With modern genetic engineering tools, it is now possible to modify microorganisms so that their production of industrially useful proteins is enhanced. By introducing genetic modifications into these organisms, we can use them as biological factories to produce large quantities of the desired protein. Bacteria with this enhanced ability can produce insulin, growth hormones, and enzymes. This approach of increasing microbial secretory protein expression has led to breakthroughs in medicine, industry, and agriculture.
Nonetheless, the traditional method of genetically engineering bacterial strains for high protein production is time-consuming as it relies on introducing genetic modifications in individual strains and evaluating the effectiveness of protein production. As an alternative, researchers sometimes rely on screening large-scale libraries to identify strains that secrete high amounts of protein. This narrows down only those strains that are the best at producing the desired protein. Unfortunately, current screening techniques rely on multiple chemical treatments and are either too slow or too complicated.
To overcome these limitations, a team of researchers has now developed a novel, high-throughput mutant strain screening method. The study was led by Tetsuya Kitaguchi, associate professor at the Tokyo Institute of Technology (Tokyo Tech), Japan, and was conducted in collaboration with Ajinomoto Co., Inc. An innovative method that combines microfluidics and versatile biosensing to robustly identify enhanced bacterial strains that produce the highest amount of a desired protein, is reported in their recently published study in Small.
The researchers first used a type of biosensor called Q-body to measure the amount of the desired protein produced by each strain. Q-bodies are artificial antibodies that fluoresce when bound to their target. In this case, they were designed to bind to the desired protein, establishing a connection between the intensity of fluorescence and levels of protein produced.
In addition, the team also devised a protocol for sorting the mutant strains based on their performance. Using microfluidic technology, tiny droplets of water containing individual bacterium and Q-bodies were introduced in an oil emulsion, taking advantage of oil and water’s mutual immiscibility. These tiny droplets were used as microscopic bacterial cultures and reactors.
After 48 hours of incubation, these oil-covered water droplets were encapsulated again in a water emulsion and sent through a flow cytometer. This device measures the fluorescence of each droplet and sorts droplets with higher fluorescence intensity.
The researchers tested their method by screening a huge library of bacterial strains created to produce FGF9, a human cytokine, and subjected to circumstances that cause random mutations. Using this method, the team identified a mutant strain that produced three times as much FGF9 as the control strain. As Kitaguchi remarks, “The entire screening process of 106 mutants was completed in approximately three days, surpassing the throughput of culture evaluation methods that use the latest automated lab instruments.”
The team hopes their proposed method will have a significant impact on the pharmaceutical industry due to its simplicity, accuracy, and versatility. Kitaguchi says, “Applying our screening method for the development of biopharmaceutical proteins may dramatically shorten the time required to establish highly productive industrial microbial strains. We thus believe that this study can contribute to the inexpensive manufacture of various biopharmaceutical proteins.”
- This press release was supported by the Tokyo Institute of Technology