The malonyl/acetyl-transferase from murine fatty acid synthase is a promiscuous engineering tool for editing polyketide scaffolds

doi:10.21203/rs.3.rs-3914462/v1

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The malonyl/acetyl-transferase from murine fatty acid synthase is a promiscuous engineering tool for editing polyketide scaffolds

https://doi.org/10.21203/rs.3.rs-3914462/v1

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published 24 Aug, 2024

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Modular polyketide synthases (PKSs) play a vital role in the biosynthesis of complex natural products with pharmaceutically relevant properties. Their modular architecture makes them an attractive target for engineering to produce platform chemicals and drugs. In this study, we demonstrate that the promiscuous malonyl/acetyl-transferase domain (MAT) from murine fatty acid synthase serves as a highly versatile tool for the production of polyketide analogs. We evaluate the relevance of the MAT domain using three modular PKSs; the short trimodular venemycin synthase (VEMS), as well as modules of the PKSs deoxyerythronolide B synthase (DEBS) and pikromycin synthase (PIKS) responsible for the production of the antibiotic precursors erythromycin and pikromycin. To assess the performance of the MAT-swapped PKSs, we analyze the protein quality and run engineered polyketide syntheses in vitro. Our experiments include the chemoenzymatic synthesis of fluorinated macrolactones. Our study showcases MAT-based reprogramming of polyketide biosynthesis as a facile option for the regioselective editing of polyketide scaffolds.

Biological sciences/Biochemistry/Enzymes/Multienzyme complexes

Biological sciences/Biochemistry/Biocatalysis

Polyketides (PKs) are natural products, some of them exhibiting strong biological activities, such as the antibiotic erythromycin^1,2 or the immunosuppressant rapamycin^3,4. They are produced by polyketide synthases (PKSs) through subsequent connection and modification of simple acyl building blocks^5,6. Multienzyme type I PKSs possess an exceptional ability to synthesize complex compounds. They divide into two types: (i) Modular PKSs consist of several modules that assemble into linear metastructures (Fig. 1)^7,8. They operate in a sequential manner with each module elongating the growing polyketide intermediate by two carbon atoms. Optionally, each module can further modify the intermediate before it is handed over to the next module. Catalytic activity in modular PKSs is provided on enzymatic domains. Essential domains for chain elongation include the acyltransferase (AT) domain, which selects the extender substrate (typically a malonyl- or methylmalonyl-CoA⁹), the ketoacyl synthase (KS) domain, which uses the extender substrate to elongate the PK intermediate, and the acyl carrier protein (ACP) which is responsible for the transport of the substrates and intermediates within and also between the modules¹⁰. If present, processing domains reduce and dehydrate the intermediate, which can introduce stereochemistry at the α- and β-position of the PK intermediate (Figs. 1A and S1). (ii) The iterative PKSs, which are monomodular systems, synthesize PKs via multiple cycles of chain elongation and processing carried out by the same module^11–14.

Typically, modular PKSs exhibit a high product fidelity which can be attributed in part to the specificity of the substrate-selecting AT domains¹⁵. Different engineering attempts have been made, to alter the products of PKSs. Among these strategies, modification of the AT domains has demonstrated great potential, as it allows editing the polyketide scaffold with chemical entities that are introduced via the substrates¹⁶. Essentially, three strategies have been applied (Fig. 1B): (i) The AT domains have been engineered in the binding pocket to modulate substrate specificity^17,18. For example, Williams and coworkers achieved the modification of macrolactones with large alkyl moieties (e.g., butyl and pentyl) by an approach combining structure-guided mutagenesis and in silico modeling¹⁹. (ii) The AT domains have been swapped with domains of different substrate specificity^20–23. An early study following this strategy was conducted on the deoxyerythronolide B synthase (DEBS) by Leadlay and coworkers, substituting the methylmalonyl-CoA (MMalCoA) specific AT domain from module 1 of DEBS1-TE with the malonyl-CoA (MalCoA) specific AT domain from module 2 of the rapamycin synthase²⁴. More recently, the substrate-specific AT domain of module 6 from DEBS was replaced by the promiscuous malonyl/acetyl-transferase (MAT) domain from the related murine fatty acid synthase (mFAS). This strategy allowed loading extender substrates that are not recognized by the native DEBS AT domain, most importantly fluorinated MalCoA analogs, yielding macrolactone backbones similar to that of solithromycin^25,26. (iii) Finally, the AT domains have been inactivated by point mutation and complemented with stand-alone trans-AT enzymes for loading the modular PKSs through protein-protein interactions^27–29. Using fluoromalonyl-CoA (FMalCoA) as substrates, Chang and coworkers recently employed this strategy to leverage the synthesis of fluorinated erythromycin analogs^26,30.

The domain-swapping strategy benefits from transferring the functional properties of the external domain to the accepting PKS module, enabling the efficient regioselective editing of the polyketide scaffold. However, the strategy is invasive, as the external domain introduces non-cognate domain-domain interfaces that can destabilize the PKS module. Furthermore, non-cognate transient transferase-ACP interactions arise during the loading of the intermodular ACP with the substrate³¹. The AT domains in modular PKSs are typically substrate selective and thus in control of the accurate selection of extender substrates supplied for the condensation reaction³². In iterative PKSs, AT domains are usually responsible for loading starter and extender substrates, which makes them inherently less specific. The MAT domain from mFAS is well characterized as highly substrate promiscuous and kinetically fast enzymatic domain, allowing a variety of substrates to be efficiently loaded^33,34. On the basis of these properties and the high structural similarity of PKSs and animal FASs³⁵, we have recently achieved the chemoenzymatic synthesis of fluorinated macrolactones by AT/MAT-swapped FAS/PKS hybrid modules²⁵. In the present work, we evaluate the scope of this approach to additional PKS modules and demonstrate that AT/MAT-swaps can be accomplished with high success rates. We engineer functional PKS modules and assembly lines that generate new polyketides, including fluorinated derivatives of precursor macrolactones of the antibiotics methymycin and pikromycin. Specifically, we were working with the recently established split venemycin PKS (split VEMS, Fig. 2) in vitro testbed³⁶, the engineered monomodular pikromycin synthase (PIKS) M5-TE³⁷, and the bimodular DEBS3 (Fig. 1).

VEMS is a short modular PKS consisting of three modules (Fig. 2A)³⁸. It utilizes the starter substrate 3,5-dihydroxybenzoic acid (DHBA) and elongates it twice using the elongation substrate MalCoA, resulting in the production of venemycin 1. Recently, we developed a split version of VEMS, where each module is located on an individual polypeptide chain, allowing recombinant expression in Escherichia coli in high yield without loss in activity (Fig. 2B)³⁶. Split VEMS is a useful engineering platform, as it permits to target the PKS-comprising modules interdependently. In this study, we aimed to introduce MAT swaps in all three modules of VEMS, allowing to target three positions of the 2-pyrone product scaffold (Fig. 2C). In this context, we tested different domain boundaries and found that the boundaries previously defined for DEBS module 6 (M6)²⁵, are most effective for VEMS as well.

Next, we used the validated boundaries to introduce AT/MAT-swaps to modules of the prominent PKSs PIKS (Figure S2) and DEBS (Figs. 1A and S1A). Four hybrid constructs were targeted, systematically exchanging the specific AT domains of the engineered PIKS M5-TE and DEBS M5-M6-TE with the promiscuous MAT domain from mFAS (Fig. 2C). The functionality of the constructs was assessed with different extender substrates, yielding C12- and C14-membered macrolactone scaffolds with site-selective modifications.

Engineering a PKS assembly line with a promiscuous loading module-

Design of MAT-swapped loading modules for split VEMS-

First, we sought to introduce the polyspecific MAT domain into the VEMS loading module (M0), thereby creating a promiscuous hybrid loading module that allows for priming PK synthesis with various acyl-CoA starter substrates. The VEMS M0 comprises an adenylation (A) domain, responsible for selection and activation of the starter substrate DHBA, which is subsequently loaded onto the ACP domain (Fig. 2A). Additionally, VEMS M0 features a nonfunctional KR domain³⁸ that is believed to play a structural role³⁹. In the design of hybrid modules, it is inevitable to generate non-cognate domain-domain interactions (DDIs). Dependent on the characteristic of the non-cognate interface, this can affect the performance of the engineered PKS. Due to the intricate nature of this issue, two different hybrid designs were evaluated (Fig. 3A): For one hybrid M0 (H1M0) the A-KR section was replaced by the MAT domain derived from mFAS and the VEMS ACP0 was further exchanged with the mFAS ACP, thereby ensuring native DDIs between MAT and ACP, both from mFAS, interacting during transacylation. In this case, a non-cognate interface arises during the translocation step between the mFAS ACP of M0 and the VEMS KS1 of the downstream M1 (Figure S3). In the second hybrid M0 (H2M0), the VEMS ACP0 was retained providing native interplay between the ACP0 and the KS1 during translocation. In this case, non-cognate DDIs occur during the transacylation between the mFAS MAT and the VEMS ACP0. The constructs were conceived based on a recent design of an mFAS-derived loading module from our lab. In this construct, the mFAS MAT domain was excised and fused to the mFAS ACP via a flexible 12-residue long linker³³. Both hybrid modules H1M0 and H2M0 feature a C-terminal DEBS-derived docking domain to enable docking to the split VEMS M1, carrying the matching docking domain N-terminally.

Evaluation of hybrid loading modules-

The two hybrid loading modules were produced in E. coli and purified. While H1M0 was purified via a single affinity tag and could not be obtained in purity, H2M0 was purified via tandem affinity chromatography and obtained in high purity, which was further validated via size exclusion chromatography (Figure S4). To assess the functionality of the hybrids loading modules, they were tested within the context of a complete assembly line (HXM0 – M1 – M2-TE, Fig. 3A). It was assumed that the MAT domain can load its C-terminal ACP (mFAS ACP for H1M0 and VEMS ACP0 for H2M0) with a starter substrate, which then primes synthesis in split VEMS M1. Next, the starter substate is elongated with MalCoA in M1 and M2-TE. The resulting triketide intermediate bound to the ACP2 of the final module M2-TE is then released under pyrone-ring formation. As a starter substrate, we decided to use acetyl-CoA (AcCoA), because it is known to be the preferred starter substrate for the MAT domain³³. AcCoA would result in the formation of triacetic acid lactone (TAL, 2), which can be analyzed by liquid chromatography-mass spectrometry (LC-MS). Notably, the expected mass could only be found using H2M0 (Fig. 3A), preserving the native ACP-KS interface of chain translocation.

Exploiting the promiscuity of the MAT domain to produce 2-pyrone derivatives-

With a functional MAT-swapped loading module in hand, we sought to exploit the reported promiscuity of the MAT domain³³ to load the VEMS-based platform with a pool of starter substrates (Fig. 3B). A total of five starter substrates (acetyl-CoA, propionyl-CoA, butyryl-CoA, crotonyl-CoA, and 4-hydroxybutyryl-CoA), known to be accepted by the MAT domain¹³³, were tested. To investigate the tolerance of the downstream modules, these starter substrates were selected to possess varying chain lengths and oxidation patterns at the α and β positions. LC-MS analysis confirmed the production of 2-pyrone compounds (2, 3, and 4) from three out of five tested starter substrates.

MAT swaps in Elongation Modules of split VEMS-

Subsequently, we sought to replace the MalCoA-specific transferases (AT1 and AT2) of VEMS elongation modules (M1 and M2-TE) with the promiscuous MAT domain. Previous studies have demonstrated that hybrid PKSs created by domain swapping frequently suffer from decreased catalytic activity and insolubility due to misfolding of the protein subunits^40,41. Due to the lack of original structural data for VEMS, we utilized computational modeling (ColabFold⁴²) to predict the structures of relevant VEMS subunits. The linker domain (LD) is of particular importance for AT/MAT-swaps, as it serves as a spacer between the KS and transferase (AT or MAT) domain. The LD is comprised of two distinct parts: LD₁, which is the part sandwiched between KS and transferase, and LD₂, which wraps back onto the KS surface and connects the transferase domain to the downstream ACP domain (Figs. 4, S5, and S6). The essential question for the AT/MAT-swap is, whether the transferase should be swapped including or excluding the LD or whether it should be cut within the LD fold. In order to increase the chance of creating a functional hybrid construct, a broad range of domain boundaries for AT/MAT-swaps was tested. We categorized boundaries into three groups depending on how the LD was treated, i.e., whether it was retained from the acceptor module (VEMS M1 or VEMS M2-TE) or derived from the donor module (mFAS) (Figures S5 and S6). In H1 constructs, the boundaries were positioned close to the core fold of the AT domain while preserving the LD (LD₁- and LD₂-part) of the accepting VEMS. Conversely, in H2 constructs, the LD was derived from the donating mFAS. Lastly, in the H3 group, the boundaries were set so that the LD₁-part originated from the acceptor module, while the LD₂-part was derived from the donor module (Fig. 4).

AT/MAT-swapped elongation module VEMS M2-TE-

For VEMS M2 TE, a total of 24 hybrid constructs M2*-TE were designed (Figure S5), comprising 6 H1-, 8 H2-, and 10 H3-constructs, and subjected to test expression in E. coli. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that just certain constructs express, albeit at insufficient quantities to allow protein isolation and analysis (Figures S7 and S8). The struggle to obtain functional hybrid constructs in adequate quantities underscores the intricate nature of the KS-AT didomain fold.

AT/MAT-swapped elongation module VEMS M1-

Similarly, as for VEMS M2-TE, different boundaries for the AT/MAT-swap in the first elongation module M1 were tested. M1 engineering was performed in the context of the full-length polypeptide VemG (M0-M1) because we assumed highest protein stability under native-like conditions. The boundaries of the best expressible hybrid construct should then be applied to the engineered M1 of split VEMS (M1). Four constructs were tested for expression in E. coli (2 of H1, 1 of H2, and 1 of H3 group, Figs. 4 and S6), of which one construct of the H1 group yielded enough protein for further analysis (Figure S9). To confirm functionality, the AT/MAT-swapped M0-M1* was assembled with M2-TE to VEMS. LC-MS analysis confirmed the formation of venemycin 1 when using the substrates DHBA and MalCoA, indicating functionality of the hybrid module (Figure S10). These boundaries were then utilized to create a stand-alone AT/MAT-swapped M1* (Figure S11) that is compatible with split VEMS. The AT/MAT-swapped hybrid module M1* was also active when used as a stand-alone module within the split VEMS testbed, as confirmed by product formation (Figure S10).

Since the polyspecific transferase offers promiscuous substrate loading, the hybrid assembly line was challenged with the non-native extender substrate MMalCoA, which can normally not be processed by split VEMS (Fig. 5). When using an extender substrate mixture of MalCoA and MMalCoA, the derivative 5-methyl-venemycin 7 was only produced by the assembly lines M0-M1* VEMS and M1* split VEMS, carrying the AT/MAT-swap, demonstrating the ability of the hybrid assembly line to incorporate non-native extender substrates. Of note, providing MalCoA and MMalCoA as extender substrates, M1* split VEMS also produced venemycin 1, as a result of loading both malonyl and methylmalonyl by the promiscuous MAT. The derivative 3,5-dimethyl-venemycin 8, which would result from the incorporation of MMalCoA by both elongation modules, could not be observed due to the substrate-specific AT of M2-TE.

Promiscuous Assembly Line with Substrate-Dependent Starting Points-

The mFAS-derived MAT domain possesses a dual functionality, meaning that it can prime but also continue fatty acid synthesis by loading the appropriate starter (AcCoA) or elongation substrate (MalCoA). This property is not found in modular PKSs, in which transferases are more diversified and either load the starter for priming synthesis or the elongation substrate for growing the polyketide intermediate. In this regard, the AT/MAT-swapped M1* offers the chance to harness M1* as an alternative starting position of polyketide synthesis. As a first test, we supplied propionyl-CoA to the hybrid M1* assembly line, which cannot be processed by split VEMS but is accepted by the MAT domain³³. Since AcCoA may arise in situ during synthesis by decarboxylation of MalCoA, we chose to work with propionyl-CoA. Confirmed by LC-MS, the hybrid split VEMS assembly line produced the respective 2-pyrone 3 (Fig. 6A). To further investigate the production of 3 by split VEMS, we systematically omitted modules of split VEMS and found that both the M1* and M2 modules were required for the production of 3 (Figure S12). Based on these data, we conclude that the production involves priming by M1*, followed by an elongation step by M1* before handing over the intermediate to M2-TE. Subsequently, M2-TE catalyzes a second elongation step and the release of compound 3. The dual functionality of the MAT domain turns split VEMS into an assembly line with two priming points that can be utilized depending on the chemical structure of the starter substrate: DHBA-derivatives are activated by M0³⁹ and forwarded to M1, while acyl-CoAs can be incorporated by MAT of M1*. Furthermore, the promiscuity of MAT in M1* enables the modulation of position 5 of the 2-pyrone scaffold when non-native extender substrates are used (Fig. 6B). We demonstrated the versatility of this assembly line by combining 5 different starter (DHBA, 3-hydroxybenzoic acid, acetyl-CoA, propionyl-CoA, and butyryl-CoA) and two extender substrates (MalCoA and MMalCoA) leading to 10 products with a 2-pyrone scaffold generated by one engineered PKS.

AT/MAT-swaps in mono-modular PIKS system-

To further interrogate the utility of the AT/MAT-swap strategy, we engineered a late-stage elongation module of the well-characterized PIKS, which is responsible for the production of the precursors of the antibiotics methymycin and pikromycin^43,44. We chose the recently engineered PIKS module 5 (PIKS M5) that is C-terminally elongated with the PIKS M6 TE domain giving an overall domain structure of KS-AT-KR-ACP-TE (termed PIKS M5-TE).³⁷ Domain boundaries for the AT/MAT-swap were chosen in accordance with the H1 boundaries previously established by Rittner and Joppe et al.²⁵ (Figure S15), which also performed best in the comprehensive hybrid design screening of VEMS. PIKS M5-TE and the AT/MAT-swapped PIKS M5*-TE were produced in E. coli, with PIKS M5*-TE yielding about 50% of the amount of the non-swapped PIKS M5-TE (Figure S16).

First, the activities of PIKS M5-TE and PIKS M5*-TE were tested chemoenzymatically by the in vitro product formation assay established by Sherman and coworkers⁴⁵ (Fig. 7). Activated PIKS pentaketide was employed as the elaborate starter substrate mimic and MMalCoA was added as the native elongation substrate of PIKS M5. The NADPH consumption of KR5 was monitored fluorometrically to evaluate the reaction progress. As confirmed by LC-MS, the hybrid module PIKS M5*-TE was able to produce the methymycin precursor 10-deoxymethynolide (10-dml, 14), although at a reduced speed of 0.18 min^− 1 compared to PIKS M5-TE (1.6 min^− 1). Next, we challenged the hybrid PIKS M5*-TE with the non-native extender substates MalCoA and fluoromethylmalonyl-CoA (FMMalCoA). The formation of the expected demethylated and fluorinated 10-dml derivatives 2-demethyl-10-dml 16 and 2-fluoro-10-dml 17 was confirmed by LC-MS. NADPH monitoring revealed the highest turnover using the extender substrate MalCoA (0.48 min^− 1), which is the preferred substrate of the MAT domain. In addition, 2-demethyl-3-oxo-10-dml 15 was identified as byproduct when incubated with MalCoA due to impaired acceptance of the demethylated intermediate by the KR5 domain such that the TE releases the non-reduced macrolactone 15⁴⁶. Unexpectedly, the formation of 2-fluoro-10-dml 17 was also observed with PIKS M5-TE, indicating that the native AT domain is “leaky” for the di-substituted FMMalCoA substrate.

AT/MAT-swaps in bimodular DEBS System-

Next, we applied the AT/MAT-swap to DEBS3, the ultimate bimodular polypeptide of the 6-deoxyerythronolide B-producing DEBS assembly line. DEBS3 comprises two covalently connected elongation modules, module 5 (M5) and module 6 (M6), each possessing the KS-AT-KR-ACP module composition and a C-terminal TE domain facilitating product release. Overall, we designed three bimodular PKS/FAS hybrid proteins by systematically replacing the DEBS AT domains (AT5 and AT6) with the MAT domain using H1 boundaries (Figure S15). In hybrid M5*-M6-TE, DEBS AT5 was replaced with the MAT domain, in hybrid M5-M6*-TE DEBS AT6 was replaced with the MAT domain, while in M5*-M6*-TE both AT domains were replaced with the MAT domain.

While hybrid M5*-M6-TE and M5-M6*-TE would allow the modification of position 4 or 2 of the C14-macrolactone product, respectively, hybrid M5*-M6*-TE simultaneously targets both positions. Recombinant production in E. coli yielded the hybrid M5-M6*-TE in high purity and similar yields to the WT DEBS3 (11 mg/L and 13 mg/L of purified protein, respectively, Figure S17). In contrary, M5*-M6-TE was obtained in lower yield (0.4 mg per liter) compared to DEBS3, and the double hybrid M5*-M6*-TE could not be isolated at all.

As before, the activated PIKS pentaketide was employed as a starter substrate and MMalCoA was added as the native elongation substrate of DEBS AT5 and AT6 (Fig. 8).³³ The reduction of NADPH by KR5 and KR6 was monitored fluorometrically to evaluate the reaction progress. DEBS3 as well as both hybrids revealed to be active and produced the C14-macrolactone 3-hydroxy-narbonolide 18 by two elongations. Of note, both hybrids, as well as native DEBS3 used as a control, produced 10-dml 14 as a byproduct. Two pathways could account for the detected 12-membered macrolactone byproduct: Production of 10-dml involves either omission of M6 after elongation by M5, followed by direct release through the TE domain of M6, resembling the natural PIKS pathway that accounts for 10-dml synthesis, or direct loading of KS6 of M6 with the PIKS pentaketide followed by elongation and processing to 10-dml, a mechanism which was previously exploited by others and us^19,25,47.

Subsequently, the functional hybrid proteins were examined for their ability to produce derivatives of 14-membered macrolactone by providing 1:1 mixtures of elongation substrates (MMalCoA/MalCoA or MMalCoA/FMMalCoA) and NADPH (Fig. 9).

While for both substrate mixtures, native DEBS3 produced WT product 3-hydroxy-narbonolide 18 only (with 10-dml 14 observed as a byproduct via module skipping, see Figure S18A & S19A, pathway (1)), the AT/MAT-swapped hybrid proteins led to more complex product outputs. M5*-M6-TE produced 4-demethyl-3-hydroxy-narbonolide 19 from the MMalCoA/MalCoA mixture as well as 4-fluoro-3-hydroxy-narbonolide 20 from the MMalCoA/FMMalCoA mixture. In MMalCoA/MalCoA-containing samples, no WT product 18 was observed in LC-MS and demethylated C-4 regioisomer 19 was the main product reflecting the efficiency of the MAT domain for MalCoA over MMalCoA. In contrast, a mixture of WT product 18 and C-4-fluorinated product 20 was observed in samples containing MMalCoA/FMMalCoA substrate mixture. The main product was compound 18, which is in line with the diminished efficiency of MAT towards FMMalCoA observed before²⁵. For MMalCoA/MalCoA substrate mixtures, byproducts 10-dml 14, 2-demethyl-3-oxo 10-dml 15 and 2-demethyl-10-dml 16 were identified from LC-MS which was attributed to either skipping of hybrid module M5* and direct loading of the starter substrate to M6 and elongation with MMalCoA (see Figure S18B, pathway (2)) or elongation with MalCoA in hybrid module M5*, optional KR5 skipping, followed by skipping of module M6 and direct TE-mediated offloading (see Figure S18B, pathway (3) for 15 and (4) for 16).

Similarly, hybrid M5-M6*-TE was subjected to mixtures containing MMalCoA/MalCoA or MMalCoA/FMMalCoA in order to prepare C-2 analogs 3-hydroxy-2-demethyl-narbonolide 21 and 2-fluoro-3-hydroxy-narbonolide 22 of WT product 18. Mass peaks attributed to the respective C-2-demethylated or C-2-fluoromethylated compounds 21 and 22 were identified. Substantial shifts in retention time of approx. 0.5 min for 3-hydroxy-2-demethyl-narbonolide 21 and 0.2 min for 2-fluoro-3-hydroxy-narbonolide 22, with respect to the C-4 regioisomers 19 and 20, indicate production of C-2 analogs. Chang and co-workers have recently shown that C-2 and C-4 regioisomers of C14 macrolactones can be distinguished based on their retention time³⁰. Hybrid M5-M6*-TE produced the demethylated C-2 regioisomer 21 as the main product in mixtures containing MMalCoA/MalCoA. No WT product 18 was observed in LC-MS but 2-demethyl-3-oxo-10 dml 15 and 2-demethyl-10-dml 16 were identified (see Figure S18D) and explained by direct loading of the starter substrate onto KS6 of the hybrid module M6*, elongation with MalCoA and offloading of the non-reduced (KR-skipping) and the reduced compound (Figure S18C, pathway (5) & (6)). The absence of 10-dml 14 in LC-MS indicated efficient translocation of the intermediate from M5 to M6*, loading of MalCoA in M6* only and no M6*-skipping. A product mix of C14-membered products 18 and 22 (with 10-dml 14 as byproduct, see Figure S19) was obtained for MMalCoA/FMMalCoA assays similar to the product mix obtained by hybrid M5*-M6-TE.

The implementation of the polyspecific MAT domain from mFAS in PKS engineering paves the way to the regioselective modification of PKS product scaffolds. In this study, we aimed to evaluate the broad applicability of AT/MAT-swaps in PKS engineering. We focused on the in vitro analysis of engineered proteins, and considered product formation as a tool to determine the functionality of the hybrid constructs. Specifically, we have expanded the AT/MAT-swap to new PKS assembly lines (split VEMS and PIKS, DEBS used previously²⁵) and interrogated the potential of the MAT’s ability in the promiscuous loading of starter and extender substrates in PKS/FAS hybrid proteins.

Successful domain swaps could be designed at different locations within the assembly lines and in modules of different domain compositions. For the first time, loading modules (VEMS M0) and non-terminal modules (VEMS M1 and DEBS M5) have been addressed for the AT/MAT-swap to incorporate non-native starter and extender substrates during PK synthesis. Remarkably, the boundaries that exhibited the best performance in the previously documented DEBS swap²⁵, consistently worked best across the tested boundaries in VEMS.

Based on our data, we can provide the following engineering guidelines for the AT/MAT-swap: (i) Prioritize the AT/MAT-exchange without flanking sequences (H1 boundaries), which in this study let to functional protein in five out of six modules, judged by yield and protein quality. Some PKS/FAS hybrid proteins with H2- and H3-designed boundaries were active in product formation assays (data not shown) but generally suffered from very low yields and poor purity. (ii). For none of the tested modules (VEMS M1 and M2-TE tested in this study, and DEBS M6-TE tested previously²⁵), alternative domain boundaries have proven to be superior. Thus, AT/MAT-swaps using different boundaries are likely to offer low chances of success. (iii) Protein quality will suffer from the AT/MAT-swap, such that monitoring protein quality is important, especially when working with complex proteins and multiple AT/MAT-swaps per polypeptide. We note that an elaborate biosensor-guided method has recently been published, in which AT swaps can be quickly evaluated in hybrid protein quality. The approach enabled screening of libraries of AT-exchanged hybrids in higher throughput, while finally identifying swap boundaries in agreement with our findings⁴⁰. As a limitation of our study, we note that our engineering rules are deviated from a dataset involving a total of six modules. The limited sample size derives from the goal of achieving a quantitative evaluation of protein performance in vitro.

With this approach, engineered PKSs can benefit from the broad substrate flexibility of the MAT domain which enables a more comprehensive expansion of the product spectrum compared to previous AT engineering efforts employing substrate-specific PKS AT domains^19,24. Overall, our study demonstrates the broad potential of employing the substrate-tolerant MAT domain for the regioselective editing of polyketide scaffolds during their biosynthesis. Of note, the accessible product scope can be limited by the tolerance of the downstream PKS domains to process the non-native intermediate. However, as a result of MAT’s promiscuity, product mixtures are obtained when subjecting substrate mixtures to the hybrid PKSs. This should not necessarily be considered disadvantageous when working with a PKS assembly line with a single swap, as the product mixture would be just moderately complex and suitable for chromatographic separation. If demanded, the AT/MAT-swap approach can be further developed by utilizing specialized MAT variants, which were recently engineered through active site mutagenesis³³. In this regard, the engineered M1* split VEMS proves to be a particularly interesting substrate tolerant assembly line. With M0 of VEMS and the MAT in M1*, it contains two entry points to the assembly lines and possesses the ability to generate product pools in response to its substrate environment. Such designs may be developed into responsive element in cell function and regulation.

In spite of the successes in AT/MAT-swaps, there are limits to the generation of FAS/PKS hybrids. We failed to obtain the hybrid modules VEMS M2*-TE and DEBS M5*-M6*-TE, indicating that not all proteins can be harnessed for AT/MAT-swapping and that more than one swap per polypeptide lowers the chance to get a functional hybrid, due to the fragile nature of the PKS module fold.

Reagents

CloneAmp HiFi PCR Premix was from Takara. Primers were synthesized by Sigma Aldrich. For DNA purification, the GeneJET Plasmid Miniprep Kit, the GeneJET Gel Extraction Kit from ThermoFisher Scientific and the Wizard® SV Gel and PCR Clean-up System from Promega Corp. were used. Cloning was performed with the In-Fusion HD Cloning Kit from Takara. NiCo21 (DE3) Competent cells were from New England BioLabs. Stellar Competent Cells were from Takara and One Shot BL21 (DE3) Cells were from ThermoFisher Scientific. All chemicals for buffer preparations were from Carl Roth GmbH (KH2PO4, K2HPO4, NaH2PO4, Na2HPO₄, glycerol, disodium ethylenediaminetetraacetic acid (EDTA), VWR Chemicals (NaCl) and AlfaAesar (imidazole). Desthiobiotin was purchased from IBA Lifesciences and biotin was from abcr GmbH. For cell cultures, LB (Lennox) and 2 xYT media were obtained from Carl Roth, Gibco™ Bacto™ Tryptone and Gibco™ Bacto™ yeast extract were purchased from ThermoFisher Scientific. Isopropyl-β-D-1-thiogalactopyranoside (IPTG), ampicillin, and carbenicillin were from Carl Roth. Spectinomycin was from Sigma-Aldrich. His60 Ni Superflow Resin was from Takara, and Strep-Tactin Sepharose columns as well as Strep-Tactin®XT 4Flow® high capacity resin were from IBA Lifesciences. For anion exchange chromatography, the HiTrapQ HP column (column volume = 5 mL) was from Cytiva. Superose™ 6 Increase 10/300 GL column from Cytiva was used for SEC polishing and protein analysis. Proteins were concentrated with Amicon® Ultra centrifugal filters from Merck Millipore. Coenzyme A, 3,5-dihydroxybenzoic acid, 3-hydroxybenzoic acid and adenosine-5’-triphosphate (ATP) were from Sigma-Aldrich. NADPH disodium salt was from F.Hoffmann-La Roche Ltd. Malonate, methylmalonate and magnesium chloride hexahydrate were from Carl Roth. Malonyl-CoA, methylmalonyl-CoA and reducing agent tris-(2-carboxyethyl) phosphine (TCEP) was from ThermoFisher Scientific. Fluoromethylmalonyl-CoA was prepared as previously described²⁵. Acetyl-, propionyl-, crotonyl-, 4-hydroxybutyryl- and butyryl-CoA were from Sigma Aldrich.

Plasmids

AT/MAT-swapped constructs of VEMS PKS were generated via In-Fusion Cloning (Takara) from previously described plasmids. The DNA encoding PIKS proteins pikAIII (M5) and pikAIV (M6) (from Streptomyces venezuelae ATCC 15439 (DSMZ)), and DEBS protein DEBS3 (from Saccharopolyspora erythraea ATCC 11635 (DSMZ)) were amplified from genomic DNA by PCR and introduced into a pET22b(+) expression vector by In-Fusion Cloning (Takara). These expression plasmids (PIKS M5 (pAR328), PIKS M6-TE (pSR006) and DEBS3 M5-M6-TE (pAR268) as well as mFAS MAT (pAR264³³) were used as a template to generate all engineered PIKS M5 and DEBS3 constructs of this study via In-Fusion Cloning (Takara). The resulting plasmids, cloning strategies, and primer sequences are further specified in Tables S1 – S4. The plasmid sequences were verified by Sanger Sequencing (Microsynth Seqlab). All protein sequences of the constructs generated in this study are listed in Tables S5 – S9. Construct designs for the boundary screening for AT/MAT-swaps in VemG M1 and VemH M2s are further illustrated in Figures S5 and S6.

Bacterial Cell Cultures

All PKS proteins were expressed and purified using similar protocols. H1M0, PIKS M5-TE- and VemG-based constructs were expressed in BAP1⁴⁸, VemH-based constructs and split VEMS M1-based constructs in NiCo21, and H2M0 and DEBS3-based constructs in BL21 cells. All proteins were expressed in the holo-form (to activate the ACP domain post-translationally with a phosphopantetheine arm). For expression in NiCo21 and BL21 cells, the construct-encoding plasmids were co-transformed with a plasmid encoding for the phosphopantetheine transferase Sfp from Bacillus subtilis (pAR357³³). Cell cultures were grown on a 2 L scale in 2x YT media (VEMS) or TB media (PIKS and DEBS) at 37°C until an OD₆₀₀ of 0.3 was reached, whereupon the temperature was adjusted to 18°C. At an OD₆₀₀ of 0.6, protein production was induced by adding 0.1 mM (VEMS) or 0.25 mM (PIKS and DEBS) IPTG, and the cells were grown for another 18 h at 140 rpm. Cells were harvested by centrifugation (5,000 x g, 15 min), resuspended in lysis buffer (50 mM sodium phosphate, 10 mM imidazole, 450 mM NaCl, 10% glycerol, pH 7.6), lysed by French Press and cell debris was removed by centrifugation (50,000 x g, 45 min.) The cell lysate was subsequently purified using affinity chromatography. All constructs contained a C-terminal His-tag. VemG-based constructs, H2M0, and hybrid constructs PIKS M5*-TE and DEBS M5*-M6-TE contained an additional N-terminal Twin-Strep-tag for tandem affinity purification.

Protein Purification: For VEMS based constructs, the purification procedure was as follows: For proteins containing just a C-terminal His-tag, the purification procedure was as follows: The supernatant was applied onto the column (5 mL Ni resin). A first wash step was performed with the above-mentioned lysis buffer (10 column volumes), followed by a second wash step with 10 column volumes of wash buffer (50 mM sodium phosphate, 25 mM imidazole, 300 mM NaCl, 10% glycerol, pH 7.6). Proteins were eluted with 6 column volumes of elution buffer (50 mM sodium phosphate, 300 mM imidazole, 10% glycerol, pH 7.6). The eluate was purified by anion exchange chromatography using a HitrapQ column on an ÄKTA FPLC system (column volume 5 mL). Buffer A consisted of 50 mM sodium phosphate, 10% glycerol, pH 7.6, whereas buffer B contained 50 mM sodium phosphate, 500 mM NaCl, 10% glycerol, pH 7.6. Protein concentrations were determined with a Nanodrop. Samples were stored as aliquots at − 80°C until further use.

For proteins containing a C-terminal His- and an N-terminal twinstrep-tag, the purification procedure was as follows: The supernatant was applied onto the first affinity chromatography column (5 mL Ni resin). Washing was performed with 5CV of lysis buffer (50 mM sodium phosphate, 10 mM imidazole, 450 mM NaCl, 10% glycerol, pH 7.6). Proteins were eluted with 2x2.5 column volumes of elution buffer (50 mM sodium phosphate, 300 mM imidazole, 10% glycerol, pH 7.6). The eluate was applied to the second affinity chromatography column (5mL strep resin) and washed with 6 CV strep-Wash buffer (50 mM sodium phosphate, 10% glycerol, pH 7.6). The target protein was eluted with 2x2.5 CV strep elution buffer (50 mM sodium phosphate, 2.5 mM desthiobiotin, 10% glycerol, pH 7.6). The eluate was purified by anion exchange chromatography using a HitrapQ column on an ÄKTA FPLC system (column volume 5 mL). Buffer A consisted of 50 mM sodium phosphate, 10% glycerol, pH 7.6, whereas buffer B contained 50 mM sodium phosphate, 500 mM NaCl, 10% glycerol, pH 7.6. Protein concentrations were determined with a Nanodrop. Samples were stored as aliquots at − 80°C until further use. VEMS-based constructs H2M0 and M1* (YZ046) were further polished and analyzed via size exclusion chromatography (SEC) using an ÄKTA FPLC system with a Superose 6 Increase 10/300 GL column from Cytiva (buffer: 50 mM sodium phosphate, 500 mM NaCl, 10% glycerol, pH 7.55). The SEC profiles are provided in Figures S4 and S11.

For PIKS and DEBS proteins, the purification procedure was as follows: The cell lysate was applied onto the column (5 mL Ni-NTA resin) and the flowthrough was discarded. The proteins were washed with (i) 5 column volumes (CV) lysis buffer (25 mL), (ii) 2 CV wash buffer (10 mL; 50 mM sodium phosphate, 30 mM imidazole, 450 mM NaCl, 10% glycerol, pH 7.6) and the target protein was eluted with 2.5 CV elution buffer (12.5 mL; 50 mM sodium phosphate, 300 mM imidazole, 450 mM NaCl, 10% glycerol, pH 7.6). Proteins bearing a Twin-Strep-tag were additionally purified via Strep-TactinXT 4Flow high capacity resins (2 mL). The eluent was transferred to the StrepTactinXT resin, washed with 5 CV strep wash buffer (10 mL; 50 mM sodium phosphate, 10 mM imidazole, 450 mM NaCl, 10% glycerol, pH 7.6) and eluted with 2.5 CV strep elution buffer (5 mL; 50 mM sodium phosphate, 50 mM biotin, 10 mM imidazole, 450 mM NaCl, 10% glycerol, pH 7.6). Proteins were further polished and analyzed by SEC using a Superose 6 Increase 10/300 GL column equilibrated with SEC buffer (250 mM potassium phosphate, 10% glycerol, pH 7.0). Target protein fractions were pooled, concentrated to 10–20 mg mL^− 1 using Amicon® Ultra centrifugal filters (100 kDa MWCO), flash frozen in liquid nitrogen and stored at -80°C until further use.

The enzymes PrpE and MatB (starter and extender substrate regeneration system) were purified as described previously^49,50. Protein concentrations were determined with a Nanodrop. Samples were stored as aliquots at − 80°C until further use.

Product Formation Assay: For VEMS-based assembly lines, reactions were carried out in a 40 µL scale. Reaction solutions were prepared using the reaction buffer (400 mM sodium phosphate, 10% glycerol and pH 7.2). The reducing agent TCEP was used in a final concentration of 5 mM. In situ extender substrate generation was performed using 10 µM MatB, 1 mM CoA, and 10 mM malonate or 5 mM malonate and 5 mM methylmalonate. ATP and MgCl₂ were provided in a final concentration of 9 mM. The starter substrates DHBA and 3-hydroxybenzoic aicd were used at a final concentration of 0.75 mM, while CoA-based starter substrates (acetyl-CoA, propionyl-CoA, crotonyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA) were used at a final concentration of 1 mM. In the reactions shown in Fig. 6A the starter substrate propionyl-CoA was generated in situ using 2 µM PrpE and 0.5 mM propionate. The enzymes constituting the assembly line were used in a final concentration of 8 µM. The reaction mixture was incubated overnight at room temperature and extracted two times with 450 µL ethyl acetate. Dried samples were reconstituted in 100 µL methanol and analyzed by HPLC-MS or HPLC-HRMS in positive mode. Components were separated over a 16 min linear gradient of acetonitrile from 5–95% in water. m/z ratio of venemycin 1 [M + H]⁺ m/z = 221.04, 4-hydroxy-6-methyl-2-pyrone 2 [M + H]⁺: m/z = 127.04, 6-ethyl-4-hydroxy-2-pyrone 3 [M + H]⁺: m/z = 141.05, 4-hydroxy-6-propyl-2-pyrone 4 [M + H]⁺: m/z = 155.07, (E)-4-hydroxy-6-propenyl-2-pyrone 5 [M + H]⁺: m/z = 153.05, and 4-hydroxy-6-(2-hydroxypropyl)-2-pyrone 6 [M + H]⁺: m/z = 171.06), 5-methyl-venemycin 7 [M + H]⁺: m/z = 235.06), 3,5-dimethyl-venemycin 8 [M + H]⁺: m/z = 249.07).

For DEBS- and PIKS-based constructs all assays were performed in SEC buffer (250 mM potassium phosphate, 10% glycerol, pH 7.0) at 25°C and a total volume of 20 µL. The assay was conducted in 384-well Small Volume HiBase Microplates (Greiner Bio-one) with ClarioStar microplate reader (BMG labtech). Settings: absorption: 348 − 20 nm, emission 476 − 20 nm, gain: 1500, focal height: 12.4 mm, flashes: 12, orbital averaging: off. The enzymes (PIKS M5-TE, PIKS M5*-TE, DEBS M5-M6-TE, DEBS M5*-M6-TE and DEBS M5-M6*-TE) were prepared as 4-fold stock solution (16 µM) (Sol 1), PIKS pentaketide was also prepared as 4-fold stock solution (4 mM) (Sol2), and the X-CoA (400 µM)/NADPH (120 µM) mixture was prepared as 2-fold stock solution (Sol 3). For testing bimodular DEBS3, Sol 3 comprised a 1:1-mixture of X-CoA substrates (MMalCoA/MalCoA and MMalCoA/FMMalCoA; total concentration: 400 µM) and NADPH (120 µM). 5 µL priming substrate (Sol 2) were mixed with 5 µL enzyme solution (Sol1) and reaction was initiated by adding 10 µL of X-CoA/NADPH mix (Sol 3). Assay components were provided at final concentrations of 4 µM (enzyme), 1 mM (PIKS pentaketide), 200 µM XCoA and 60 µM NADPH. Reaction progress was monitored fluorometrically for 30 min and converted into concentrations using a NADPH calibration curve. Afterwards, reaction mixtures were immediately extracted with ethyl acetate (3x 300 µL), organic solvent was removed in a SpeedVac in vacuo and dried samples were subsequently dissolved in 50 µL methanol and centrifuged at 20 000 x g for 20 min. 40 µL supernatant was measured on HPLC-ESI-MS using the Ultimate 3000 LC (Dionex) system equipped with a Acquity UPLC BEH C18 (2.1 x 50 mm, particle size 1.7 µm, Waters) and connected to an AmaZonX (Bruker). m/z ratios of macrolactone compounds: 14 [M + H-H₂O]: m/z = 279.19, 15 [M + H-H₂O]: m/z = 263.14, 16 [M + H-H₂O]: m/z = 265,15, 17 [M + H-H₂O]: m/z = 297.15, 18 [M + H-H₂O]: m/z = 337.19; [M + Na]: m/z = 377.19, 19 [M + H-H₂O]: m/z = 323.25; 20 [M + H-H₂O]: m/z = 355.20; [M + Na]: m/z = 395.23, 21 [M + H-H₂O]: m/z = 323.25; [M + Na]: m/z = 363.25, and 22 [M + H-H₂O]: m/z = 355.20; [M + Na]: m/z = 395.23.

Bioinformatical Analysis: Structure predictions were performed with ColabFold⁴² using default settings. The structures were predicted without template information. MSA options were set as follows: MMseqs2 (UniRef + Environmental) was chosen as MSA mode and unpaired + paired as pair mode, and the model type was set to auto. Amino acid sequences used for structure prediction are provided in Table S10. AlphaFold error estimates are provided in Figures S20 and S21.

Funding Sources:

Support for this work was received from the DFG grant GR3854/6 − 1 and 10 − 1.

Authors contribution:

L.B., S.R., and M.G. wrote the manuscript. For VEMS-based constructs, L.B. conceived and supervised the project and designed the constructs. L.B. and Y.Z. cloned and purified the constructs. L.B. analyzed the data. For PIKS- and DEBS-based constructs, S.R. conceived the project, designed, cloned and purified the constructs, and analyzed the data. M.G. designed the research and analyzed the data.

Acknowledgments:

We thank H. Bode for use of their LC-MS instruments. We thank M. Joppe for starting the project with us (DEBS construct design) and Prof. D. Sherman for providing the substrate PIKS pentaketide.

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The malonyl/acetyl-transferase from murine fatty acid synthase is a promiscuous engineering tool for editing polyketide scaffolds

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Abstract

Figures

Introduction

Results and Discussion

Engineering a PKS assembly line with a promiscuous loading module-

MAT swaps in Elongation Modules of split VEMS-

AT/MAT-swaps in mono-modular PIKS system-

AT/MAT-swaps in bimodular DEBS System-

Conclusion

Methods

Declarations

Funding Sources:

Authors contribution:

Acknowledgments:

References

Additional Declarations

Supplementary Files

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