Abstract
Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.
Highlights
Microbes acquire new phenotypes via horizontal gene transfer, a process that reshapes microbial evolution and ecology
We constructed four strains of E. coli MG1655 that each lack a conditionally-essential Fe-S enzyme: NadA, IspG
The activities of the heterologous enzymes were tested using a complementation assay: growth in selective media indicates that the heterologous enzyme is functional when expressed in E. coli (Figure 1B, 1C)
Summary
Microbes acquire new phenotypes via horizontal gene transfer, a process that reshapes microbial evolution and ecology. Gene transfer is an everyday laboratory technique that enables expression of heterologous proteins and engineered biosynthetic pathways. Obstacles against foreign gene expression, which include restriction-modification systems and differences in codon usage, can impede horizontal gene transfer from influencing host phenotype. In the lab, such genetic obstacles are routinely avoided to ensure heterologous protein expression, e.g. by using strains that lack genome defence systems or by optimizing codon usage. Heterologous proteins must be expressed and must retain their activity to affect the host phenotype or to function as part of an engineered pathway. Unlike obstacles to heterologous protein expression, the obstacles that prevent heterologous protein activity are rarely studied 1
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