboratory groups. Mine was in an animal physiology group conducting research on carp—and especially their muscle tissue. That was the first time I really thought, ‘Wow, research is cool!’ So, in a way, fish got me hooked on research.”

The ability of carp to survive under ice for extended periods is attributed to alcoholic fermentation. The research practical investigated this phenomenon by isolating mitochondria and conducting enzyme assays. During his MSc studies in biology, his major research project focused on plant molecular biology, investigating the mechanism by which Agrobacterium can transfer DNA to plants.

“A second research project, on inducing bacteria to produce organic acids, got me even more excited. Unlike the waiting periods of months in other research areas, here I felt I obtained new results every day. This really sparked my interest in research as a potential career path.”

Extremely acidic environments

Jack Pronk completed his MSc in biology in 1986 before earning his PhD in microbiology from TU Delft in 1991.

“As a PhD student, I studied Thiobacillus bacteria, and especially peculiar acid-loving species thriving at pH values as low as 1.6—truly extreme conditions. So extreme that stainless steel parts of the expensive cultivation set-ups sometimes started to dissolve.”

Acidithiobacillus ferrooxidans, an acidophilic bacterium, is crucial in bioleaching metal ores by oxidising ferrous iron and sulfur compounds found in the ores. This process solubilises metals such as copper, gold, and uranium, facilitating their extraction.

“These acid-loving bacteria can be used for recovering metal from ores, but they also play a role in natural environments where sulfur-containing minerals and/or iron are present. My PhD project sought to understand their physiology better.”

One surprise emerging from the PhD project was that Acidithiobacillus ferrooxidans can not only grow on sulfur compounds and iron but also on formic acid—a compound that is very toxic under acidic conditions. By using a controlled feeding strategy, much higher culture densities could be obtained with formic acid than with sulfur compounds or iron.

“The most intriguing outcome of my PhD research was the revelation that Acidithiobacillus ferrooxidans, traditionally known for oxidising ferrous iron, also possesses the capability to reduce ferric iron, enabling respiration and growth in anaerobic conditions. This unexpected flexibility in microbial behavior, discovered during exploration, deeply resonated with me and has since become a driving force in my career.”

Couldn’t have been at a better time

Jack Pronk transitioned into yeast research through a stroke of luck and strategic planning. He was due to go into military service and had already been called up, but his services were needed elsewhere.

“During my PhD studies, I acquired the license to be a radiological safety officer. At the time, our department had a large isotope laboratory, and at the end of my PhD, I was the only person with that qualification. This gave my PhD supervisor and then head of the department, Gijs Kuenen, the ammunition for a letter to the Ministry of Defence.”

He got out, and just at that moment, there was a vacancy for an assistant professor in the TU Delft yeast group, led by Hans van Dijken. This marked the beginning of his journey into the world of yeast physiology and molecular biology.

"Reflecting on this turn of events," Jack Pronk remarked, "I fell headlong into yeast research. And it couldn’t have been at a better time, really."

A stroke of luck

In the mid-1990s, two pivotal advancements occurred in yeast research. First, the complete genome sequence of the yeast and model eukaryote Saccharomyces cerevisiae was published in 1996, thus facilitating its precise genetic modification. Second, the field of yeast metabolic engineering started to take off.

"At the time, our lab specialized in growing microorganisms under tightly controlled conditions in chemostats. These bioreactors were invaluable for both fundamental research and quantitatively studying industrial process conditions. Combining this with yeast genetic engineering, uncommon then, likely caught the attention of Stanford University colleagues, pioneers in Affymetrix (now Applied Biosystems) microarray usage."

Affymetrix microarray technology revolutionized genetic research in the late 1990s by enabling the simultaneous analysis of the expression of thousands of genes. By embedding DNA probes on chips, it simplified the previously slow process of gene-by-gene analysis.

"The colleagues at Stanford had devised a very elegant experiment combining chemostats and microarrays and asked, 'Can we send someone over to run the cultures?' This stroke of luck enabled us to use and subsequently acquire a microarray set-up early on, marking a pivotal moment in our research capabilities."

Keep their machinery running

The new set-up enabled Jack Pronk and colleagues to significantly advance their understanding of yeast physiology and metabolism. The studies revealed how these tiny organisms manage their energy and grow under various conditions, especially focusing on their interactions with oxygen and utilization of various nutrients.

Understanding these processes in yeast is not just about academic curiosity. Large-scale industrial processes for making bread, beer, and wine—but also biofuels and chemicals—depend on optimally adapting the yeast cells' metabolic machinery to the relevant process conditions.

"Beer brewing and industrial production of ethanol rely on the ability of some yeast cells to grow in the complete absence of oxygen. Throughout my career, I have been fascinated by the question of why only very few of the thousands of known yeast species master this trick."

The potential of genomics for science and innovation had not gone unnoticed in politics.

"At the turn of the century, we had a forward-thinking government that recognized the potential of genomics for addressing major societal challenges. They established the Netherlands Genomics Initiative."

Burnout and comeback

By setting up multiple virtual research centers, in which researchers from different research institutes, universities, and industry partners collaborated, the Government of the Netherlands aimed to advance genomics research and its application. One such initiative was the Kluyver Centre for Genomics of Industrial Fermentation, tasked with revolutionizing fermentation processes for producing food, pharmaceuticals, biofuels, and biochemicals.

“I had just been appointed Antoni van Leeuwenhoek Professor of Industrial Microbiology at TU Delft. Colleagues insisted that I would be a good leader for this initiative. Encouraged by ‘You can do it,’ I took on the task. I remain grateful for their trust and glad I did not dodge the responsibility. It did, however, help to precipitate a rather traumatic learning experience.”

Like many in academia, Jack Pronk had unconsciously been struggling with imposter syndrome for a long time. A burnout turned out to be the most valuable learning experience of his career.

Reflecting on past experiences, I often dismissed compliments on my conference presentations, believing they were merely polite gestures. Despite achieving full professorship at 35, I harbored doubts about my abilities, fearing that luck had played a significant role and that my shortcomings would eventually be exposed. Now, I realize how this insecurity can drive individuals to exceed expectations, yet it can also lead to unhealthy perfectionism, in which a person feels that they must constantly give more than 100% to avoid being uncovered. 

A lot of Bach, fishing, support from colleagues who had gone through the same experience, and professional help enabled Jack Pronk to come back stronger.

"I learned not to continually measure myself against unrealistic targets or others but to also appreciate what goes well. It taught me the importance of getting to know my 'user manual,' which I think is a lifelong learning challenge for us all."

Critical hurdle in scaling up

His comeback proved to be an extraordinary success. Jack Pronk led the Kluyver Centre for Genomics of Industrial Fermentation for 12 years as Scientific Director, and his research at TU Delft moved into new directions. At the turn of the century, the increasing focus on the fossil fuel–sparked climate crisis led to a surge in interest in bioethanol as an alternative to conventional transport fuels. Addressing the challenges related to bioethanol production became one of his objectives.

One significant challenge in producing bioethanol was to use crude plant-based materials as feedstock. While traditional yeast strains, such as Saccharomyces cerevisiae, could efficiently ferment 6-carbon (hexose) sugars from fruits, vegetables, and grains, they struggled with the 5-carbon (pentose) sugars commonly present in lignocellulosic biomass from agricultural residues, wood, and grasses. 

This type of biomass contains substantial amounts of the two pentose sugars xylose and arabinose, but yeast do not harbor the metabolic pathways needed to convert these sugars into ethanol.

“This limitation impeded the efficient conversion of agricultural residues, such as straw and leftover corn stalks, into ethanol, presenting a critical hurdle in scaling up bioethanol production from non-food sources.”

Indian elephant dung

Earlier attempts to solve the pentose challenge largely relied on introducing two new enzymes in the yeast metabolism: xylose reductase and xylitol dehydrogenase. Despite many elegant studies, this two-enzyme solution was challenging to regulate and control. Jack Pronk and colleagues were convinced that the answer might comprise a completely different kind of enzyme when he met fellow microbiologist Huub Op den Camp from Radboud University Nijmegen.

“At the time, Huub was working with a fungus he had isolated from elephant dung. He was not involved in research on pentoses, but when Huub described a specific piece of DNA from the fungus, I realized it might hold the key to our problem. Together, we demonstrated that introducing the fungal gene into Saccharomyces cerevisiae solved this missing link.”

In the anaerobic fungus Piromyces sp. strain E2, isolated from the faeces of an Indian elephant, Jack Pronk and his collaborators found what many had been seeking—a xylose isomerase gene that would work well in yeast.

“This result initiated years of research, which increasingly also involved collaboration with the Dutch company DSM. Through this process, Saccharomyces cerevisiae was adapted for processing the pentose sugars from agricultural waste streams.”

Still great potential

By around 2010, the modified yeast could quickly convert all relevant sugars from plant residues into ethanol, paving the way for more sustainable biofuel production technologies and minimizing reliance on food crops as feedstock.

“Only four years later, DSM and the United States ethanol-producing company POET together opened a dedicated, full-scale plant for producing fuel ethanol from corn residues. The plant ran for a number of years until declining oil prices and a changing political context forced production to be interrupted.”

Despite this setback, Jack Pronk believes that the production of ethanol from agricultural residues will make a comeback. Ethanol is not only useful as a transport fuel but also as a precursor for compounds ranging from ethylene to aviation fuel.

“I see great potential for ethanol, produced by low-emission technologies, as a generic feedstock for producing food protein, pharmaceuticals, and fine chemicals. There remains huge potential for its production from agricultural residues. I am convinced that genetically modified microorganisms, be they yeasts, bacteria, or fungi, will enable cost-effective and sustainable ethanol production from these feedstocks.”

Weird and wonderful fungi

Equally important, the xylose isomerase example has proven that these kinds of productions are scalable. With the ability to engineer yeast, it is now straightforward to develop strains for the production of other valuable products from lignocellulosic biomass. However, a basic understanding of yeast physiology and growth remains equally important. In 2016, Jack Pronk received a European Research Council (ERC) Advanced Grant to investigate and eliminate the oxygen requirements in yeasts.

“We sought inspiration in the genomes of an evolutionarily ancient group of fungi, the Neocallimastigomycetes, or neos. These weird and wonderful fungi live in the oxygen-free guts of large herbivores. During evolution, their massive genomes have acquired a host of bacterial genes, as well as modifications in their own genes, that have helped them adapt to life in the absence of oxygen.”

Growth of Saccharomyces yeasts without oxygen requires special anaerobic growth factors: molecules such as sterols, unsaturated fatty acids, and vitamins. Investigating Neocallimastigomycetes’ methods helped Jack Pronk’s team to reduce these needs, enhancing the robustness of Saccharomyces yeasts in anaerobic processes and expanding their suitability across yeast species.

“Neos serve as a striking example of nature’s ability to carry out genetic modification through the transfer of DNA across species. This process, a testament to natural evolution, happens on a scale that’s almost hard to believe. Without playing down the amazing possibilities of synthetic biology, we have only just begun to explore the options offered by microbial biodiversity.”

Brewing and antibiotics

Over the past decade, Jack Pronk and his team took another significant step towards creating a more efficient and sustainable method for producing ethanol that is now applied at full scale in the ethanol industry in the United States. For this invention, they called on help from his old acquaintances, the Thiobacillus bacteria. However, it was not the metal-eating variety but a relative, Thiobacillus denitrificans, from which the researchers borrowed a gene to fine-tune ethanol production using yeast.

“Integrating plant-like CO2-fixation enzymes—active in the Calvin cycle—into a yeast enabled more efficient conversion of CO2 to ethanol by minimizing the unwanted by-product glycerol created during yeast growth. This way, we can create an even more economical production process and reduce environmental impact by co-utilizing some CO2.”

Over the years, Jack Pronk’s research and knowledge about yeast have benefited far more industries than just bioethanol production. His collaboration with DSM and other partners has resulted in 17 patents and spans various sectors, including the production of important chemicals such as lactate and pyruvate, brewing, and antibiotics. In 2018, his career took another new turn.

“The Dean stepped into my office and said, ‘Your head of department is leaving. We need an interim; would you be okay to do this?’ And my answer was, ‘Yes, of course.’ He then asked if I wanted to be head of department for a longer period. My less helpful answer was, ‘No way.’”

Six years later, Jack Pronk is still Head of the Department of Biotechnology at TU Delft. Asked to explain how his career took the twists and turns it did, he replies:

“Humans think they are rational. We are anything but rational, but we excel at rationalizing after the fact. This holds especially in describing career paths, in which choices seem logical in hindsight but are often driven by deeper motivations. At crossroads, advice from family, friends, and mentors, both in and outside science, has been invaluable, inspiring me and shaping decisions already taking shape subconsciously.”

We should stick this in yeast

Jack Pronk’s career exemplifies this notion through his pioneering research in industrial microbiology, particularly his work with Saccharomyces yeast in producing biobased fuels and chemicals.

“Our dream is to use microbial processes to replace existing chemical, fossil-based processes, thereby creating sustainable alternatives to traditional ones. Especially in academic research, it is important to acknowledge the unpredictability of which research line will ultimately make it to full-scale application. Our role is to continually explore and generate new options and, in the process, train new generations of scientists.”

To pass on knowledge, Jack Pronk has always been passionate about teaching and mentoring the researchers of tomorrow—and even as time has become more scarce because of his role as head of the department, he still gives priority to teaching the first-year students.

A remarkable example of Jack Pronk’s teaching philosophy in action occurred during a lecture when a spontaneous new insight emerged while teaching.