Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host
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Curated by eLife
Evaluation Summary:
Three of Nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms? This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study provides insights on the phylogenetic and biosynthetic limitations concerning formation of functional heterologously expressed Fe-S proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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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.
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Evaluation Summary:
Three of Nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms? This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study …
Evaluation Summary:
Three of Nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms? This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study provides insights on the phylogenetic and biosynthetic limitations concerning formation of functional heterologously expressed Fe-S proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Three of nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms. This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. …
Reviewer #1 (Public Review):
Three of nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms. This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study described in the manuscript provides considerable insight into the question. It is shown that for the case of at least one model Fe-S protein (NadA) heterologous expression of an active species sometimes also requires the heterologous co-expression of the native Fe-S cluster assembly machinery. Whether or not compatible co-expression of the heterologous Fe-S cluster machinery for expression of active NadA in E. coli is required is correlated with phylogenic distance. This same phylogenic correlation, however, could not be established for the active/inactive heterologous expression in E. coli of a different Fe-S protein (IspG). Instead, in some cases, the production of active heterologously expressed IspG required the heterologous co-expression of electron transfer proteins necessary for IspG turnover. The important conclusion of the work is that heterologous production of Fe-S proteins in E. coli is complicated and could require either heterologous co-expression of Fe-S cluster machinery or heterologous expression of compatible electron transfer partners, and perhaps, in certain cases, heterologous expression of both assembly and electron transfer systems. This reviewer found the work to be thorough and compelling. The conclusions should be of considerable interest to a very wide readership because there are so many biochemical processes that rely on Fe-S protein and their experimental analysis is very often stymied by an inability to produce them in active forms using heterologous gene expression. There is also great interest on this topic from the biotechnological perspective because many industrial processes are limited by an inability to heterologously produce active Fe-S proteins that are currently rate-limiting. Along these lines the authors provide an interesting proof of concept by demonstrating an ability to boost production of 2-methyl tryptophan by circumventing the Fe-S protein dependent bottle neck.
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Reviewer #2 (Public Review):
D'Angelo et al. carry out a comprehensive screen to determine which members of two enzyme families can be heterologously expressed in E. coli in an active form. The two enzymes used for the study, NadA (quinolinate synthase) and IspG (4-hydroxy-3-methylbut-2-enyl-diphosphate synthase) both require an iron-sulfur (Fe-S) cluster cofactor for activity and Fe-S enzymes are notoriously difficult to express in non-native organisms. The authors are attempting to determine which Fe-S cluster biosynthesis (maturation) pathways are necessary to help produce fully activated NadA and IspG. They utilize an elegant genetic complementation assay for the initial activity screen, followed by more comprehensive proteomic analysis, and then iterative addition of alternative Fe-S cluster biosynthesis pathways and partner …
Reviewer #2 (Public Review):
D'Angelo et al. carry out a comprehensive screen to determine which members of two enzyme families can be heterologously expressed in E. coli in an active form. The two enzymes used for the study, NadA (quinolinate synthase) and IspG (4-hydroxy-3-methylbut-2-enyl-diphosphate synthase) both require an iron-sulfur (Fe-S) cluster cofactor for activity and Fe-S enzymes are notoriously difficult to express in non-native organisms. The authors are attempting to determine which Fe-S cluster biosynthesis (maturation) pathways are necessary to help produce fully activated NadA and IspG. They utilize an elegant genetic complementation assay for the initial activity screen, followed by more comprehensive proteomic analysis, and then iterative addition of alternative Fe-S cluster biosynthesis pathways and partner proteins to help activate recalcitrant NadA or IspG orthologues. NadA demonstrated a relatively straightforward behavior. It was matured to the active form by phylogenetically related Fe-S cluster biosynthesis pathways. NadA orthologues more distant from E. coli required co-expression of an Fe-S cluster biosynthesis pathway from organisms more closely related to their native species.
The results with IspG were more complex. Only a 8 of 47 IspG proteins tested were active in the complementation assay. There did not seem to be a clear relationship between successful expression of active IspG in E. coli and the phylogenetic relatedness of the native organism to E. coli. Furthermore, co-expression of heterologous Fe-S cluster biosynthetic pathways did not significantly improve IspG activation. Subsequently, the authors also co-expressed an electron transfer partner protein with each IspG in order to help reduce the oxidized IspG Fe-S cluster formed during its reaction cycle, thereby assisting IspG with turnover in vivo. Significantly, they found that co-expression of a heterologous electron transfer partner protein resulted in a marked improvement in activation of many IspG proteins in E. coli. They interpreted these results to indicate that IspG proteins were likely maturing properly to the Fe-S form but required the appropriate electron transfer partner for multiple turnovers (and full activity). They further probed the question of what defines an "appropriate" electron transfer protein using a somewhat tangential approach. They analyzed which specific ferredoxin proteins, when co-expressed heterologously, could allow turnover of the cobalamin-dependent rSAM methyltransferase TsrM, from Streptomyces laurentii. They discovered that an appropriate electron transfer protein for this enzyme must have the necessary reduction potential to catalyze the reduction of Co(II) to Co(I) (-730 mV vs Ag/AgCl). From this, one could extrapolate that IspG may face a similar electrochemical/thermodynamic hurdle if it is expressed in the absence of its appropriate redox partner.
The conclusions of the paper are supported by the data presented. This work also provides one of the few systematic analyses of factors that influence heterologous Fe-S protein expression. However, there were several experiments that could have improved the manuscript and helped tease more information out of the studies performed.
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Reviewer #3 (Public Review):
Summary:
In this study, the authors use synthetic biology to study the challenges with programming cells using non-native proteins containing iron-sulfur cluster cofactors. The study focuses on two different challenges in synthetic biology. The first challenge is focused on understanding what controls the activity of iron-sulfur cluster dependent enzymes. The authors find that functional expression can depend upon evolutionary distance from the host being programmed and expression of iron-sulfur cluster machinery. The second challenge focuses on understanding what controls the activity of iron-sulfur cluster containing protein electron carriers within synthetic electron transfer pathways. The first aspect of this study screened large numbers of iron sulfur enzymes for function in the cellular assay, while …
Reviewer #3 (Public Review):
Summary:
In this study, the authors use synthetic biology to study the challenges with programming cells using non-native proteins containing iron-sulfur cluster cofactors. The study focuses on two different challenges in synthetic biology. The first challenge is focused on understanding what controls the activity of iron-sulfur cluster dependent enzymes. The authors find that functional expression can depend upon evolutionary distance from the host being programmed and expression of iron-sulfur cluster machinery. The second challenge focuses on understanding what controls the activity of iron-sulfur cluster containing protein electron carriers within synthetic electron transfer pathways. The first aspect of this study screened large numbers of iron sulfur enzymes for function in the cellular assay, while the second aspect assessed the function of three ferredoxins in a synthetic electron transfer pathway.
Major strengths:
The use of cellular assays to study iron-sulfur cluster enzymes and electron transfer proteins is critical to developing synthetic biology rules for programming cells that are useful for green chemistry, bioelectronics, and synthetic biology. A great deal of effort in synthetic biology has focused on developing design rules for protein expression, such as developing models to predict transcription and translation initiation. However, models are lacking for adding in considerations of iron-sulfur cluster biogenesis (or other cofactors). This study seeks to better understand what governs expression of functional iron-sulfur enzymes and electron carriers. By expressing large numbers of enzyme homologs from across the tree of life within cellular selections, this study uses a high-throughput approach to assess metallocluster biogenesis. This aspect of the study is a major strength within the context of the iron-sulfur enzyme focus.
Major weaknesses:
As one considers using cellular assays to study challenges with iron-sulfur cluster biogenesis, there are several challenges to consider beyond cofactor biogenesis. All of the proteins being studied are expressed from a promoter and ribosomal binding site (RBS), which can lead to variability in protein accumulation prior to iron-sulfur cluster biogenesis. In fact, gene sequences proximal to the RBS can affect translation initiation as illustrated in the many applications of the RBS calculator. Note that the context of an RBS site placed next to divergent iron-sulfur proteins is expected to depend upon evolutionary distance of those proteins so suitable expression controls in cellular assays should be used to benchmark protein expression.
Additional challenges with cellular assays include the need for a protein to fold within the cell, such as melting temperatures and interactions with chaperones. Recent studies of iron-sulfur cluster containing electron transfer proteins have revealed that changing the temperature of a cellular assay can affect the observed function of iron-sulfur cluster containing proteins. This can arise because those proteins evolved to function at temperature below the standard growth temperature of E. coli, or because they evolved to function with chaperones in addition to iron-sulfur cluster assembly proteins as noted. This study notes the diverse organisms used as sources of enzymes, but it is not clear how the optimal growth temperatures of those organisms relates to the conditions used for cellular assays.
Oxygen has been important to the evolution of iron-sulfur proteins and cluster biogenesis machinery. The complementation observed could depend upon oxygen and iron-sulfur cluster biogenesis, especially if the enzymes being tested are orthologs or paralogs from organisms that grow under anaerobic conditions. Understanding more characteristics about the sources of those enzymes such as growth conditions, iron-sulfur cluster assembly systems, and their divergence seems critical to interpreting cellular assay data. Finally, it isn't clear if expression of non-native iron-sulfur biogenesis systems rescues complementation because of specificity of that system versus the lack of regulation over the expression of the system.
Conclusions supported by results:
In the abstract, the authors conclude" Our results clarify how incompatibilities with foreign Fe-S and electron transfer networks each impede functional heterologous expression and provide insight into how these barriers can be overcome for engineering Fe-S enzyme-dependent biosynthetic pathways." The results assessing complementation of non-native enzymes in E. coli show that a subset can be rescued by expressing a non-native iron-sulfur cluster biogenesis system. However, it is not clear how much mechanistic insight is provided, since the study does not consider different mechanisms that could influence the observed trends, such as variation in translation initiation rates, thermostabilities of enzymes in the context of the cellular assay, oxygen sensitivity, and the lack of regulated expression of the iron-sulfur biogenesis systems. From the results, it isn't clear how a synthetic biologist should choose different iron-sulfur cluster assembly systems to include in their cellular programming.
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