Nanotechnology

A new high-throughput method for screening protein-secreting microbial strains

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May 30, 2023

(Nanowerk News) A unique method to screen large-scale libraries for industrially useful bacterial strains was recently developed by Tokyo Tech researchers. This simple approach combines biosensors and microfluids to quickly identify mutant strains that secrete large amounts of industrially useful proteins, opening the door for more applications, such as low-cost biopharmaceuticals.

With the tools of modern genetic engineering, it is now possible to modify microorganisms so that the production of industrially useful proteins—such as those used in biopharmaceuticals—is increased. 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 hormone, and enzymes. This approach to increasing the expression of microbial secretory proteins has resulted in breakthroughs in medicine, industry, and agriculture.

Nonetheless, the traditional methods of genetically engineered bacterial strains for high protein production are very time consuming. This is because it relies on introducing genetic modifications in individual strains and evaluating the effectiveness of protein production. As an alternative, researchers sometimes rely on large-scale library screenings to identify strains that secrete high amounts of the protein. This allows the extraction of only those strains that are best at producing the desired protein. Unfortunately, current screening techniques rely on a variety of chemical treatments and are either too slow or too complicated.

To overcome these limitations, the research team has now developed a new high-yield mutant strain screening method. This study was led by Associate Professor Tetsuya Kitaguchi from the Tokyo Institute of Technology (Tokyo Tech), Japan, and was carried out in collaboration with Ajinomoto Co., Inc. The innovative method, which combines microfluidics and versatile biosensing to quickly identify enhanced bacterial strains that produce the highest amounts of the desired protein, is reported in their study published in the journal Small (“Efficient Microfluidic Screening Method Using Fluorescent Immunosensors for Recombinant Protein Secretion”).

To this end, the researchers first used a type of biosensor called a Q-body to measure the desired amount of protein each strain produces. The Q-body is an artificial antibody that glows when it binds to its target. In this case, they are designed to bind to the protein of interest, establishing a relationship between fluorescence intensity and production of the target protein.

In addition, the team also devised a clever protocol to sort mutant strains based on their performance (Figure 1). Using microfluidic technology, tiny water droplets containing individual bacteria and Q-bodies are introduced in the oil emulsion, taking advantage of the mutual immiscibility of oil and water. These tiny droplets are used as microscopic bacterial cultures and reactors. microbial strain screening Figure 1. An overview of the screening approach. The proposed method involves using water microdroplets as small bacterial cultures and bioreactors and then sorting them according to their fluorescence intensity. Q-bodies are designed to become fluorescent upon binding to a target protein. Thus, the fluorescence intensity is directly related to the desired amount of protein that can be produced by each bacterial strain. (Image: Tokyo Technology)

The proposed method involves using water microdroplets as small bacterial cultures and bioreactors and then sorting them according to their fluorescence intensity. Q-bodies are designed to become fluorescent upon binding to a target protein. Thus, the fluorescence intensity is directly related to the desired amount of protein that can be produced by each bacterial strain.

After 48 hours of incubation, these oil-covered water droplets were repackaged, in an aqueous emulsion, and sent via a flow cytometer. This device uses a laser and a detector to measure the fluorescence of each individual droplet. After this, it uses a sorting mechanism to separate the droplets with higher fluorescence intensity.

The researchers tested their method by screening a large library of bacterial strains made to produce FGF9, a human cytokine, and subjected to a state that causes random mutations. Using this method, the team was able to identify a mutant strain that produced three times more FGF9 than the control strain (Figure 2). As dr. Kitaguchi, “The entire screening process of 106 mutants was completed in approximately three days, surpassing the output of culture evaluation methods using the latest automated lab instruments.” Finding the best performing strains among mutant bacteria Figure 2. Finding the strain with the best performance among the mutant bacteria. The plots show a comparison of the amount of desired protein produced between 24 strains randomly selected before sorting and 24 strains selected from the best 50 performers. The best performing strain produced three times more of the desired protein compared to the control strain, demonstrating the potential of the proposed approach. (Image: Tokyo Technology)

Going forward, the team has high hopes; they hoped their proposed method would have a significant impact on the pharmaceutical industry because of its simplicity, accuracy, and versatility. Dr. Kitaguchi said, “Applying our screening method to the development of biopharmaceutical proteins can dramatically shorten the time required to establish highly productive industrial microbial strains. Therefore, we believe that this research can contribute to the creation of a variety of inexpensive biopharmaceutical proteins.”



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