First images of a microorganism's drug manufacturing line

Researchers at the University of Michigan (UM) have obtained the first three-dimensional snapshots of the ‘assembly line’ within microorganisms that naturally produces antibiotics and other drugs. Their research has been published in two articles in the journal Nature.

Georgios Skiniotis, David Sherman, Janet Smith and Kristina Håkansson described the structure of a central enzyme in the assembly process that creates polyketides, a broad class of diverse and bioactive chemical compounds that comprises some of the most important antibiotics, antifungal agents, cancer chemotherapeutics and immunomodulators in wide clinical use.

“Ultimately, understanding the details of polyketide creation can allow us to successfully design and engineer systems for the production of novel products with high medicinal value,” said Skiniotis, the corresponding author on both papers.

David Sherman, Georgios Skiniotis and Janet Smith in the cryo-EM suite in the Life Sciences Institute at the University of Michigan. Image credit: Erin Grimm, Life Sciences Institute.

Nearly two-thirds of all drugs currently used are derived partially or entirely from a natural source. But the natural process used by microorganisms to create the medicines has been a “black box”, said Smith, who added, “We knew something went in and something else came out, but we didn’t know what happened inside.”

“There were lots of models proposed and lots of debate and disagreement, but very little evidence,” noted Sherman. “Over 15 years we were able to characterise the basic biochemical features of this remarkable biochemical assembly line, but high-resolution features remained beyond our grasp - until now.”

The polyketide synthase (PKS) enzyme was derived from a type of terrestrial bacteria called Streptomyces venezuelae, which produces pikromycin, an antibiotic precursor to the widely prescribed erythromycin. The researchers describe the enzymatic steps of the fifth cycle in a six-cycle biological assembly-line process that produces pikromycin. The structure was not what they had anticipated.

“What surprised us was that a tiny protein called acyl carrier protein, or ACP, moved sequentially to different sites within the PKS factory, adding additional chemical components - and each step was determined by the cargo it acquired in the previous step,” Smith said. “Understanding the role of ACP radically changes how we understand the entire structure and process.”

Cryo-EM structure of the central enzyme in the assembly process that creates polyketides, with the acyl carrier protein protein (ACP) in orange. Image credit: Somnath Dutta, Life Sciences Institute.

The discovery was made possible through the Skiniotis lab’s application of cryogenic electron microscopy (cryo-EM) visualisation, a technology that enables researchers to obtain high-resolution snapshots of protein complexes after rapidly freezing them. The visualisations provided the first images of the complete structure and of the changes and movement of the ACP protein that builds the chemical end product. Final confirmation of assembly-line function and product formation came from advanced mass spectrometry experiments in the Håkansson lab.

“We had low expectations because of the size and flexibility of the enzyme,” Skiniotis said. “But when we finally figured it out and could see the ACP, it was like a switch was flipped.”

Sherman noted the urgency of the need for new drugs, in light of the growing threat of antibiotic resistance. “Now that we can see how the machine is working, we can understand why previous attempts to engineer these natural factories to make drugs have failed - and how we might be able to do it better,” he said.

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