Versatile selective evolutionary pressure using synthetic defect in universal metabolism

Synthetic defects in NAD⁺ regeneration abolishing anaerobic growth can be rescued by metabolically isolated foreign enzyme-substrate pairs

To implement the artificial-selection system, a genetically-modified E. coli strain (strain AL) unable to regenerate NAD⁺ during anaerobic fermentation was constructed by inactivating the genes encoding the ethanol pathway (bifunctional aldehyde-alcohol dehydrogenase, adhE) and, to eliminate a potential mutational escape route⁷, the lactate pathway (lactate dehydrogenase, ldhA). When cultured aerobically in a minimal medium, AL cells grew similarly to WT cells. However, they were unable to grow anaerobically in the same medium, indicating the expected impairment of anaerobic fermentation (Fig. 1c). We validated this strain by genetic complementation, through a transformation with a plasmid bearing the WT adhE gene, which allowed anaerobic growth similar to WT cells, reaching a maximum optical density at 600 nm (OD₆₀₀) of 0.8 by 24−36 h (Fig. 1d). ¹H–NMR spectra of the fermentation broth confirmed the profile of metabolites was similar to that of the parental strain (except for the expected absence of lactate, Supplementary Table 1).

We attempted to restore anaerobic growth of AL cells using exogenous enzyme-substrate pairs. In each case, the substrate was not expected to be natively produced or consumed by E. coli, so the main link to the E. coli metabolic network was through NAD. Three NADH-dependent oxidoreductases were tested: acetoin reductases from Bacillus subtilis (BDHA)¹⁶ and Klebsiella pneumoniae (BUDC)¹⁷, and alcohol dehydrogenase from Thermus sp. ATN1 (TADH)¹⁸ (Fig. 1e). AL cells transformed with any of the exogenous reductases were able to grow anaerobically in minimal medium only if also supplemented with the corresponding substrate (acetoin for BDHA and BUDC, and cyclohexanone or 3-methylcyclohexanone for TADH) (Fig. 1f−h). The substrates alone did not restore growth except for acetoin, with which growth was eventually observed, probably due to the activity of an endogenous acetoin reductase^11,15. The consumption of substrates and generation of corresponding reduced products was confirmed in all three cases using ¹H-NMR (Supplementary Table 1). These results with cyclohexanone and 3-methylcyclohexanone indicate that NAD⁺ regeneration alone, without any effects of carbon flux with metabolism, is enough to rescue AL cells from their metabolic defect.

High-throughput artificial selection outperforms computational design of cofactor specificity reversal

NAD(P)-dependent oxidoreductases have been extensively engineered to make them more suitable for specific applications, targeting properties such as kinetic parameters, substrate specificity, or cofactor preference^19,20,21,22. Switching the cofactor preference of NADP-dependent enzymes to NAD is of particular interest due to the lower cost and higher stability of NAD, and the higher efficiency of cell-free NAD recycling systems^23,24, for which low-cost co-substrates are available. Substrate selectivities, and modifications to them, are not generally computationally predictable. NAD/NADP selectivity is a partial exception, as the many known examples and a common structural motif enabled the development of a computational design tool, CSR-SALAD, which represents the state of the art in cofactor-selectivity reversal^25,26.

Alcohol dehydrogenases (ADHs), also known as ketoreductases (KREDs), are of great industrial interest primarily due to their ability to perform an asymmetric reduction of aldehydes and ketones. Thanks to their regioselectivity and stereoselectivity, ADHs can be used not only to produce enantiomerically pure alcohols, but also other types of compounds, such as γ- and δ-lactones through the desymmetrisation of meso-diols^27,28. The primary-secondary alcohol dehydrogenase of Clostridium beijerinckii (CBADH) reduces this organism’s waste acetone to isopropanol, but is strictly NADP-dependent, which is unusual in anaerobic fermentation²⁹ (Fig. 2a). An efficient NAD-utilizing acetone reductase variant of CBADH would be useful in industrial biotechnology and cofactor recycling, so we set out to evolve one.

CSR-SALAD was used to analyze CBADH, predict residues critical for cofactor preference, and design a cofactor preference reversal strategy. The software suggested 567 protein variants encoded by a library of 648 genetic variants of positions G198 (substitutions to DEGKNRS), S199 (to DGHILNRSV), and Y218 (to ADFINSTVY), located at the cofactor binding pocket close to the 2′ phosphate group of NADP (Fig. 2b). CSR-SALAD assumes reversal would cause substantial loss of activity, and proposed subsequent rounds of site-saturation mutagenesis and screening to attempt to recover activity, initially prioritizing I175 and R200. A previous manual analysis of the CBADH crystal structure proposed substitutions of residues G198, S199, R200, and Y218 to reverse cofactor preference²⁹, but neither the specific substitutions proposed nor saturation mutagenesis of S199 yielded any NADH-dependent variants of the closely-related Clostridium autoethanogenum ADH³⁰. Here we applied the proposed substitutions to CBADH, constructing variants CBADH_R1 (S199G, R200G, and Y218F) and CBADH_R2 (G198D, S199G, R200G, and Y218F), but plasmids encoding these variants did not restore anaerobic growth of AL cells in minimal medium supplemented with 15 mM acetone, indicating low or absent NADH-dependent activity.

We performed full combinatorial saturation mutagenesis of all four target residues of CBADH (G198, S199, R200, and Y218) replacing each codon with NNN to yield a library (CBADH_Lib) of 16.8 million unique genetic variants encoding 160,000 unique protein variants. Every variant proposed by CSR-SALAD was included, comprising only 0.4% of the library. By including R200, the activity loss that CSR-SALAD envisages as an emergent problem to be solved by subsequent recovery is addressed simultaneously during cofactor preference reversal.

To select for CBADH variants able to accept NAD, AL cells were transformed with the library, and pools of variants were incubated anaerobically in a minimal medium supplemented with 15 mM acetone, so that any variant with NADH-dependent acetone-reducing activity would lead to a regeneration of NAD⁺, restoring growth. For this library and each similar case in this study, multiple independent transformations (six for this library) were used to obtain complete library coverage (approximately 18 million transformant clones in this case) and samples were sequenced to confirm diverse codon incorporation. Clones from each transformation were pooled and subjected to selection separately. Growth was observed in three of the variant pools (Fig. 2c) so these were each subcultured under the same conditions once, then plasmid DNA was extracted and sequenced, revealing the presence of the same CBADH variant in each case (CBADH_S) with G198D, S199Y, and Y218P substitutions. Interestingly, NAD-dependent mouse class II alcohol dehydrogenase (ADH2)³¹, which is distantly related to CBADH, contains D227 and P247 which are equivalent positions to 198 and 218 in CBADH. CBADH_S was not among the 567 variants proposed by CSR-SALAD. The selection of the same variant in three independent experiments indicates the superiority of this variant, the strength of the artificial selection pressure, and its utility to isolate variants. Transforming AL cells with isolated plasmid encoding CBADH_S enabled fast anaerobic growth in a medium containing acetone (Fig. 2d). Acetone consumption and corresponding isopropanol generation were confirmed by ¹H-NMR (Supplementary Table 1). Unlike the parent enzyme CBADH_WT, purified CBADH_S was unable to oxidize isopropanol using oxidized NADP (NADP⁺) as the cofactor, but could do so with NAD⁺ (K_m = 17.49 mM, k_cat = 333 min⁻¹ and k_cat/K_m = 316.67 M⁻¹ s⁻¹, Supplementary Fig. 1 and Supplementary Table 2).

Eliminating transhydrogenase activities provides strict cofactor selection allowing exceptional reversal of cofactor preference

Unexpectedly, we observed that AL cells were able to slowly grow anaerobically in a medium with acetone when transformed with the parent enzyme CBADH_WT, despite its strict NADP-dependence (Fig. 2c, d). We hypothesized that acetone-dependent anaerobic growth recovery by CBADH_WT could be mediated by the activity of one or both of E. coli’s two transhydrogenases, PNTA and STHA, which could use the NADP⁺ produced by CBADH_WT to oxidize NADH. To test this hypothesis, we generated E. coli strains with knockout mutations of transhydrogenase genes pntA (ALP strain), sthA (ALS strain), or both (ALPS strain) in addition to knockout mutations of adhE and ldhA. We then tested the ability of CBADH_WT and CBADH_S to restore anaerobic growth of ALP, ALS, and ALPS cells. The ALP and ALS strains, each with one intact transhydrogenase gene, were able to grow anaerobically in a medium with acetone when transformed with either NADP-dependent CBADH_WT or NAD-dependent CBADH_S (Supplementary Fig. 2). However, only NAD-dependent CBADH_S could support acetone-dependent anaerobic growth of ALPS cells, which lack either intact transhydrogenase gene (Fig. 2f and Supplementary Fig. 2). This demonstrates that transhydrogenases are indeed responsible for the acetone-dependent recovery of anaerobic growth by CBADH_WT, and that either STHA or PNTA alone is sufficient to generate the required NAD⁺ to sustain anaerobic growth. Interestingly, this is thought to be the non-physiological direction for the membrane-bound, ‘energy-linked’ transhydrogenase PntA³². Due to its inability to grow anaerobically when transformed with an NADP-dependent oxidoreductase, the transhydrogenase-free ALPS strain provides a more stringent selection system than the AL, ALP, or ALS strains, strictly requiring NAD-dependent oxidoreductase activity to restore growth.

To validate the ALPS strain as a selection host, we used it to evolve NAD-dependent variants of TBADH, an NADP-dependent alcohol dehydrogenase from Thermoanaerobacter brockii closely related to CBADH²⁹. Similarly to CBADH_WT, WT TBADH was able to support the anaerobic growth of AL cells in a medium supplemented with acetone, but not of ALPS cells (Fig. 2g). We performed full combinatorial saturation mutagenesis of residues G198, S199, R200 and Y218 of TBADH to generate a library (TBADH_Lib) of 16.8 million unique genetic variants (Fig. 2e). A similar selection procedure as for CBADH was followed but using the ALPS strain, and two different variants were identified, TBADH_S1 and TBADH_S2. TBADH_S1 had substitutions G198S, S199K, R200P, and Y218V. Surprisingly, TBADH_S2 contained a duplication of residues 191−241, in addition to substitutions in the targeted residues, both in the positions of the original sequence and in the corresponding positions of the duplication (Supplementary Fig. 3). The substitutions were G198H, S199R, R200A, Y218M, G198’A, and R200’K (198’ and 200’ denote positions 249 and 251 of TBADH_S2, the positions in the duplication equivalent to the original 198 and 200 residues). Since the duplication was not intentionally introduced into the variant library by design, it presumably arose through a rare event during the PCR, ligation or within cells, highlighting the potential of the selection system to isolate variants with desired properties even when these are rare and outside the intended design space.

TBADH_S1 and TBADH_S2 were both able to restore the anaerobic growth of ALPS cells in a medium with acetone (Fig. 2g), and ¹H-NMR of the fermentation broth confirmed the production of isopropanol (Supplementary Table 1). When the enzymatic activity was assayed, TBADH_S1 could oxidize isopropanol only with NAD⁺, certifying the reversal of cofactor preference (Supplementary Fig. 1 and Supplementary Table 2). The k_cat of TBADH_S1 for the oxidation of isopropanol with NAD⁺ was 112 ± 5.7 min⁻¹, 4.5 times lower than the k_cat of TBADH_WT for the same reaction with NADP⁺. However, the K_m of TBADH_S1 for isopropanol was 3.74 ± 0.54 mM, a 32-fold decrease (improvement) compared to the K_m of TBADH_WT for isopropanol in the presence of NADP⁺. Overall, the catalytic efficiency (k_cat/K_m) of TBADH_S1 for the oxidation of isopropanol (k_cat/K_m = 496.67 M⁻¹ s⁻¹) was more than seven times greater than that of TBADH_WT with NADP⁺ (k_cat/K_m = 70 M⁻¹ s⁻¹). This is, to our knowledge, the highest relative catalytic efficiency obtained in any case of cofactor specificity reversal for an alcohol dehydrogenase, and the best for the reversal of preference from NADP to NAD for any enzyme. In contrast, TBADH_S2 was able to oxidize isopropanol both with NAD⁺ and NADP⁺ (in the presence of NAD⁺, K_m = 22.07 mM, k_cat = 238.5 min⁻¹ and k_cat/K_m = 180 M⁻¹ s⁻¹; in the presence of NADP⁺, K_m = 55.15 mM, k_cat = 231.4 min⁻¹ and k_cat/K_m = 70 M⁻¹ s⁻¹; Supplementary Fig. 1, Supplementary Table 2). A decrease in k_cat compared to TBADH_WT was observed, but the K_m for isopropanol decreased (improved) in the presence of both cofactors.

We crystallized CBADH_S and TBADH_S1 in order to elucidate the structural basis for the observed reversal of cofactor preference. The resulting maps showed clear density for NAD⁺ in the case of CBADH_S, but only partial occupancy for TBADH_S1 (Supplementary Fig. 4). In both cases, the size of the cofactor-binding pocket is reduced due to the substitution of residues 198 and 199 by others with bulkier side chains (Fig. 3), which sterically prevents the binding of the 2′ phosphate of NADP. In the case of CBADH_S, this effect is further enhanced by the presence of an aspartate residue at position 198, which would also prevent NADP binding through electrostatic repulsion between the side-chain carboxylate group and the 2′ phosphate of NADP. Another common feature of both CBADH_S and TBADH_S1 is the substitution of Y218. The side chain of Y218 is known to undergo a 120° rotation in the WT enzymes to allow stacking to the adenine moiety of NADP and the formation of a hydrogen bond with the 2′ phosphate through its hydroxyl group²⁹. As shown by the CBADH_S and TBADH_S1 structures, the side chains of the substituted residues are not close enough to the 2′ phosphate to interact with it, and would only be able to form hydrophobic interactions with the adenine moiety, if any (Fig. 3).

Simultaneous optimization of multiple kinetic parameters by high-throughput artificial selection

Next, we applied the artificial selection system to an imine reductase (IRED). IREDs are of great industrial interest thanks to their ability to catalyze the asymmetric reduction of imines and the reductive amination of ketones, both of which yield chiral amines, fundamental building blocks in the pharmaceutical and agrichemical industries^33,34. All known natural IREDs are NADP dependent³⁵, so there is great interest in developing NAD-dependent variants due to the lower cost and higher efficiency of NAD-regeneration systems. The most active NAD-dependent IRED thus far is MsIRED_C, a variant of Myxococcus stipitatus IRED (MsIRED), which is able to reduce 2-methyl-1-pyrroline amongst other substrates (Fig. 4a). MsIRED_C was obtained through several rounds of mutagenesis and screening³⁶. We aimed to obtain a superior NAD-dependent variant of MsIRED through a faster and simpler workflow by applying the artificial selection system.

CSR-SALAD predicted residues N32, R33, T34, and K37 to be critical determinants of cofactor specificity (Fig. 4b). Thus, we performed full combinatorial saturation mutagenesis of these residues to generate a library (MsIRED_Lib) of 16.8 million unique genetic variants. AL cells transformed with the library were able to grow anaerobically in medium with 2-methyl-1-pyrroline (Fig. 4c), and the same MsIRED variant (MsIRED_S) was identified from three independent transformations of the library, comprising residue substitutions N32D, R33V, T34R, and K37R. We analyzed the fermentation broth of AL cells transformed with MsIRED_S (Fig. 4d) by ¹H-NMR, confirming the consumption of 2-methyl-1-pyrroline and the production of the corresponding amine, 2-methylpyrrolidine (Supplementary Table 1). The enzymatic activities of MsIRED_C and MsIRED_S were compared (for MsIRED_C, K_m = 34.06 mM, k_cat = 161.17 min⁻¹ and K_i = 4.94 mM; for MsIRED_S, K_m = 19.57 mM, k_cat = 78.1 min⁻¹ and K_i = 11.42 mM; Supplementary Fig. 5, Supplementary Table 2). Both variants were able to reduce 2-methyl-1-pyrroline only with NADH and showed substrate inhibition, as shown by the decrease in activity at the highest concentrations of substrate. MsIRED_S performed better than MsIRED_C at all tested substrate concentrations (Fig. 4e), partly due to the higher value of the substrate inhibition constant K_i, which indicates a relief of substrate inhibition in MsIRED_S compared to MsIRED_C. To our knowledge, MsIRED_S has the highest NAD-dependent IRED activity yet reported. These results highlight the ability of the artificial selection system to obtain variant enzymes where multiple kinetic parameters are enhanced simultaneously, resulting in enzymes with optimal activity towards the desired substrate. Furthermore, while identification of MsIRED_C by Borlinghaus and Nestl³⁶ required multiple rounds of mutagenesis and screening, we obtained the superior NAD-dependent variant MsIRED_S in a single round.

Chemically-directed evolution of enzyme variants with modified chemoselectivity and positional selectivity

We aimed to apply the artificial selection pressure to target the evolution of enzyme activity towards non-native chemicals. Nitroreductases can synthesize aromatic hydroxylamines or amines (pharmaceutical and agrichemical precursors) from low-cost and readily available nitroaromatics³⁷, can activate nitroaromatic anticancer prodrugs^38,39, and can be used for bioremediation of soils contaminated with explosives such as TNT^40,41. However, these applications often require the tailoring of natural nitroreductases to improve their catalytic properties. We targeted the classic Enterobacter cloacae nitroreductase NfsB (EntNfsB), a type-I nitroreductase able to reduce several nitroaromatic compounds, including 4-nitrobenzoic acid (4-NBA), and which can accept NAD and NADP as cofactors⁴². We characterized the activity of the WT enzyme, EntNfsB_WT, with 4-NBA, 4-nitrobenzyl alcohol (4-NBALC), and 2-nitrobenzoic acid (2-NBA), finding that it was able to reduce 4-NBA and 4-NBALC but not 2-NBA (Fig. 5a, Supplementary Fig. 6, and Supplementary Table 2). The activity towards 4-NBALC was, however, considerably lower than towards 4-NBA, which prevented the accurate determination of kinetic parameters.

We performed full combinatorial saturation mutagenesis of residues S40, T41, Y68, and F124 of EntNfsB (generating a library (EntNfsB_Lib) of 16.8 million unique genetic variants), chosen based on their proximity to the substrate-binding pocket and their lack of direct contact with the essential FMN⁴³ (Fig. 5b). AL cells were transformed with the library and cultured anaerobically in a minimal medium supplemented with 2-NBA or 4-NBALC (Fig. 5c, d). We identified a different variant from the cells grown in the presence of each of the substrates. The variant selected in the cultures with 2-NBA (EntNfsB_S1) contained substitutions S40A, T41I, and F124A, while the variant selected in the cultures with 4-NBALC (EntNfsB_S2) contained T41L, Y68L, and F124L substitutions. In order to determine the product generated by the EnfNfsB variants, the fermentation broth of AL cells transformed with plasmids expressing either EntNfsB_S1 or EntNfsB_S2 and grown anaerobically in minimal medium with 2-NBA or 4-NBALC (Fig. 5e, f) respectively was characterized by ¹H-NMR (Supplementary Table 1). Interestingly, the observed product signals matched those of the aromatic amines corresponding to 2-NBA or 4-NBALC, despite a previous report indicating that EntNfsB_WT can only reduce 4-NBA to 4-hydroxylaminobenzoic acid, and not all the way to the amine 4-aminobenzoic acid⁴². That report only assayed the in vivo activity up to 24 h. It is possible that the highly reducing conditions inside AL cells, combined with longer periods of incubation under anaerobic conditions, allow for the formation of the amine products. Enzymatic assays confirmed that EntNfsB_S1 was able to reduce 2-NBA (K_m = 0.808 mM, k_cat = 28.4 min⁻¹ and k_cat/K_m = 585 M⁻¹ s⁻¹), and that EntNfsB_S2 could reduce 4-NBALC more efficiently than WT EntNfsB, allowing determination of kinetic parameters (K_m = 1.111 mM, k_cat = 205.0 min⁻¹ and k_cat/K_m = 3075 M⁻¹ s⁻¹, Supplementary Fig. 6, Supplementary Table 2). Both variants retained the ability to reduce 4-NBA. Therefore, EntNfsB_S1 is active on both 2-NBA and 4-NBA, unlike any previously reported nitroreductase. The ability to engineer nitroreductase selectivity and promiscuity in this way could prove useful in applications including activation of anticancer prodrugs with multiple nitro groups, and bioremediation of soils contaminated with nitroaromatics, where a complex mixture of different compounds is often found, and therefore the ability to act on multiple isomers is desirable. The isolation of EntNfsB_S1 and EntNfsB_S2 demonstrates chemically-directed evolution as a powerful application of the artificial selection system, using an external supply of target substrates to direct both improvement of an existing poor activity, and acquisition of an activity with a non-native substrate.

Coupling artificial selection to genetic design optimization yields the best-performing synthetic isopropanol pathway

We anticipated that the artificial selection system should be readily applicable to more complex systems than individual enzymes, such as metabolic pathways. Furthermore, the selection pressure should act not only on the sequences of enzymes, but in general on any genetically-encoded trait that can be linked to the generation of NAD⁺, such as regulatory sequences controlling gene expression. These sequences, particularly promoters and ribosome binding sites (RBSs), are important in the design and optimization of heterologous/synthetic metabolic pathways, as they control the amount of each enzyme that is produced. To maximize production, high yet balanced flux across the pathway is required, while avoiding the accumulation of intermediates^44,45 and expression-associated metabolic burden⁴⁶. Finding the best combination of regulatory elements for a specific pathway is not trivial, often requiring multiple rounds of laborious and time-consuming screening.

Isopropanol is a widely used solvent, additive, and platform chemical with a large market, conventionally manufactured by petrochemical routes. We sought to develop an isopropanol production pathway and prototype whole-cell biocatalyst superior to the natural and engineered examples reported previously⁴⁷ (Fig. 6a). A library (MP_Lib) of variants of pathway-encoding plasmids, differing in the regulatory elements (promoters and RBSs) controlling each gene, was constructed by combinatorial DNA assembly using the Start-Stop Assembly⁴⁸ system (Fig. 6b), giving a library of 60.4 million different variants. While only one enzyme in the pathway (CBADH) directly generates NADP⁺ (converted to NAD⁺ by transhydrogenases), the rate depends on the flux through the entire pathway, which in turn depends on a combination of regulatory genetic elements giving optimal expression of all enzymes, without causing problematic overexpression. This coupling allows the artificial selection pressure to be applied to the identification of optimal genetic designs.

AL cells were transformed with MP_Lib and grown anaerobically on minimal medium agar plates, with gluconate as a carbon source instead of glucose, because the maximum theoretical yield of isopropanol from gluconate is greater. Colonies of transformed AL cells were visible after 65 h of anaerobic incubation. We verified the ability of 10 colonies to grow anaerobically after inoculating them into minimal medium liquid cultures, and confirmed the presence of isopropanol in the fermentation broth by ¹H-NMR (Supplementary Table 1).

We compared the combinations of regulatory elements and the isopropanol titer of 10 ‘S’ variants obtained by artificial selection with those of 10 ‘R’ variants picked from the library at random without artificial selection. We found only two different combinations of regulatory elements among the S variants (MP_S1 and MP_S2), whereas the 10R variants were all different, and included defective variants lacking one or more of the genes, which can arise during DNA assembly (Fig. 6b). In these minimal culture conditions, the S variants gave an average isopropanol titer of 4.97 mM after 17 h of incubation, which was significantly greater (t(9) = 9.22 and p = 1.54 × 10⁻⁸, or excluding defective variants t(9) = 6.82 and p = 4.19 × 10⁻⁶) than the average of 0.60 mM produced by the R variants (Fig. 6b). This indicates that the artificial selection pressure had acted both to eliminate defective variants and to favour specific combinations of genetic elements leading to maximized production of isopropanol.

We compared the performance of the best isopropanol pathway variant MP_S1 with the best isopropanol pathway reported previously, by Hanai and coworkers⁴⁷, using the same growth medium and culture conditions. The previous best 43.5% of maximum theoretical isopropanol yield during the production phase (with 45 mM titre) was surpassed here by WT E. coli cells transformed with MP_S1, which achieved 56% at the same point (with 62 mM titre), the highest isopropanol yield reported so far for any organism, including both native producers and engineered strains.