Microbial Genome Mining
Discovery of new antibiotics has slowed so much that “we could run out antibiotics to treat life-threatening infection very soon,” said senior researcher Lijiang Song, Ph.D., experimental officer in mass spectrometry at the University of Warwick. “This is why the American infectious disease society proposed that we need to discover 10 new antibiotics by 2020, which many people think is overambitious if not impossible. The main reason has been the high rate of re-discovery of known compounds.
“We have addressed this problem by employing high-resolution mass spectrometry to do de-replication in the modern microbial genome mining approach for novel natural products. This means we can identify known compounds very early on and shift our focus to true novel compounds.
“We have successfully identified and characterized more than 30 new compounds in the last five years and patented two families of novel bioactive microbial polyketide natural products (as antibiotics or anticancer agents.)”
Dr. Song’s methodology was created “to analyze the genome sequence first to identify potential interesting secondary metabolite gene clusters, such as PKS, NRPS, where one can even predict whole or part of the structure of the secondary metabolite if made.
“We then make knock-out mutants and carry out comparative metabolite profiling with LC-UV-MS/MS for wild-type and knock-out mutants to identify novel targets, followed by structure elucidation with MS and NMR.”
Dr. Song labels HR-MS/MS as “probably the most efficient way to do de-replication while high-resolution and high-accurate MS is probably the most sensitive (nanogram or even lower) and reliable way to do de-replication.”
With the emergence of high-speed, high-resolution MS, particularly the Bruker MaXis UHR-Q-TOF, said Dr. Song, it has become possible to screen a large number of samples for novel bioactive natural products by using UHPLC coupling with HR-MS/MS and still identify novel compounds quickly.
Dr. Song predicts that this approach will find its way into most natural product-based drug discovery programs. “Apparently, the current 40–60,000 resolution is good enough for most of the things we analyze, but it is not enough to distinguish sulphur isotope, which is one of the element contained in many natural products.
“I would like to think the resolution of Q-TOF HRMS will reach 100,000 in a few years time. By then HRMS/MS will become even more useful. Though many may argue that FT-ICR is better, the running cost for FT-ICR turned out to be too high for most of the labs. Their extreme high resolution (millions) is probably most suited for more specific applications.”