The molecular biology laboratory has changed dramatically over the last 30 years, owing to the vast improvements in techniques and technologies that have paved the way for countless groundbreaking discoveries. Engineering biology is one area that has evolved tremendously, as it has proven to be a powerful approach to finding sustainable alternatives to finite resources. However, it can still take huge amounts of time, money, and effort to develop optimal microorganisms and bioprocesses for efficient and scalable production. This article discusses the importance of combining the right tools and knowledge to engineer microbes more efficiently and predictably for use in industrial bioprocesses.
The engineering biology minefield
Microbes have evolved over millions of years to ensure that they are the fittest to survive in their environment, learning to cope with constant and changeable pressures. While we are still learning the vast intricacies of biology, all living organisms share a drive for survival. This common attribute makes the re-engineering of natural biological processes more complicated, as any genetic changes we make, from deleting or upregulating genes to modifying post-translational modifications, forces microbes into survival mode. As the high product yields that are required for industrial biotechnology are often cytotoxic, microbes will find ways to expel or loop out the inserted DNA, giving that cell a selective advantage that could lead to a non-producing mutant taking over a culture and significantly reducing product yield. As a result, the identification of stable, high performing microbes is vital in the development of an effective bioprocess.
Limitations of traditional methods
Most traditional industrial bioengineering methods relied on either blasting organisms with UV light or treating them with chemicals to cause the DNA within the cell to mutate and rearrange. This scattergun approach can work, but it is non-targeted, making it highly unpredictable. The alternative, more refined approach was to create specific modifications, such as inserting or deleting a single gene of interest, but this made it a slow and iterative technique. Furthermore, these methods did not feed into the learning cycle; researchers didn’t know why an approach failed and would simply cast it aside and start again. These limited technologies were used long before engineering biology came to the fore as a viable tool for industrial biotechnology, where even constructing one engineered microbe was considered a success. However, through the advent of robotics and fine-tuned techniques, and the accumulation of decades of knowledge, thousands of bespoke engineered microbes can be constructed in a week.
Applying an engineering ethos
Albeit slow and costly, the successes of those early years made the power of industrial biotechnology clear. However, as scientists wanted to do increasingly complex things, such as modifying microbes to make a chemical usually derived from petroleum, generalized approaches were no longer fit for purpose. Applying engineering biology principles to industrial biotechnology has started to overcome these challenges and significantly sped up project timelines. Workflows are now structured to include a design stage that feeds into the building phase, then test and learn, with subsequent discoveries fed back into the next round of design. This replaces the iterative approach with a well-defined cycle, avoids repeating mistakes through constant learning and, ultimately, saves time and money.
This engineering-style approach, and the speed and accuracy that is now achievable, has been enabled by the parallel development of technology. Thanks to modern engineering biology, scientists can now make and test hundreds or thousands of genetic constructs instead of just one. Laboratory automation has further amplified this approach. A scientist at a bench manually building one genetic construct is slow, but liquid handling robots perform these repetitive and time-consuming tasks with ease, meaning labs can now screen thousands of genes in the same timeframe.
Technology is redundant without the know-how
The underpinning technologies are available but knowing how to apply them efficiently and effectively to each microbe is crucial. Traditionally, Escherichia coli and Saccharomyces cerevisiae were used as hosts for most engineering biology projects, but whole panels of microbes—including other yeasts and bacteria, both Gram-negative and Gram-positive—are now often used to find the best host for a given project. However, the genome of each microbe is different, and the approach must be tailored to each host; editing the genome of E. coli, for example, is completely different to that of Bacillus subtilis. Even something as fundamental as transporting DNA into the cell needs to be carefully considered. Therefore, there is no universal solution, and each project must be personalized to the needs of the target product and microbe.
Safety in numbers and diversity
Combining expertise is vital, and a multi-disciplinary team—including molecular biologists, biochemists, synthetic chemists, and more—is needed to carry a project to fruition. A molecular biologist, for example, is effective at building strains to perform at a milliliter scale, but likely wouldn’t know how the same strain would perform at 10,000 liters. The design and build must be executed with upscaling in mind, which involves working closely with a fermentation team. If a microbe is engineered to generate a lot of product, but its growth is hampered in the fermenter, then you must go back to the design and build phase, thus losing time and money. For this reason, external collaborations with companies or academia can be essential, and knowing when in-house efforts need to be supported through sharing of resources and expertise is vital to success.
The landscape of the molecular biology lab has changed dramatically in recent years, not only in terms of the equipment and techniques used but also the personnel driving its evolution. This has opened up the benefits of engineering biology to a wide variety of applications, from the production of biofuels and fine chemicals to pharmaceutical products, including vaccine development. Like the scientific method itself, this field will continue to be developed and refined, making bioengineering processes more efficient to drive future discoveries and developments.