Keeping an eye on microbes

RALEIGH, N.C.--Sensors are hardly a new feature in the industries of biotechnology and pharmaceuticals, as these devices enable scientists and physicians to track everything from patient biometrics to the delivery of therapeutics. But a new class of sensors is now underway with an aim at tracking antibiotic molecules—specifically those resulting from microbes.

This work, undertaken by a team of researchers from North Carolina State University, was published in ACS Synthetic Biology in a paper titled “Development of transcription factor-based designer macrolide biosensors for metabolic engineering and synthetic biology.”

The antibiotic molecules in the spotlight for this work are known as macrolides. As noted in the paper's abstract, “Macrolides are a large group of natural products that display broad and potent biological activities and are biosynthesized by type I polyketide synthases (PKSs) and associated enzymatic machinery.”

According to the LiverTox website, courtesy of the U.S. National Library of Medicine, “Five macrolide antibiotics are currently available for use in the United States: erythromycin, clarithromycin, azithromycin, fidaxomicin and telithromycin, the latter being a related ketolide. Erythromycin was initially isolated in 1952 from Streptomyces erythreus; the other macrolide antibiotics are semisynthetic derivatives.” S. erythreus is a kind of bacteria found in soil. These antibiotics are bacteriostatic, which means they stop bacteria from growing and reproducing rather than killing them.

Unfortunately, the downside of this subset of natural products is that the microbes capable of generating antibiotic macrolides only produce small amounts of a limited number of antibiotics.

“Our ultimate goal is to engineer microbes to make new versions of these antibiotics for our use, which will drastically reduce the amount of time and money necessary for new drug testing and development,” Gavin Williams, associate professor of bio-organic chemistry at NC State and corresponding author of the ACS paper, said in a press release. “In order to do that, we first need to be able to detect the antibiotic molecules of interest produced by the microbes.”

The tactic of choice was a protein known as MphR, a naturally occurring molecular switch. As noted in a press release penned by NCSU's Tracey Peake, in E. coli, MphR is capable of detecting the presence of macrolide antibiotics secreted by microbes that are attacking the E. coli bacteria. Upon sensing an antibiotic, MphR activates a resistance mechanism to cancel out the effect of the antibiotic.

The team generated a library of MphR protein variants and screened them to see which one could turn on the production of a fluorescent green protein when in the presence of a target macrolide. The resulting variants were tested against erythromycin, which MphR can recognize, and some were found to improve the detection ability tenfold. The ACS paper abstract reports that “[T]he promiscuous macrolide sensing transcription factor MphR is a powerful platform for engineering variants with tailored properties. We identified variants that displayed improved sensitivity towards erythromycin, tailored the inducer specificity and significantly improved sensitivity to macrolides that were very poor inducers of the wild-type MphR biosensor.”

As noted on the Williams lab page of the NCSU website, “Often, the ability to access analogues of natural products for drug discovery and medicinal chemistry are restricted by our limited ability to manipulate natural product biosynthesis in microbes … To address this major limitation, we have created genetically encoded biosensors that produce a fluorescent signal in the presence of the target natural product. These designer biosensors enable ultra-high throughput approaches to engineering natural product biosynthesis, and are being used to screen the productivity of thousands of pathway variants, thus bypassing our limited ability to rationally reprogram complex biosynthetic pathways.”

“Essentially we have co-opted and evolved the MphR sensor system, increasing its sensitivity in recognizing the molecules that we’re interested in,” Williams explained. “We know that we can tailor this biosensor and that it will detect the molecules we’re interested in, which will enable us to screen millions of different strains quickly. This is the first step toward high-throughput engineering of antibiotics, where we create vast libraries of genetically modified strains and variants of microbes in order to find the few strains and variants that produce the desired molecule in the desired yield.”