Cross-species transfer of Pseudomonas iModulons into E. coli

To initiate the project and prior to implementing cross-species iModulon transfer, we refactored a known cellular function within the original host as a proof of concept. Successful homologous refactoring and complementation of E. colis branched-chain amino acid (BCAA) metabolism was achieved (Supplementary Note, section1 and Supplementary Fig.1) to demonstrate identification, reconstruction, and transfer of genetic constituent of a biological function based on iModulon (i, ii, and iii). This motivated us to investigate the potential for transferring biological functions across species. Among the available species with iModulon structures in iModulonDB12, Pseudomonas is well-known for its versatile metabolism to degrade and utilize diverse compounds, including aromatics19,20,21. First, we chose to reconstruct and transfer a simple bioconversion process from Pseudomonas putida15 to E. coli in order to examine iModulons capability to rapidly identify genes associated with specific functions.

The VanR iModulon that is responsible for vanillate (VA) transport and conversion into protocatechuate (PCA) was chosen for our first cross-species iModulon transfer (Fig.1A). It comprises three genes with annotated functions, vanA, vanB, vanK, and predicted porin-like galP-IV(Fig.1B) in two converging operons (Fig.1C). Notably, the iModulon exactly matches with the genes for the vanillate transport and metabolism22,23. Four genes, vanA, vanB, galP, and vanK are functionally annotated to encode for vanillate O-demethylase oxidoreductase complex, outer-membrane porin, and a major facilitator superfamily transporter, respectively22. Although the function of the outer membrane OprD-domain containing galP-IV has never been addressed, it is hypothesized that it facilitates the diffusion of the ligand through the outer membrane23,24. Since the mechanism of VanR regulation has not been established, the four genes constituting the VanR iModulon were cloned and heterologously expressed under the control of IPTG-inducible Trc promoter on a plasmid, pVanR_iM (Fig.1D). When refactoring iModulons for heterologous expression, we tried to preserve native genetic arrangement, for VanR and following iModulons if possible, to ensure optimal expression levels of the gene members as demonstrated elsewhere25,26.

A Vanillate transport and conversion in P. putida. OM outer membrane. CM cytoplasmic membrane. B iModulon weights of genes in P. putida. Four genes (green circles) with high weighting constitute the VanR iModulon. Gray lines indicate thresholds for determining iModulon membership. Gray circles identify genes not in the iModulon. C Graphical representation of vanR locus on the P. putida chromosome. D The VanR iModulon was refactored in a single operon under the control of trc promoter (PTrc), resulting in the pVanR_iM plasmid. Shades show genetic rearrangement for cloning purposes. E Vanillate (VA) conversion of E. coli carrying empty or pVanR_iM plasmid into protocatechuate (PCA). Gray circles, green diamonds, and orange triangles indicate cell density, VA, and PCA levels of the culture, respectively. Measurements from E. coli carrying empty or pVanR_iM plasmid are represented by hollow or filled symbols, respectively. Data were presented as mean valuesSD. Error bars indicate the SD of three replicate cultures. Source data are provided as a Source Data file.

E. coli carrying pVanR_iM converted VA into PCA up to 15.34mg/l passively diffused to the supernatant27,28 during 48h of fermentation in M9 glucose (4g/l) medium supplemented with 100mg/l VA, while the negative control carrying empty plasmid did not metabolize any VA (Fig.1E). This first cross-species iModulon transplantation illustrates the rapid identification of enzymes required for biotransformation by ICA. Furthermore, iModulon engraftment provided a rapid way to biochemically verify a predicted pathway in a heterologous host.

Next, we chose to transfer an ampicillin resistance function of Pseudomonas aeruginosa to E. coli. P. aeruginosa displays beta-lactam resistance with endogenous beta-lactamase, AmpC, and has an iModulon involved in the inducible ampicillin resistance16. Activity levels of the AmpC iModulon are highly induced against beta-lactam challenge, but not under other antibiotic treatments (Supplementary Fig.2). In the previous iModulon engraftment examples, genes comprising an iModulon matched with the predicted genes necessary for building the desired function. However, identifying all the genes necessary to build a biological function may not be trivial, given previous characterization efforts. Many iModulons contain genes whose functions are unknown or are seemingly unrelated to the overall function being transferred.

The AmpC iModulon comprises class C beta-lactamase encoded by the ampC gene29 that serves as a core for the functionality and six lesser characterized auxiliary genes, carO (PA0320), creD (PA0465), PA0466, PA0467, PA4111, and PA4112 (Fig.2A). The seven iModulon genes are distributed across three genomic loci separated by over 4Mb. P. aeruginosa readily becomes resistant to ampicillin by transcriptional activation of ampC30. However, it is not known if the resistance trait is carried by this single gene. To examine if this resistance function is transferable across species, the constituent genes were refactored into a single operon (Fig.2B). In addition, we constructed a plasmid that contained beta-lactamase alone to address any involvement of auxiliary factors in the function.

A iModulon weights of genes in P. aeruginosa. Seven genes constitute the AmpC iModulon (blue circles). Gray lines indicate thresholds for determining iModulon membership. Gray circles identify genes not in the iModulon. B Refactoring the P. aeruginosa AmpC iModulon on bacterial artificial chromosome (BAC). Genes are expressed with the trc promoter (PTrc). Shades show genetic rearrangement for cloning purposes. C Dose-kill curves of P. aeruginosa and E. coli carrying empty BAC, BAC_ampC, or BAC_AmpC_iM. Data were presented as mean valuesSD. Error bars indicate the SD of biological replicates (n=3). Note that the range of ampicillin concentration (Amp) is different, due to the huge difference in ampicillin tolerance. D Cell density of cultures treated with different ampicillin concentrations after 10h of incubation. Data for P. aeruginosa and E. coli carrying empty BAC, BAC_ampC, or BAC_AmpC_iM are in orange, gray, light blue, and blue, respectively. Arrows indicate the minimum inhibitory concentration (MIC). Data were presented as mean valuesSD. Error bars indicate the SD of biological replicates (n=3). Source data are provided as a Source Data file.

Ampicillin disc diffusion assay revealed that E. coli carrying the AmpC iModulon or ampC gene were resistant to ampicillin, while E. coli carrying empty plasmid were not (Supplementary Fig.3). The source of AmpC iModulon, P. aeruginosa, showed ampicillin resistance with the minimum inhibitory concentration (MIC) of 2048g/ml (Fig.2C). The MIC of ampicillin for laboratory E. coli strain MG1655 with empty plasmid was 16g/ml, which is comparable to previous reports31,32 (Fig.2C, D). E. coli strain with the P. aeruginosa beta-lactamase showed a dramatic increase in ampicillin resistance with an MIC of 1024g/ml, while it was lower than that of the original host (Fig.2D). Strikingly, E. coli harboring the entire AmpC iModulon, six auxiliary genes in addition to ampC, had an MIC of 4096g/ml, which was four times higher than that with ampC alone (Fig.2D).

Although little is known about the molecular function of auxiliary genes, they were required to completely replicate the ampicillin resistance characteristics of P. aeruginosa. Previous reports have shown a decrease in beta-lactam resistance of the inner membrane protein creD knockout mutant of P. aeruginosa33 and growth enhancement of E. coli by endogenous creD overproduction (shares 37.4% sequence identity; BLOSUM62)34. Although the function of CreD is still elusive, reports indicate its relevance in biofilm development in P. aeruginosa35 and envelope integrity in Stenotrophomonas maltophilia36. Additionally, calcium-regulated oligonucleotide/oligosaccharide binding (OB)-fold protein CarO has been reported to be related to susceptibility to various stresses in bacteria37. Also, it shares similarity with Salmonella enterica stress-related protein VisP (38% sequence identity), which binds to peptidoglycan and inhibits the lipid A modifying enzyme LpxO38. Since lipid A is an anchor of lipopolysaccharide to the outer membrane and affects the properties of the outer membrane, expression of carO might be beneficial for cells to maintain structural integrity under cell wall deficient conditions induced by beta-lactam39.

Engrafting Pseudomonas iModulons to E. coli highlighted critical properties of iModulon gene membership. Harnessing only core genes for transferred cellular function may not be sufficient, as auxiliary genes may be needed to reconstruct an optimal function. Full iModulon gene membership helps to recreate the targeted cellular function, even without a complete understanding of the molecular function of all the genes involved.

As illustrated by the AmpC case, we further investigated the iModulon-based transfer of cellular traits and compared it to the alternative conventional methods. The 2,3-butanediol (2,3-BDO) iModulon was chosen to examine the role of iModulon genes of unknown functions. 2,3-BDO is a byproduct of bacterial fermentation processes that can be produced by a variety of microorganisms, including Pseudomonas species40,41,42. In Pseudomonas, 2,3-BDO can serve as a carbon and energy source and is degraded by enzymes in the 2,3-BDO catabolic pathway42. This catabolic pathway involves the conversion of 2,3-BDO into acetoin, which is further converted into acetaldehyde and acetyl-CoA by butanediol dehydrogenase and acetoin dehydrogenase, respectively (Fig.3A).

A A pathway responsible for 2,3-BDO utilization. B Scatter plot shows weights of genes in P. putida to AcoR iModulon. Gray lines indicate thresholds for determining iModulon membership. Five genes constitute the AcoR iModulon (orange circles). Gray circles identify genes not in the iModulon. Black circles are three neighboring genes. C Genomic structure of the AcoR iModulon. Orange shade shows predicted operonic structure. Genes in the iModulon are in orange. Arrows indicate three different plasmid constructs for cross-species transfer. D 2,3-BDO degradation by P. putida. The formation of acetoin was negligible. Blue and yellow boxes represent 2,3-BDO and acetoin in the culture medium. Red circles show cell density. Dots indicate individual data points. Data were presented as mean valuesSD. Error bars indicate the SD of the three biological replicates. E 2,3-BDO and acetoin degradation by E. coli carrying empty plasmid or one of the three constructs. 2,3-BDO was added at the start of the culture and the remaining amount and acetoin formation was measured. Blue and yellow boxes represent 2,3-BDO and acetoin in the culture medium. Red circles show cell density. Dots indicate individual data points. Data were presented as mean valuesSD. Error bars indicate SD of the three biological replicates. Source data are provided as a Source Data file.

We transferred the 2,3-BDO iModulon of P. putida (called the AcoR iModulon15) to E. coli. The AcoR iModulon comprises acoABC (encoding acetoin dehydrogenase complex), bdhA (encoding 2,3-BDO dehydrogenase), and a gene acoX (Fig.3B). AcoX encodes for a protein of unknown function and co-exists with acetoin-utilizing genes in various bacteria41,43. Operon prediction also suggests that the transcriptional unit contains acoX and two other hypothetical proteins (PP_0550 and PP_0551) in addition to characterized metabolic enzymes, acoABC-bdhA (Fig.3C)18,44.

To examine which genes are required for recreating the 2,3-BDO catabolic pathway, we built three different plasmid based on (1) operonic structure (Op353; acoXABC-bdhA-PP_0551-PP_0550), (2) iModulon structure (acoXABC-bdhA), and (3) four genes encoding enzymes predicted to be sufficient for converting 2,3-BDO into acetaldehyde and acetyl-CoA based on current gene annotations (pathway; acoABC-bdhA) (Fig.3C). 2,3-BDO dehydrogenase activities of the source organism and E. coli strains carrying the three plasmids individually were examined during 96h of batch cultivation in LB medium supplemented with 2g/l of 2,3-BDO. The original strain, P. putida KT2440, showed 2,3-BDO utilization with a negligible level of acetoin (Fig.3D). The negative control, E. coli MG1655 carrying an empty plasmid converted 0.77g/l of 2,3-BDO into acetoin, possibly due to endogenous promiscuous alcohol dehydrogenase activity (Fig.3E). On the other hand, the plasmids based on the pathway, operonic structure, and iModulon showed higher conversion of 2,3-BDO with amounts of 1.36, 1.75, and 1.96g/l, respectively (Fig.3E).

Interestingly, the strains showed varying levels of acetoin dehydrogenase activity. First, all the 2,3-BDO consumed by the negative control resulted in roughly the equimolar amount of acetoin; not surprising since there is no acetoin dehydrogenase introduced. The strain carrying the functional gene annotation-based pathway plasmid did not further convert acetoin into downstream products, even though it contained genes encoding for the acetoin dehydrogenase complex. Second, strains with the full operon or AcoR iModulon not only consumed more than 1.7g/l of 2,3-BDO, but there was only a small amount of acetoin left in the medium, indicating conversion of acetoin by acetoin dehydrogenase. The difference between annotation-based and iModulon-based plasmid is the presence of acoX (Fig.3C), a gene encoding a predicted small molecule kinase that has been reported to have no acetoin, NAD, or pyruvate kinase activity45. However, acoX was critical for acetoin dehydrogenase activity.

Although the acoX product has no known function in acetoin metabolism, it is conserved and colocalizes on the genome with the acetoin dehydrogenase in several acetoin-utilizing bacteria from multiple phyla, such as P. aeruginosa (76% sequence identity) and Clostridium magnum (32% sequence identity)42. However, there is no significant match of AcoX from the BLASTP search on other acetoin-utilizing bacteria such as Bacillus subtilis, Klebsiella pneumoniae, and Pelobacter carbinolicus. Therefore, the requirement of AcoX in acetoin metabolism is species-specific and could not be determined by analyzing the genome sequence context.

When the iModulon and operonic constructs were compared, the iModulon construct performed better than the operonic construct for 2,3-BDO degradation (Fig.3E). Two additional genes in the operonic construct encode the predicted membrane occupation and recognition nexus (MORN) domain-containing peptidase and a NAD(P)-binding oxidoreductase, whose relation with 2,3-BDO metabolism is unknown. These two genes were irrelevant for function. Instead, expression of the hypothetical proteins reduced 2,3-BDO degradation, possibly by imposing an unnecessary transcriptional burden on the cell. The iModulon gene membership provided information on the necessary genes to support a 2,3-BDO catabolic process that would not have been found using only functional gene annotation. This example illustrates the unique advantages of using the iModulon structure for cross-species transfer of the full genetic basis for a desired integrated function.

Lastly, we chose the MdcR iModulon from P. aeruginosa16 to transfer into E. coli that, again, comprises genes identical to a reported set for malonate transport and utilization. The MdcR iModulon comprises seven subunits of malonate decarboxylase complex21 and two putative membrane proteins, MadL-MadM (Fig.4A and Supplementary Fig.4). Although the function of these membrane proteins have not been elucidated in P. aeruginosa, MadL and MadM have 71 and 81% of sequence homology to malonate transporters in Malonomonas rubra46, respectively, suggesting a potential malonate uptake function. These genes are encoded in a single operon on the P. aeruginosa genome, and thus the entire operon was subjected to cross-species transfer.

A Malonate catabolic pathway in P. aeruginosa. B Genetic structure of the malonate catabolic operon of P. aeruginosa cloned in a heterologous expression plasmid, pMdcR_iM. Brown genes constitute MdcR iModulon. C Malonate utilization of P. aeruginosa, E. coli carrying empty plasmid, and pMdcR iM. Cells were incubated for up to 72h in M9 malonate (2g/l) media. Circles and diamonds show cell density and malonate concentration in culture, respectively. Green, gray, and brown lines represent P. aeruginosa, E. coli carrying empty plasmid, and pMdcR iM plasmid, respectively. Data were presented as mean valuesSD. Error bars indicate the SD of three replicated cultures. D Growth rates of E. coli carrying the MdcR iModulon over the course of evolution. Dashed lines are moving averages of three individual ALE lineages. Growth rates for each ALE lineage are colored differently. E Malonate utilization and growth of three evolved populations. Circles represent cell density, with the solid circles being extracellular malonate concentrations. Measurements for each ALE lineage are colored differently. Data were presented as mean values of two replicated cultures. F Growth rates of clones isolated from malonate-evolved populations in M9 malonate medium. Data were presented as mean values of two replicated cultures. Dots show individual data points. Strain names are given as AX.IY. X is the ALE lineage number and Y is an arbitrary identifying number for the clonal isolate from the same ALE lineage. G Adaptive mutations in the ALE endpoint clones that are not present in the parental strain. fs, frameshift mutation. The heatmap shows allele frequencies colored as in the provided color key. H Plasmid-to-chromosome copy number ratio (P/C ratio) and expression level of mdcA of unevolved parent strain and evolved clones. Blue and orange boxes represent the P/C ratio and mdcA expression level, respectively. Data were presented as mean values in two biologically replicated cultures. Dots are individual data points, each of which is composed of two technical duplicates. Source data are provided as a Source Data file.

The operon was cloned and heterologously expressed under the control of a Trc promoter on a plasmid, named pMdcR_iM (Fig.4B). Malonate is a non-native nutrient for E. coli, thus it is expected that a strain with the pMdcR_iM alone would then enable growth in M9 malonate medium as the breakdown product of the pathway, acetate, can support growth47. We experimented with varying levels of expression using different concentrations of the inducer (IPTG) to activate the MdcR iModulon. E. coli could slowly utilize (doubling time of 11.20.6h; over the course of 72h of fermentation in M9 malonate medium) malonate as a carbon source only at weak expression level (Fig.4C). In contrast to complete utilization of malonate by P. aeruginosa within 12h of fermentation (Fig.4C), the observed slow utilization by E. coli suggests a potential metabolic imbalance in E. coli, perturbed by and unable to accommodate the malonate pathway.

Therefore, we implemented adaptive laboratory evolution to allow E. coli to rebalance and optimize its metabolism with malonate as a substrate. The E. coli strain carrying the pMdcR_iM was grown in an M9 malonate medium and evolved using serial passaging that imposes growth rate selection pressure (Fig.4D) on an automated ALEbot48. After 21 passages, populations showed faster growth with a short lag phase compared to their ancestor (Fig.4E). The evolved populations fully consumed malonate within 40 hrs. Subsequently, three clones were isolated from each replicate evolved population, and they all displayed a faster growth rate than the ancestor (Fig.4F).

To understand the genetic bases of improved growth, we resequenced the genome of the evolved clones (Supplementary Table1). All the evolved clones carried mutations on DNA polymerase I, encoded by polA, which is required for plasmid maintenance (Fig.4G)49. Previous studies reported a change of plasmid copy number induced by polA mutation50. Quantitative measurement of plasmid copy number indicated a reduction of plasmid copy number, which led to a reduction of MdcR iModulon expression (Fig.4H). Thus, the initial metabolic failure was likely due to the sub-optimal expression of the MdcR iModulon (Supplementary Note, section2), which could be optimized by ALE.

Engraftment of the MdcR iModulon, in addition to three other iModulons, demonstrated cross-species iModulon transfer as a rapid way of creating new functionality in bacteria with minimal engineering. We found that the overall behavior of the iModulon interferes with the host factors that require modifications to optimally support the system. This optimization could be rapidly achieved by ALE that identified few genetic changes in the host, while the transferred genes acquired no adaptive mutations.

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