A user manual for yeast’s genetic switches

When introducing genes into yeast to make it produce drugs and other useful substances, it is also necessary to reliably switch the production on or off. A Kobe University team found three gene regulation design principles that provide a flexible guideline for the effective control of microbiological production.

It’s said that DNA is the blueprint of life, telling our cells what to produce. But DNA also contains the switches telling those cells when to produce something and how much of it. Therefore, when introducing new genes into cells to produce useful chemicals such as drugs or raw materials for chemical production, it is also necessary to include a genetic switch, a piece of DNA called a “promoter,” that tells the cells to start production as needed. Kobe University bioengineer TOMINAGA Masahiro says: “The problem is that these promoters cannot be used in a plug-and-play manner unless researchers deeply understand how they interact with other genetic elements. Indeed, there are not so many cases in which researchers use artificial promoters to precisely control the cellular production and achieve their research purpose.” Sometimes the production is too low, sometimes it is “leaky,” meaning that it cannot be turned off at will. This is especially true for bioengineering yeast, which is more complex in its genetic regulation compared to bacteria. But this increased complexity also enables its use to produce many useful chemicals.

As experts in modifying yeast cells, Tominaga and colleagues from the team led by ISHII Jun took a systematic approach to working out how to design effective promoters. “We came up with the idea that by carefully describing our process of improving a prototype promoter, we could prepare a ‘user manual’ for how to achieve high-performance and precise control so that these genetic systems could be more widely used,” Tominaga explains.

In a paper now published in the journal Nature Communications, they describe three design principles for yeast promoters. First, if researchers not only need large amounts of the product but also the ability to switch the production on or off at will, they should introduce multiple copies of the regulatory elements enabling this within the promoter. This reduces leakiness and increases the productivity. Second, the distance between promoter elements should be as small as possible to enhance the productivity even more. And third, the promoter should be insulated from surrounding DNA by including extra DNA before it to further reduce leakiness. Tominaga says: “We showed that a promoter’s performance can be improved more than 100-fold by simply modifying its surrounding sequence. This is the first study to clearly propose a solution to the problem why potent yeast promoters work in some environments and not in others.”

The Kobe University bioengineers demonstrated the usefulness of their system by showcasing the production of two pharmaceutically useful proteins, so-called “biologics.” Not only could they produce these two biologics in separate yeast strains but also in the same strain and with the ability to independently control which biologic is produced at any time. The latter is important because it has potential applications in hospitals, as the team explains in the study: “In addition to the conventional fermentation of single biologics, the rapid and single-dose production of multiple biologics with a single yeast strain at the point of care is crucial for emergencies that require production speed and flexibility rather than purity and productivity.” They also achieved the notoriously difficult production of a coronavirus protein that can be used for the production of treatments, further showcasing both the usefulness and the flexibility of their design principles.

Tominaga explains his wider outlook on the implications of this study: “Synthetic biology advocates creating new biological functions by rewriting genome sequences. The reality is however that we are often confused by unexpected changes resulting from our edits. We hope that our study is the first step towards the ability to design every single base in the genome with clear intentions.”

This research was funded by the Japan Agency for Medical Research and Development (grants JP21ae0121002, JP21ae0121005, JP21ae0121006, JP21ae0121007, JP20ae0101055 and JP20ae0101060), the Japan Science and Technology Agency (grants JPMJCR21N2 and JPMJGX23B4) and the Japan Society for the Promotion of Science (grants JP23K26469, JP23H01776 and JP18K14374). It was conducted in collaboration with researchers from the Pharma Foods International Co. Ltd and National Institute of Health Sciences.

Kobe University is a national university with roots dating back to the Kobe Higher Commercial School founded in 1902. It is now one of Japan’s leading comprehensive research universities with nearly 16,000 students and nearly 1,700 faculty in 10 faculties and schools and 15 graduate schools. Combining the social and natural sciences to cultivate leaders with an interdisciplinary perspective, Kobe University creates knowledge and fosters innovation to address society’s challenges.