A Conversation with Randy Berka

INDUSTRIAL BIOTECHNOLOGY: Dr. Berka, what would you describe as the two or three most significant advances or changes related to fungal research that you have seen since you the start of your research career focusing on fungi?

RANDY BERKA: Fungal research is certainly an area that has seen accelerated development over the last 10 to 15 years, and I would preface my comments with the disclaimer that my career has been spent exclusively within the industrial biotech sector, so I might have a somewhat limited perspective regarding the academic side of fungal research. Back when I started working with fungi in the mid-1980s, our toolbox was fairly meager compared with well-studied species such as Saccharomyces cerevisiae. Methods for doing simple things like DNA-mediated transformation were only available for two model species of fungi at that time: Neurospora crassa and Aspergillus nidulans. So people who worked with industrial fungi had to work out very basic techniques for doing transformations, gene knockouts, and building vector components for gene expression and protein production. This all took a long time, and there are still tools that need to be developed or improved. But in the last decade I think we have seen more rapid development of the tools involved in fungal research, particularly for the species that are agriculturally, industrially, or medically important.

I think this has happened as a result of several things that have taken place more or less in parallel. The first is a rekindling of interest in using fungi and fungal enzymes to break down plant biomass to fermentable sugars. That has been part of the ongoing quest for clean, renewable energy and sustainable sources of carbon for key building block chemicals. The second development is the application of low-cost, high-throughput DNA sequencing technologies. Genomics is now revealing the genomic blueprints of many species, and this has accelerated the pace of fungal research in many areas, not just for biofuels, but for agriculturally and medically important fungi as well. It has also provided new insights into the tremendous chemical and biological diversity of these organisms.

An area in which fungal research is advancing rapidly and still needs to advance further is what I often refer to as agro-mycology: learning enough about fungal pathogens to control them effectively, as well as uncovering the secrets of how beneficial fungi in soil can be used to increase food production for the world. This aspect of fungal research is growing rapidly. With the world's population increasing, arable land being constant, and water supplies dwindling, I think a lot of emphasis will be placed on using microorganisms in sustainable agriculture, and that would include fungi: the microrhizal fungi, phosphate-solubilizing fungi, and fungi that improve plant nutrition and the efficiency of water utilization.

Genomics has propelled this area. We wanted to sequence fungal genomes 20 years ago, but the technology was not available to do it cost effectively. It was not practical to sequence a genome when it cost a million dollars to do so. Now, when you can sequence a genome for a thousand dollars or even less, it really opens the door for people. The same applies to the field of synthetic biology. The cost of making synthetic DNA at present is still a bit high. If the cost starts to plummet, the way sequencing costs have plummeted, then you are going to start to see synthetic biology being applied not only to fungi, but to all kinds of microorganisms in new ways. Right now I think cost is a barrier to applying synthetic biology, at least at large scale.

IB: How has fungal genomics transformed mycology?

RANDY BERKA: Fungal genomics has greatly accelerated the pace of fungal research. Over the past 5 years it has progressed more than during the previous 20 years of the pregenomics era combined. Genomics has been the great facilitator and opened a lot of new windows for exploring many fungal species. When I first started attending the fungal genetics conferences at Asilomar in the mid-1980s, there were about 150-200 attendees, but that number has grown to more than 900 attendees in recent years, with people on the waiting list to attend.

Genomics has become the foundation on which fungal research is moving forward, not just in industrial research but in many different areas, whether medical mycology, plant pathogenesis, ecology, cell biology, or genetics. There is no doubt that genomics has been a driving force behind fungal research and productive collaborations between fungal biologists worldwide. I have been fortunate to be involved in some of those collaborations, mainly through the JGI Community Sequencing Program. It has been a lot of fun to witness how members of the fungal community, both from academia and industry, have come together, sharing a common interest, to do science that probably would not have happened without that shared foundation of genomics. At meetings you will see the Trichoderma and the Aspergillus communities, for example, coming together. Everybody is now following the same playbook, and the different research groups are borrowing from each other.

IB: What types of biotechnological challenges are currently best served by fungi? Why?

RANDY BERKA: One of the biggest challenges we still face is converting plant biomass material to renewable fuels and biochemicals. These products can replace those currently derived from petroleum, which of course is not a renewable resource. Since fungi and their enzymes already break down biomass efficiently in nature and because these enzymes can be produced in large quantities in industrial fermentation, I think they are particularly well-suited to address this challenge. As I mentioned, I also feel that certain soil fungi are good candidates to address key challenges in agriculture related to pest control, plant fertility, and enhanced crop performance. However, we still have much to learn about fungi and what they do naturally in the soil. Investigators are learning a lot from metagenomics about bacterial communities in soil, but I think we have barely begun to learn about the fungal communities: their roles in the microbiome; how they interact or communicate with plants or other organisms in the environment.

IB: What kinds of new industrial biotechnology applications do you envision for fungi in the future?

RANDY BERKA: It is always difficult to try and look into the crystal ball and predict what may happen in the future, especially in a day and age when things are moving and changing so quickly. I think there is little doubt that fungi will be used to produce a whole constellation of new enzymes for a variety of industrial applications. That would include new enzymes for existing applications as well as novel enzymes for new processes. I think we will begin to use our planet's biodiversity to discover new enzymes, biochemicals, and metabolites that can be deployed innovatively to bring about more sustainable solutions and alternatives to the less environmentally friendly processes that we currently use. I think it is also safe to say that fungal research will continue to focus on improving the productivity and economics of the fungal production strains used to make enzymes and biochemicals as we learn to improve the efficiency of carbon utilization, maximizing the flow of the input carbon to the desired products, and minimizing carbon loss to byproducts and things like CO₂. When we learn to do that, we can begin to exploit the incredible biochemical capacity and versatility of fungi to produce new molecules more economically. That speaks to the combined use of genomics, systems biology, and synthetic biology, as well as biochemical engineering to reach the enzyme and biochemical yields that approach the theoretical maximum.

IB: Are there specific gaps in the available technology that create barriers to achieving these goals? How can they be overcome?

RANDY BERKA: There are definitely technology gaps impeding progress, particularly with industrial fungi. For example, with the exception of a few model organisms such as Neurospora, transformation efficiencies are still too low for most species of interest in industry, and that prevents us from employing some of the techniques such as genome-wide RNAi screens to reveal new pathways, discover bottlenecks to overcome to improve productivity, and do rapid strain improvement. In addition, the poor transformation efficiencies and low frequency of homologous recombination events in these organisms limit the exchange of nucleic acid sequences and prevent us from doing allele replacement to associate single nucleotide polymorphisms (SNPs) in genomes with corresponding phenotypes. When you are looking at a pedigree of strains that have been developed through years and years of mutagenesis, of course you uncover many SNPs, most of which are irrelevant to the improved phenotype, but some of which could be very useful for developing future, improved strains. You have to be able to find the “gold nuggets” in the huge slag heap of mutations that have occurred. We can certainly generate that long list of SNPs from comparative genomics data, but following up on hundreds of SNPs in filamentous fungi is certainly not as convenient as it is in something like yeast.

Lastly, I think an obstacle that needs to be overcome for industrial strain improvement is the lack of methods for sexual crossing of strains. That presents a barrier for rapid strain improvement and getting rid of unwanted background mutations, although strides are being made to overcome that in some industrial species, such as Trichoderma reesei.

IB: What types of applications does Novozymes perform that use fungi?

RANDY BERKA: Novozymes uses fungi as workhorse production organisms to manufacture a variety of enzymes for various industrial applications. For example, fungal enzymes are used for degradation of plant polysaccharides like starch and cellulose in conventional and advanced biofuel processes. Additional industrial applications include detergents, brewing, baking, textiles, food, animal feed, and more. Novozymes also uses fungal strains for agricultural applications such as biofertility. Most recently, we reported metabolic engineering of the fungus Aspergillus oryzae to produce high levels of malic acid, which is a dicarboxylic acid used in the food and beverage industry. That is a good example of how a bulk chemical precursor currently produced from petroleum can be generated from renewable resources by fungal fermentation.

The role of fungi, fungal enzymes, and fungal metabolites is increasing as the tools to make products more economical continue to expand. In other words, as the productivity of the organisms to make a specific enzyme improves, it creates opportunities to apply those enzymes in other industries, where it might not previously have been economically feasible. So we are seeing an expansion of fungal enzyme technology into areas where maybe 15 years ago it was not cost effective to do so, because of the development of tools to improve the strains, the economics, and the manufacturing. It is an integrated process from start to finish.

IB: How have the goals and directions of fungal research at Novozymes evolved in recent years?

RANDY BERKA: The basic goal of fungal research at Novozymes, which focuses on providing innovation for our customers and developing cost-effective solutions, has not changed in the 21 years I have been with the company. I think the application of genomics and other omics tools has provided us with the ability to identify bottlenecks and perform better strain improvement than 10 or 15 years ago, which opens up new opportunities for our customers as well.

IB: What do you find most interesting about working with fungi?

RANDY BERKA: Over my 30-year career in biotechnology and working with fungi, I learned that it is not a one-size-fits-all area and you have to approach each application individually. I have developed my own biases for what I think the best organism is for any particular application. So I would say that the most interesting aspect for me is the biological and biochemical diversity of these organisms. That never ceases to amaze me! They have had a tremendous impact on industrial biotechnology, starting with the first biotechnology patent in the US, filed by Jokichi Takamine in 1894 for fungal amylase. People started developing large-scale tank fermentation to produce citric acid and penicillin in the 1930s and 1940s.

Today fungal enzymes are being used for biofuels and renewable chemicals research and production, and all of this would not have been possible without years of development of large scale fermentation processes. As a result, I am an avid proponent of the biorefinery concept and a future biobased society, in which many of the products we use in our everyday lives are manufactured using environmentally safe biological processes. I am fairly confident that sustainable bioagriculture and enzyme technologies will be linchpins to new world economies based on renewable raw materials.