The Hidden Potential of Enzyme Promiscuity
Evolution in the laboratory can turn a familiar enzyme into a potential hotspot of innovation in synthetic biology
A team of researchers led by Prof. Tobias Erb at the Max Planck Institute in Marburg has discovered that a well-known enzyme is surprisingly flexible. After experimental evolution in the laboratory, succinate semialdehyde dehydrogenase (SAD) was able to function in two metabolically distinct processes - the formation of vitamins and the breakdown of sugars - in addition to its established role as a detoxifier. The results demonstrate the flexibility of 'promiscuous' enzymes. They also illustrate the hidden potential of enzyme promiscuity for applications in synthetic biology.
Although enzymes are usually known for their specificity, many have the ability to act on multiple substrates, a property that may play a key role in the evolution of enzymes. However, these promiscuous activities are often weak under physiological conditions and therefore remain undetected. To reveal and exploit them, researchers turn to adaptive laboratory evolution (ALE), which induces various genomic mutations. While most ALE studies have focused on how multiple enzymes can compensate for a single metabolic deficiency, the extent to which a single enzyme can compensate for different metabolic deficiencies has not been investigated.
Tobias Erb and his team aim to understand how biochemical networks function and harness this knowledge to build entirely new pathways. Their approach reflects a dual goal in synthetic biology: to deepen our understanding of biological processes while engineering novel biological functions and products. Enzyme promiscuity is a starting point both in nature and in the laboratory to create new reactions and pathways.
Laboratory evolution boosts the side activities of enzymes
Their studies focused on succinate semialdehyde dehydrogenase (SAD), an enzyme previously known for its role in aldehyde detoxification. To their surprise, when subjected to ALE, the enzyme independently compensated for the loss of two unrelated metabolic processes: vitamin B6 biosynthesis and glycolysis, in E. coli. The hidden abilities had been intensified by the laboratory evolution and thus became visible.
"We weren’t actively trying to overcome these deficiencies, but when we saw the results of directed evolution, we were astonished by the plasticity of SAD's active site, which enables it to function in such distinct metabolic processes," says Hai He, first author of the study published in Nature Communications.
Building on this discovery, the researchers employed a variety of techniques to investigate further. They used a combination of ALE, biochemical analysis, structural investigation, and omics approaches—including whole genome sequencing, metabolomics, and proteomics—to uncover the evolutionary mechanisms behind SAD’s new roles.
The promiscuity of enzymes is a valuable resource for future innovations
The researchers found that ALE had enhanced the enzyme’s hidden capabilities in several ways: by increasing various mutations in its active site, which boosted its activities; by altering direct and indirect regulatory elements, which raised the abundance of the SAD protein; and by increasing the gene's copy number, further elevating SAD levels.
The study not only uncovered regulatory links in metabolism but also highlighted the potential of enzyme flexibility as a driving force in metabolic evolution and as a valuable resource for biotechnological innovation.
"Understanding how these mechanisms are intertwined at the metabolic and regulatory levels is a complex challenge," says Tobias Erb. "Here, especially with regard to synthetic pathways that offer more sustainable routes or new products, a systematic exploration of underground metabolic pathways is necessary to drive future innovations in enzyme engineering."
