Structure-Guided Biochemical Analysis of Quorum Signal Synthase Specificities

Abstract

Many bacteria use membrane-diffusible small molecule quorum signals to coordinate gene transcription in response to changes in cell density, known as quorum sensing (QS). Among these, acyl-homoserine lactones (AHL) are widely distributed in Proteobacteria and are involved in controlling the expression of virulence genes and biofilm formation in pathogens, such as Pseudomonas aeruginosa. AHL molecules are specifically biosynthesized by the cognate LuxI type AHL synthases using S-adenosylmethionine (SAM) and either acyl carrier protein (ACP)- or CoA-coupled fatty acids through a two-step reaction. Here, we characterize a CoA-dependent LuxI synthase from Rhodopseudomonas palustris that utilizes an aryl-CoA substrate that is environmentally derived, specifically p-coumaric acid. We leverage structures of this aryl-CoA-dependent synthase, along with our prior studies of an acyl-CoA-dependent synthase, to identify residues that confer substrate chain specificity in these enzymes. We test our predictions by carrying out biochemical, kinetic, and structural characterization of representative AHL signal synthases. Our studies provide an understanding of various AHL synthases that may be deployed in synthetic biological applications and inform on the design of specific small molecule therapeutics that can restrict virulence by targeting quorum signaling.

Graphical Abstract

INTRODUCTION

Cellular communication is a widespread phenomenon in nature, and examples include bacterial quorum sensing (QS),^1–3 fungal communication through pheromones,^4–6 small molecule-mediated interactions between microbes and their hosts,^7–9 and cellular communication in multicellular eukaryotes.¹⁰ QS systems have been shown to influence bacterial swarming, the secretion of exoenzymes, biofilm formation, and genetic competence.^1–3 In some marine bacteria, bioluminescence is induced at high cell densities via QS systems.¹¹ Bacterial QS signal (also known as autoinducer) synthases, receptors, and cognate promoters are the crucial elements of the QS circuit.^12,13 QS communication also represents a valuable means to engineer novel signaling circuits in bacteria,¹³ which can regulate phenomena such as bistable behavior, pulse response, spatiotemporal control of gene expression, and population control.^3,14

Currently, most of our understanding of QS is largely based on studies of Gram-negative N-acyl-homoserine lactone (AHL) systems,¹³ which represent one class of bacterial signaling molecules. Other important QS systems include those that use oligopeptides (such as autoinducing peptides or AIPs and ComX, among others) or the furanosyl borate diester autoinducer-2 (AI-2) as signals. Many Gram-negative and Gram-positive strains produce the AI-2 signals, which is thought to be an identical product, 4,5-dihyroxy-2,3-petanedione, that is in chemical equilibrium with several furanones.^15,16 The oligopeptide system is predominantly used by Gram-positive bacteria.^17,18 In the case of AIPs, the signal is a ribosomally synthesized and post-translationally modified peptide,¹⁹ whose chemical structure is defined by presence of a cyclic ester or thioester.^20,21 The ComX peptide is defined by both a leader sequence and a post-translational modification, namely, prenylation and cyclization.²²

Several pathogenic bacteria utilize AHL QS systems to control the expression of virulence genes and biofilm formation.^23–25 Hence, targeting QS may be used as a means to provide protection against pathogens that rely on QS to initiate biofilm formation.¹³ Since disruption of QS would presumably only impede the expression of virulence genes, without affecting viability, there is less selective pressure for the development of multidrug resistance.^26,27 Given the significant differences in the chemical structures of the signals, and in its perception by the cognate receptor across phyla, therapeutics that target QS are expected to have narrow spectrum activity.²⁶ Such target-specific agents could exert antivirulence activity without disruption of the host microflora.²⁸ The AHL signaling circuit has also been successfully used as a part of the synthetic biology toolkit for several applications, including whole-cell microbial biosensors for pathogen diagnostics and therapeutics,^29,30 cancer therapy,³¹ as well as plant pathogen biocontrol^32,33 and prevention of biofouling.^3,13

AHL molecules are synthesized by cognate LuxI-type signal synthases, using as precursors S-adenosylmethionine (SAM) and fatty acids conjugated to either acyl carrier protein (acylACP) or coenzyme A (acyl-CoA).^34–39 Subsequently, the specific AHL signal binds to a corresponding LuxR-type regulator to activate expression of QS-dependent genes.^25,40 The LuxR receptors have coevolved with their cognate LuxI synthases, and the specificity of the AHL signal is determined by the acyl chain length and branching (Figure 1A).^41–44 Characterized signal molecules possess fatty acyl chains that differ in chain length, and in the degree of saturation, and/or oxidation state at the β-carbon.²⁵

Figure 1.

(A) Chemical structures of AHL signals produced by different proteobacteria. (B) Phylogenetic tree of 71 CoA-dependent quorum signal synthases. The Uniprot ID and enzyme names are labeled in different colors based on their cluster codes in sequence similarity network (SSN). Sequences from cluster 1a including BjaI are labeled in red. Sequences from cluster 1b including RpaI and BraI are labeled in blue. Sequences from cluster 1c including MesI and MaqI are labeled in green. Sequences from cluster 2 including MplI are labeled in purple.

In 2008, Harwood and colleagues identified aryl-homoserine lactones (aryl-HSL) as a new class of AHL signal.³⁹ So far, only two aryl-HSLs have been characterized, p-coumaroyl-HSL (pC-HSL) from Rhodopseudomonas palustris and cinnamoyl-HSL from Bradyrhizobium ORS278, the production of which is catalyzed by the LuxI homologues, RpaI and BraI, respectively.^37,39 Both RpaI and BraI are unusual CoA-dependent AHL synthases that use an acidic substrate that is imported from the environment. The third known class of CoA-dependent AHL synthase is BjaI, which catalyzes the formation of a branched-chain fatty acyl-HSL isovaleryl-HSL (IV-HSL).^38,45 These CoA-dependent enzymes constitute a new subfamily of AHL synthases that utilize a substrate derived from the environment, rather than from cellular pools.^37–39 The growing interest of AHL signaling in synthetic biology and as a therapeutic target require further understanding of the molecular basis of the substrate specificity of AHL synthases. To this end, we present biochemical, structural biological, and kinetic studies of a representative subset of these aryl- and alkyl-CoA-dependent AHL synthases.

RESULTS AND DISSCUSSION

Structure of an Aryl-CoA-Dependent AHL Synthase.

We recently reported the biochemical characterization of BjaI from B. japonicum, which showed that the preference for an acyl-CoA, rather than an acyl-ACP substrate, is established through two Trp residues that provide an “indole platform” that is critical for CoA binding.⁴⁵ A structure-based classification of CoA-dependent AHL synthases identified more than 70 CoA-dependent AHL synthases. At the sequence level, these newly identified signal synthases could be grouped into four clusters, which we termed as clusters 1a, 1b, 1c, and 2.⁴⁵ The acyl specificities of members of the clusters other than 1a (i.e., BjaI) have yet to be characterized (Figure 1B).

Sequences found in cluster 1b include those encoding for the known aryl-HSL synthases, RpaI (pC-HSL) and BraI (cinnamoyl-HSL). In order to understand the rationale for the aryl-CoA substrate specificity of these enzymes, we determined the 1.85 Å resolution structure of RpaI in complex with CoA and p-coumaroyl S-adenosyl-L-homocysteine (pC-SAH; Figure 2A, Table S1). Clear and continuous electron density can be visualized for the pC-SAH, which was produced in situ by incubation of the enzyme with SAH and pC-CoA (Figure 2B). The canonical “indole platform” in RpaI, comprising Trp 146 and 147, matches with that of BjaI in both 3D structure and primary sequence (Figures 3A and B, Figure S1).⁴⁵ The CoA binding pocket is lined with numerous hydrophobic residues, including Ile 28, Tyr29, Trp35, Val106, and Trp147 (Figure S1).

Figure 2.

(A) Ribbon diagram of RpaI in complex with pC-SAH (in yellow) and CoA (in gray), and secondary structure elements demarcated with helices colored in turquoise, strands colored in salmon, and coils colored in tan. (B) Simulated annealing difference Fourier maps (F_o – F_c) of RpaI complexes contoured to 2.5σ (blue) showing the bound pC-SAH. The coordinates of the ligand were omitted prior to map calculations, and the final refined coordinates are superimposed.

Figure 3.

(A) The closeup view of pC group binding site pocket of RpaI complex structure showing the key interaction and the key residues. (B) Michaelis–Menten curves obtained by measuring CoA production over varying concentrations of pC-CoA (RpaI) and cinnamoyl-CoA (BolI). (C) WebLogo frequency plots for the acyl-group binding pockets of cluster 1b,⁴⁶ with the numbers representing the residues in RpaI.

In the RpaI cocrystal structure, a number of small hydrophobic residues form the p-coumarate binding pocket, and these include Leu101, Ile104, Ile141, Leu143, Trp147, and Leu151 (Figure 3A,B). In addition, RpaI contains a hydrophilic residue (Gln124), the side chain of which is within hydrogen bonding distance (2.7 Å) of the hydroxyl group of p-coumarate. The corresponding residue in BjaI (Met119) is displaced from the active site and does not make contact with the substrate isovaleryl group. A hydrophobic residue in BraI (Leu125) serves a similar function to the hydrophilic Gln124 of RpaI (Figure 3C and Figure S2), reflecting the difference in specificity between the p-coumarate and cinnamate substrates used by RpaI and BraI, respectively. Last, BraI possesses a Phe152 in place of Leu151 of RpaI, which constrains the binding pocket to accommodate the smaller cinnamate group. Thus, enzymes in cluster 1b can be easily separated into two subgroups, one of which takes pC-CoA as substrate (like RpaI), and the other uses cinnamoyl-CoA as a substrate (like BraI). In the latter cluster, a putative acyl-HSL synthase from Bradyrhizobium oligotrophicum S58, named BolI, possesses a Leu125 in place of Gln124 (Table S2). In order to demonstrate that BolI does indeed utilize cinnamoyl-CoA as a substrate, as predicted by the structure-based sequence analysis, we carried out kinetic studies of recombinant BolI. Our kinetics analysis yields a K_m of 3.0 μM for BolI using cinnamoyl-CoA, which is comparable with the K_m of 2.6 μM for RpaI against pC-CoA as a substrate (Figure 3B and Figure S3).

Substrate Specificity of Clusters 1c and 2.

On the basis of the observation that a difference in the specificities between aryl vs alkyl fatty acid substrates may be reflected at the sequence level, we sought to understand the substrate preference for representative sequences from clusters 1c and 2 (Table S2). Toward this goal, we first determined the structure of MesI from cluster 1c to 1.93 Å resolution. As we were unable to obtain a structure of any member from cluster 2, we utilized the structural data reported here to generate a homology model of MplI from cluster 2 (Figure 4). A comparison of the MesI ligand pocket shows that the pocket volume is smaller than that in RpaI. A notable difference between the binding pockets is the replacement of Leu101 of RpaI with Trp103 in MesI. An equivalent Trp102 is present in the sequence of MplI suggesting that enzymes from clusters 1c and 2 likely do not accommodate aryl-CoA. This assertion is supported by their extremely low catalytic efficiency using pC-CoA as a substrate, with a k_cat/K_m of about 20.0 M⁻¹ s⁻¹ for MesI and 30.0 M⁻¹ s⁻¹ for MplI (Table 1; Figures S4 and S5). The structure of BjaI, which utilized isovaleryl-CoA, shows Trp101 at the same location, further lending credence to the notion that cluster 1c and 2 enzymes function on linear or branched acyl-CoA substrates.

Figure 4.

Primary sequence alignment of the AHL synthases RpaI, BjaI, MesI, and MplI, which represent the four clusters of CoA-dependent AHL synthases. Residues involved in forming hydrophobic CoA binding pocket were marked in a red rectangle. The “indole platform” residues were marked in a green rectangle. The residues important for acyl group binding were marked in a blue rectangle.

Table 1.

Steady State Kinetic Parameters for MesI and MplI

	MesI			MplI
Acyl-CoA	kcat (s−1) × 10−3	Km (M) × 10−6	kcat/Km (M−1 s−1) × 102	kcat (s−1) × 10−3	Km (M) × 10−6	kcat/Km (M−1 s−1) × 102
isobutyryl-CoA	17.0 ± 0.6	33.9 ± 3.9	5.0 ± 0.6	6.2 ± 0.3	6.2 ± 1.2	10.0 ± 2.0
butyryl-CoA	9.9 ± 0.6	21.7 ± 4.4	4.6 ± 1.0	9.1 ± 0.5	0.5 ± 0.1	19.5 ± 3.6
isovaleryl-CoA	2.2 ± 0.1	34.4 ± 4.8	0.6 ± 0.1	2.5 ± 0.1	1.8 ± 0.192	14.3 ± 1.6
3-methylvaleryl-CoA	1.9 ± 0.1	14.3 ± 2.2	1.4 ± 0.2	3.5 ± 0.2	10.2 ± 1.6	3.4 ± 0.6
4-methylvaleryl-CoA	2.7 ± 0.2	18.8 ± 4.0	1.5 ± 0.3	3.0 ± 0.2	12.6 ± 3.4	2.4 ± 0.7
3-cyclopropylbutyryl-CoA	1.0 ± 0.1	23.8 ± 7.9	0.4 ± 0.2	6.9 ± 0.3	20.2 ± 3.0	3.4 ± 0.5
hexanoyl-CoA	9.3 ± 0.3	4.3 ± 0.6	21.7 ± 3.2	14.4 ± 1.1	5.0 ± 1.3	29.0 ± 7.8
5-methylhexanoyl-CoA	8.4 ± 0.3	10.3 ± 1.4	8.2 ± 1.2	6.1 ± 0.4	16.3 ± 2.9	3.7 ± 0.7
octanoyl-CoA	4.8 ± 0.1	7.3 ± 1.0	6.6 ± 1.0	11.5 ± 0.6	0.9 ± 0.3	131.1 ± 39.2
decanoyl-CoA	1.1 ± 0.1	6.3 ± 1.0	1.7 ± 0.3	6.5 ± 0.4	0.7 ± 0.2	99.7 ± 30.5
lauroyl-CoA	1.7 ± 0.1	9.1 ± 1.1	1.9 ± 0.2	25.4 ± 3.1	0.4 ± 0.2	587.5 ± 235.0
tetradecanoyl-CoA				2.4 ± 0.1	0.7 ± 0.1	37.1 ± 7.6
palmitoyl-CoA				1.8 ± 0.1	4.9 ± 1.0	3.6 ± 0.7
p-coumaroyl-CoA	1.7 ± 0.1	120.2 ± 18.5	0.2 ± 0.0	1.0 ± 0.1	36.2 ± 4.7	0.3 ± 0.1

The biggest difference between the respective putative acyl-chain binding pockets of cluster 1a (BjaI) and cluster 1c (MesI) signal synthases is the replacement of large hydrophobic residues (Phe147 and Met139 in BjaI) with smaller aliphatic residues (Leu153 and Cys145, respectively in MesI). On the basis of the larger binding site cavity in MesI, we theorized that the enzyme would likely utilize a longer, unbranched acyl chain substrate. In order to test this theory, we carried out kinetic studies of MesI using acyl-CoAs with different lengths of acyl chains (Table 1). MesI showed the highest catalytic efficiency using hexanoyl-CoA (C6), relative to that using shorter (C3/C4/C5) or longer (C8/C10/C12) CoA-linked substrates. There is a notable decrease in catalytic efficiency using a branched substrate; for example, the catalytic efficiency for 5-methylhexanoyl-CoA was 2.6 fold less than for hexanoyl-CoA, and for IV-CoA it was 7.5 fold less than for butyryl-CoA. These data support the notion that MesI and close homologues function on linear acyl-CoA substrates, specifically hexanoyl-CoA. In cluster 1c (Figure S6), 6 out of 13 AHL synthases share the identical five residues with MesI that are involved in the formation of acyl group binding pocket, including Trp103, Phe106, Cys145, Trp149, and Leu153, suggesting similar substrate preferences for enzymes across the entire cluster.

With the above structure–function correlations in hand, we speculate that the acyl-CoA specificities of cluster 1c sequences that contain variances at key residue may be predictive (Figure 5A and B; Figure S6). For example, sequence UniProt ID A0A0J6T9G4 has Ala and Val in place of Cys145 and Leu153 found in MesI, respectively, which indicates that it may use heptanoyl-CoA (C7) as a substrate, considering it contains a one carbon shorter Val rather than Leu. Another sequence (UniProt ID: B8I9S4) has Ala in place of Leu153, which suggests it may take the even longer C8-CoA as the preferred substrate. This homologue also contains a Thr residue in place of the Cys145 found in MesI. Signal synthases that utilized a 3-oxo-containing substrate similarly have an equivalent Thr142 (LasI) and Thr140 (EsaI), suggesting that this sequence may utilize a 3-oxo-acyl donor. Three other class 1c sequences (UniProt ID: H0I157, A0A0K6HEL7, and A0A0K2DFB2) have four identical key residues, but an Ile residue at the position of Phe106 of MesI, which suggests that they may use branched hexanoyl-CoA as the acyl donor.

Figure 5.

(A) The structural alignment of MesI and BjaI, with BjaI residues in purple and MesI residues in cyan, and MesI residues numbering are labled in blue. (B) WebLogo frequency plots for the predicted acyl-group binding pockets of cluster 1c,⁴⁶ with the numbers representing the residues in MesI. (C) The structural alignment of MplI and BjaI, with BjaI residues in purple and MplI residues in gray, and MplI residues are labled in blue. (D) WebLogo frequency plots for the predicted acyl-group binding pockets of cluster 2, with the numbers representing the residues in MplI.

The last AHL synthase in cluster 1c is from Methylobacterium aquaticum (UniProt ID: A0A0C6FYQ0), which we have named MaqI. MaqI shows the lowest similarity to MesI regarding the five key residues involved in acyl group binding. MaqI contains Cys, Leu, and Val, respectively, in place of Trp103, Phe106, and Cys145 of MesI. Of these changes, the replacement of Trp103 with a Cys would create a pocket that may accommodate a larger/longer acyl group. In order to validate this expectation, we carried out high-resolution LC-MS analysis of MaqI using various acyl-CoAs and SAM, which demonstrated that this enzyme can catalyze the formation of long chain acyl-HSL, using linear C6-, C10-, and C12-, but not cyclic p-coumaroate-, CoA thioesters as substrates (Figure S7). To further investigate its acyl-chain length limit, we performed kinetics studies for lauroyl- (C12) and palmitoyl-CoA (C16), which showed that lauroyl-CoA (C12) is a better substrate with about 8 times higher catalytic efficiency than palmitoyl-CoA (C16; Figure S3). Hence, the likely physiological acyl donor of MaqI is C12- or a closely related acyl CoA.

Having addressed the substrate scope of sequences in cluster 1c, we next turned our attention to the CoA-dependent signal synthases that fall in cluster 2. In general, these sequences are indicative of enzymes with acyl-CoA binding pockets that are largely similar to that in BjaI but, as in the case with cluster 1c enzymes, have small aliphatic substitutions. Most notably, in the sequence of MplI, the Tyr104 that is located at the periphery of the acyl-binding pocket in BjaI, is replaced with Thr105. This substitution creates a tunnel, which may allow for accommodation of a very long acyl chain (Figure 5C). To test this prediction, we carried out kinetic studies of MplI against a panel of acyl-CoAs with various acyl chain lengths from C3 to C14 (Table 1; Figures S4 and S8). These studies supported this hypothesis as MplI showed the highest catalytic efficiency against lauroyl-CoA (C12), with a k_cat/K_m of 58.8 × 10³ M⁻¹ s⁻¹, suggesting longer acyl-CoA might be the physiological substrates for cluster 2 enzymes. Within this cluster 2, there is a total of 25 AHL synthases, and 20 of them share identical residues with MplI, which are involved with the acyl group binding (Figures 5D and S9), which indicate similar acyl donor specificity. The validation of sequence-based substrate scope predictions outlined here demonstrates that it may be feasible to predict the scope of any CoA-dependent AHL synthase using the strategy outlined above. Such sequence-based identification of substrate scope is of critical importance in both in vivo biotechnological efforts (so as to limit cross-talk from endogenous fatty acyl-CoAs) as well as for the design of therapeutics that target the quorum-signaling pathway.

Structure-Based Engineering of LuxI Synthases.

The structure–function studies described have allowed identification of AHL synthases with different specificities including those for aryl-, branched alkyl-, linear alkyl-, and 3-oxo alkyl-CoA. For some applications, it may be necessary to use a non-native AHL synthase whose specificities are tuned to one molecule. We theorized that with knowledge of the key residues in LuxI homologues that are involved in the substrate recognition, we should be able to engineer a synthase that produces a non-natural AHL signal, which may be of use for synthetic biology.

Our structural data on aryl-CoA specific RpaI showed that Gln124 determines the identity of the pC group as an acyl donor due to the hydrogen bonding interactions between the 4-hydroxyl of pC and the side chain of Gln124. In BraI, which uses cinnamoyl-CoA lacking the substitution at the 4 position of the aryl ring as a substrate, the equivalent residue is Leu125. On the basis of this observation, we reasoned that the Gln124→Ala variant of RpaI might be able to use the artificial methylated pC-CoA (Me-pC-CoA; 4-OMe-cinnamoyl-CoA), as a substrate (Table S3). While wild-type RpaI only produced less than 10% Me-pC-HSL when incubated with a mixture of pC-CoA and Me-pC-CoA in a 1:1 ratio (Figures 6A and S10), the Gln124→Ala variant of RpaI produced over 80% Me-pC-HSL under the same reaction conditions. Hence, this small, directed change in the donor-binding pocket allowed for the engineering of an AHL synthase with a novel substrate profile.

Figure 6.

Relative catalytic efficacy of wild-type and engineered variants of RpaI (A), MesI (B), BjaI (C), and MplI (D) using various acyl-CoAs as substrates. The identity of each LuxI AHL syntetases was labeled on top of the corresponding bar graph, and each variant was shown on the X axis. The Y axis represented the ratio of production of different AHLs, which were shown in different colors.

Having demonstrated that this structure–function approach may be utilized for the engineering of synthases that utilize non-natural aryl-CoA substrates, we next focused attention on manipulating the substrate tolerance for acyl-CoAs. We reasoned that such efforts might afford AHL synthases whose substrate profiles do not overlap with cognate fatty acid pools, facilitating in vivo biotechnological applications in any given organism of interest. We first examined MesI (cluster 1c) that prefers C6-CoA as an acyl donor to other length acyl-CoAs (Table 1). For example, this enzyme produced more than 60% C6-HSL, but only about 21% C4-HSL and 17% C8-HSL, when incubated with a 1:1:1 ratio of C4-, C6-, and C8-CoAs (Figures 6B and S11).

We determined the crystal structure of MesI to 1.93 Å resolution, and a structural comparison with BjaI shows that Leu153 of MesI is situated at the end of the acid chain. We speculated that this residue likely restricts chain length specificity to C6-CoA, as BjaI, which can only accommodate a C4 substrate (i.e., isovaleryl-CoA), contains Phe147 at the equivalent position (Figure 5A). In order to test this hypothesis, we generated a Leu153→Ala variant of MesI in order to increase the acyl chain length specificity to beyond a C6. As expected, this variant produced about 70% C8-HSL and 30% C6-HSL when incubated with stoichiometric concentrations with the corresponding acyl-CoAs (Figure 6B). Conversely, the Leu153→Phe variant of MesI produced over 65% C4-HSL and less than 35% C6-HSL, similar to the substrate profile of BjaI. Next, we generated a Phe147→Tyr variant of BjaI that shows preference for the production of linear C4-HSL (~80%) over the native branched IV-HSL (~20%; Figures 6C and S12). Presumably, the hydroxyl group of Tyr147 occupies the position of the branched methyl group in the native isovalerate substrate (Figure 5C). Last, we focused on MplI from cluster 2 that uses longer chain acyl CoA substrates (such as C12-CoA), presumably due to the replacement of Thr105 at a location that is normally occupied by a larger aromatic residue. Consequently, the Thr105→Tyr variant of MplI prefers the production of C4-HSL (~ 70%) to C12-HSL (~ 30%; Figures 6D and S13). These studies demonstrate that both the chain length and branching specificity of the CoA substrate may be manipulated through judicious alterations at a few positions in the active site of these AHL synthases, establishing the framework for the engineering of “designer” AHL synthases for synthetic biological applications.

CONCLUSION

Here, we describe the crystal structures of two classes of CoA-dependent AHL synthases. In combination with our previously determined structure of a aryl-CoA-dependent AHL synthase, we were able to utilize the structural data to identify residues that likely confer substrate specificity among the different classes of these enzymes. We provide support for our predictions by carrying out biochemical, and kinetic, characterization of representation AHL signal synthases. These studies should provide a framework for the design of target-specific agents that exert antivirulence activity by modulation of the QS circuitry.

Due to their substrate specificity and low metabolic cost, AHL QS systems have been successfully used as synthetic biology tools, as well as for the design of whole-cell microbial biosensors that can target pathogens and tumor cells. Our engineering studies presented here provide such a framework toward rational design of LuxI synthases for the production of versatile AHLs with acyl chains of different sizes and lengths. Moreover, as both the ACP-dependent and CoA-dependent LuxI synthases use the same mechanism to synthesize the AHL molecules, the substrate scope prediction method obtained from the CoA-dependent type can be potentially applied in the ACP-dependent enzymes. Last, knowledge of the substrate scope for the signal synthase will also inform the specificity of the cognate receptor, providing multiple targets for the design of therapeutics that target signaling only in specific QS clades.