Biotech

Tuning architectural organization of eukaryotic P450 system to boost bioproduction in Escherichia coli

Time Stamp: November 18, 2024 7:00 PM
Source Node: 747163

The biosynthetic performance of plant CYP73A5 affected by the tandem architecture

To test the effect of the architecture of tandem fused chimeras on the biosynthetic performance of eukaryotic P450 in E. coli, we targeted the plant-specific CYP73A subfamily (Fig. 2a), which catalyzes the formation of p-coumaric acid from trans-cinnamic acid in the plant phenylpropanoid pathway²⁷. Considering the different codon preference between the host and plants, we synthesized the E. coli codon-optimized coding sequences for Arabidopsis thaliana CYP73A5^Δ2-28 and ATR2^Δ2-77 (i.e. A. thaliana CPR2). Then, employing a flexible peptide linker²⁴ as an inserted hinge, we fused CYP73A5^Δ2-28 to either the N-terminus or the C-terminus of ATR2^Δ2-77, and thus constructed two types of tandem fused chimeras: (i) CYP73A5^Δ2-28-L-ATR2^Δ2-77 and (ii) ATR2^Δ2-77-L-CYP73A5^Δ2-28 (Fig. 2b).

**Fig. 2: Effects of the chimeric architecture on the biosynthetic performance of eukaryotic P450 enzyme.**

Subsequently, the biosynthetic performance of both tandem fused chimeras was determined in a recombinant E. coli strain containing A. thaliana phenylalanine ammonia-lyase 1 (AthPAL1), which can efficiently convert intracellular phenylalanine to trans-cinnamic acid in E. coli²⁸ (Fig. 2a). The recombinant E. coli strain without the chimera could not biosynthesize p-coumaric acid but accumulated 87.51 mg L⁻¹ trans-cinnamic acid at 48 h post-induction (hpi) (Fig. 2c), resulting in severe cell growth inhibition (Supplementary Fig. 1). As shown in Fig. 2c, at 48 hpi, the recombinant strain with chimera (i) produced 33.05 mg L⁻¹ p-coumaric acid, which was 2.02-fold more than that of the recombinant strain with chimera (ii). The biomass-specific productivity of p-coumaric acid for the strains harboring chimera (i) or (ii) was 86 and 43 μg (g DW h)⁻¹, respectively (Supplementary Table 1). In addition, in the medium of the recombinant strains with tandem fused chimera, trans-cinnamic acid was surplus in the range of 30.25 to 67.90 mg L⁻¹ at 48 hpi (Fig. 2c).

It should be noted that the tandem fused chimera (i) CYP73A5^Δ2-28-L-ATR2^Δ2-77 had a similar arrangement of the catalytic domains to the bacterial CYP102A1, in which the P450 heme domain is also located in the N-terminus²⁹. The result suggested that a nature-mimicking architecture for organizing eukaryotic P450 system would promote the biosynthetic performance of eukaryotic P450s even in a prokaryotic organism.

Architectural alternatives designed for plant CYP73A5 and ATR2 in E. coli

To expand the architectures for eukaryotic P450 system in E. coli, self-assembled peptide bio-machinery was harnessed to serve as a self-folded dual-track bridge to organize CYP73A5^Δ2-28 and ATR2^Δ2-77 in space at the post-translational level (Supplementary Fig. 2). Initially, a unique peptide ligation system SpySystem³⁰, in which the side chains of Lys34 in SpyCatcher peptide and Asp7 in SpyTag peptide can post-translationally form an isopeptide bond, was adopted. By fusing SpyCatcher to CYP73A5^Δ2-28 and SpyTag to ATR2^Δ2-77 at either the N-terminus or the C-terminus, a set of heterodimers with distinct architecture could be generated due to the side chain self-assembly of SpySystem. As indicated in Fig. 3a, four CYP73A5^Δ2-28-ATR2^Δ2-77 heterodimers with different linkage architectures were created, of which all showed a molecular weight (MW) of about 130 KDa (Calculated MW = 139.7 KDa) (Supplementary Fig. 3). The target bands with the desired size were further identified by LC-MS/MS, showing that the matched peptides covered the full-length amino acid sequences in a range of 78%–85%. Moreover, all of the four components, i.e. SpyCatcher, SpyTag, CYP73A5^Δ2-28 and ATR2^Δ2-77, were verified in each sample of interest (Supplementary Figs. 4–7), indicating that the reconstructed CYP73A5 and ATR2 were post-translationally assembled into a covalent heterodimer with a designed linkage architecture by virtue of the SpySystem. Heterodimer (I), CYP73A5^Δ2-28SpyCatcher-SpyTagATR2^Δ2-77, and heterodimer (II), ATR2^Δ2-77SpyTag-SpyCatcherCYP73A5^Δ2-28, were assumed to possess a similar architecture to the tandem fused chimeras (i) CYP73A5^Δ2-28-L-ATR2^Δ2-77 and (ii) ATR2^Δ2-77-L-CYP73A5^Δ2-28 (Fig. 2b), respectively. It’s worth noting that a C-termini-bridged heterodimer (III), CYP73A5^Δ2-28SpyCatcher-ATR2^Δ2-77SpyTag, and an N-termini-bridged heterodimer (IV), SpyCatcherCYP73A5^Δ2-28-SpyTagATR2^Δ2-77, were also achieved, and the latter in AthPAL1-expressing E. coli was confirmed via Western blotting of the denatured whole-cell extracts (Supplementary Fig. 8 lane 1).

Fig. 3: Architectural organization of plant CYP73A5^Δ2-28 and ATR2^Δ2-77 based on self-assembled peptide bio-machinery in *E. coli.*

Subsequently, the biosynthetic performance of these four heterodimers was tested in AthPAL1-expressing E. coli via fermentation as the tandem fused chimeras (Fig. 3b upper panel). Consistent with the results of the two tandem fused chimeras (Fig. 2c), the titer of p-coumaric acid with heterodimer (I) was higher than that with heterodimer (II), reaching 66.17 and 19.35 mg L⁻¹ at 48 hpi, respectively. The strain with C-termini-bridged heterodimer (III) biosynthesized 28.14 mg L⁻¹ p-coumaric acid, only 42.53% of the strain with heterodimer (I). As expected, the p-coumaric acid titer of the strain with N-termini-bridged heterodimer (IV) was dramatically elevated to 169.85 mg L⁻¹. And the biomass-specific productivity of p-coumaric acid for the strains harboring heterodimer (I), (II), (III) or (IV) was 192, 52, 85 and 563 μg (g DW h)⁻¹, respectively (Supplementary Table 1).

Meanwhile, the accumulation of the P450 substrate in the medium was measured (Fig. 3b lower panel). The accumulation of trans-cinnamic acid was in an inverse proportion to the production of p-coumaric acid regarding to each heterodimer, especially at 24 hpi. The results suggested that, in the AthPAL1-expressing strains, the titer of p-coumaric acid could mirror the biosynthetic performance of the reconstructed CYP73A5, which was also in agree with the order of the biomass-specific productivity, i.e. heterodimer (IV) > heterodimer (I) > heterodimer (III) > heterodimer (II). The N-termini-bridged heterodimer (IV), showing the highest p-coumaric acid output, may deploy a resemblant architecture to the membrane-associated N-terminus-guided colocalization of CYP73A5 and ATR2 on the ER membrane in plant cells³¹ (Figs. 1a and 3a). Hence, this peptide-based N-termini-bridged heterodimer of P450 and CPR might be considered as a mimic counterpart of eukaryotic membrane-bound P450 system. The in vivo results of the four heterodimers suggested that the architecture of protein assemblies would be exert an effect on the enzymatic activity of the reconstructed eukaryotic P450 system.

Enzyme kinetic assays of SpySystem-mediated CYP73A5-ATR2 heterodimers

To demonstrate the contribution of the architecture to the enzymatic activities of the reconstructed CYP73A5 enzymes, we further purified the SpySystem-appended CYP73A5^Δ2-28 and ATR2^Δ2-77 to depict the enzyme kinetics in vitro. SpyCatcherCYP73A5^Δ2-28 and SpyTagATR2^Δ^2-77 were labeled with hexahistidine at N-terminus, while CYP73A5^Δ2-28SpyCatcher and ATR2^Δ^2-77SpyTag were tagged with hexahistidine at C-terminus for polyhistidine affinity chromatography. To circumvent the potential effect of the histidine tag on enzymatic activity, the hexahistidine tag was sandwiched by a glycyl-seryl-seryl linker at N-terminus and a seryl-seryl-glycyl linker at C-terminus. As a control, CYP73A5^Δ2-28 and ATR2^Δ2-77 were respectively tagged with hexahistidine at N-terminus and also purified for enzyme kinetic assays.

The kinetic parameters of SpySystem-mediated CYP73A5-ATR2 heterodimers as well as the free-floating individuals (CYP73A5^Δ2-28 and ATR2^Δ2-77) were quantified as the rate of p-coumaric acid generation in the presence of trans-cinnamic acid as specific substrate and NADPH as electron source. When at increasing concentrations of the substrate trans-cinnamic acid (Supplementary Fig. 9), the K_m values of reconstructed CYP73A5 in the four heterodimers were all lower than the concentration of trans-cinnamic acid accumulated in the medium (Table 1, Fig. 3b lower panel), indicating that the in vivo trans-cinnamic acid might not be the limited factor for the bioproduction of p-coumaric acid. The k_cat values of SpyCatcher-appended CYP73A5 in the heterodimer (I), (II), (III) and (IV) were 11.00-, 4.72-, 1.11- and 48.94-fold higher than that of the free-floating truncated CYP73A5, respectively (Table 1). The results confirmed that both the CYP102A1-like architecture and the N-termini-bridged architecture significantly improved the in vitro turnover number of the reconstructed CYP73A5, especially the nature-mimicking architecture of heterodimer (IV), which were consistent with much higher in vivo titer of p-coumaric acid observed in the strains harboring heterodimer (I) or (IV) (Fig. 3b upper panel).

Table 1 Enzymatic assay of SpySystem-mediated CYP73A5^Δ2-28-ATR2^Δ2-77 heterodimers and the free-floating CYP73A5^Δ2-28 and ATR2^Δ2-77 with trans-cinnamic acid

Full size table

The enzymatic activities of the heterodimers were also assayed at increasing concentrations of the electron donor NADPH (Supplementary Fig. 10). As shown in Table 2, heterodimer (II) and (III), containing ATR2^Δ2-77SpyTag component, presented a significantly reduced K_m for NADPH, indicating that appending the peptide-based scaffold at the ATR2 C-terminus containing the NADPH-binding domain¹⁹ had a considerable effect on the electron donor access. In terms of the catalytic efficiency (k_cat/K_m), the heterodimer (I), (II), (III) and (IV) were 2.84-, 2.76-, 0.52- and 10.88-fold of the dissociative form, respectively (Table 2). The results revealed that the nature-mimicking architecture of heterodimer (IV) strongly boosted the catalytic efficiency of reconstructed CYP73A5-ATR2 heterodimer. It should be noted that the fitting Hill coefficients were all greater than one, indicating that the NADPH-driven p-coumaric acid generation in the reconstructed CYP73A5-ATR2 system was a positive synergistic reaction, regardless of the architecture.

Table 2 Enzymatic assay of SpySystem-mediated CYP73A5^Δ2-28-ATR2^Δ2-77 heterodimers and the free-floating CYP73A5^Δ2-28 and ATR2^Δ2-77 at increasing concentration of NADPH

Full size table

As is evident from Tables 1 and 2, the turnover number and catalytic efficiency of heterodimer (IV) with the N-termini-bridged architecture were 4.45 and 3.83 times, respectively, higher than those observed in heterodimer (I) with the CYP102A1-like architecture. Thus, the in vitro quantitative results clearly illustrated that the nature-mimicking N-termini-bridged architecture of heterodimer (IV) contributed to the reconstructed eukaryotic P450 system the highest turnover number and catalytic efficiency, even though the self-assembled bio-machinery for architecture generation, the peptide-based SpySystem herein, had an influence on substrate access and electron transfer between CYP73A5^Δ2-28 and ATR2^Δ2-77.

Configurational modulation of N-termini-bridged CYP73A5-ATR2 heterodimer

To further explore whether the biosynthetic performance was affected by the architectural configuration of the N-termini-bridged heterodimer, we next swapped the N-terminal peptide appendixes between CYP73A5^Δ2-28 and ATR2^Δ2-77. Hence, an alternative N-termini-bridged heterodimer (V), SpyTagCYP73A5^Δ2-28-SpyCatcherATR2^Δ2-77, was created with a different configuration from the N-termini-bridged heterodimer (IV), SpyCatcherCYP73A5^Δ2-28-SpyTagATR2^Δ2-77, mentioned above (Supplementary Fig. 8 lane 2; Supplementary Fig. 11). Then, the biosynthetic performance of heterodimer (V) was compared to that of heterodimer (IV) in AthPAL1-expressing E. coli. As shown in Fig. 4 panel 1, the titer of p-coumaric acid with heterodimer (V) was diminished to 83.64 mg L⁻¹ at 48 hpi, which was merely 49.24% of that with heterodimer (IV). The biomass-specific productivity of p-coumaric acid for the strain harboring heterodimer (V) was 50.80% of that observed for the strain with heterodimer (IV) (Fig. 4). The results indicated that the architectural configuration of the N-termini-bridged CYP73A5^Δ2-28-ATR2^Δ2-77 heterodimer based on SpySystem exerted a significant effect on the biosynthetic performance of the reconstructed eukaryotic P450 system in E. coli.

**Fig. 4: Effects of the N-terminal peptide appendixes of CYP73A5^Δ2-28 and ATR2^Δ2-77 on the bioproduction of p-coumaric acid in *E. coli*.**

Robust performance of N-termini-bridged CYP73A5-ATR2 heterodimer

To investigate the effect of the peptide scaffold on the biosynthetic performance of the N-termini-bridged heterodimers of CYP73A5^Δ2-28 and ATR2^Δ2-77, an alternative peptide ligation system SnoopSystem, of which SnoopCatcher and SnoopTag are able to form an intermolecular isopeptide bond spontaneously and post-translationally³², was applied. By pairwise appending SnoopCatcher peptide and SnoopTag peptide at both N-termini of CYP73A5^Δ2-28 and ATR2^Δ2-77 to substitute SpySystem scaffold, two N-termini-bridged heterodimers, i.e. heterodimer (VI), SnoopCatherCYP73A5^Δ2-28-SnoopTagATR2^Δ2-77, and heterodimer (VII), SnoopTagCYP73A5^Δ2-28-SnoopCatherATR2^Δ2-77, were further created (Supplementary Fig. 8 lane 3 and 4; Supplementary Fig. 12).

The biosynthetic performance of N-termini-bridged heterodimers (VI) and (VII) was also assessed in AthPAL1-expressing E. coli. The titer of p-coumaric acid with heterodimer (VI) was 146.97 mg L⁻¹ while that with heterodimer (VII) was 169.33 mg L⁻¹ at 48 hpi (Fig. 4 panel 2). The biomass-specific productivity of p-coumaric acid for the strains harboring heterodimer (VI) or (VII) was 465 and 557 μg (g DW h)⁻¹, respectively (Fig. 4). Quantitatively, the highest in vivo output of N-termini-bridged heterodimers based on SnoopSystem was comparable to that of heterodimers based on SpySystem (Fig. 4 panel 1 and 2). These results confirmed that the biosynthetic performance of N-termini-bridged CYP73A5^Δ2-28-ATR2^Δ2-77 heterodimers were robust, regardless of the covalent self-assembled peptide bio-machineries SpySystem or SnoopSystem employed in our experiments. Notably, unlike the N-terminal SpySystem swapping leading to a dramatical gap in the titer of p-coumaric acid (Fig. 4 panel 1), the N-terminal SnoopSystem swapping resulted in a much lower titer margin (Fig. 4 panel 2). The result suggested that SnoopSystem-based N-termini reconstruction of CYP73A5^Δ2-28 and ATR2^Δ2-77 might endow the N-termini-bridged heterodimers with an enhanced structural fitness.

Superior performance of N-termini-bridged CYP73A5-ATR2 heterodimer

Considering that there are protein-protein interactions between P450 and its redox partner in eukaryotic cells^{33,34,35,36,37}, we were wondering that whether altering both N-terminus-appended peptides of CYP73A5^Δ2-28 and ATR2^Δ2-77 affect the biosynthetic performance of the heterologous P450 in E. coli. We started with the free-floating NTA-truncated CYP73A5^Δ2-28 and ATR2^Δ2-77. While both truncated individuals were co-expressed in AthPAL1-expressing E. coli, the titer of p-coumaric acid was 75.16 mg L⁻¹ at 48 hpi (Fig. 4 panel 3 M). Subsequently, we appended both CYP73A5^Δ2-28 and ATR2^Δ2-77 at N-terminus with an octapeptide 8RP, which has been optimized at both codon level and amino acid level for the expression of eukaryotic P450s in E. coli^{38, 39} and is usually applied as an N-terminal modification element in P450-involved bioproduction of chemicals^{12, 14, 15}. As shown in Fig. 4 panel 3, the N-terminal 8RP modification brought the titer of p-coumaric acid up to 90.29 mg L⁻¹, 1.2-fold of that obtained with the NTA-truncated individuals. These results indicated that the biosynthetic performance of the free-floating CYP73A5 and ATR2 with NTA truncation or N-terminal 8RP modification were merely 44.25% and 53.16%, respectively, of the biosynthetic ability of the SpySystem-mediated N-termini-bridged heterodimer (IV) (Fig. 3b).

We further attempted to tether CYP73A5^Δ2-28 and ATR2^Δ2-77 as a noncovalent N-termini-bridged heterodimer by taking advantage of the mouse SH3 domain and its high-affinity peptide ligand (SH3lig) (K_d = 0.1 μM)⁴⁰. By N-terminal appending and swapping, two adhesive N-termini-bridged heterodimers were supposed to form post-translationally and named as heterodimer (VIII), SH3ligCYP73A5^Δ2-28/SH3ATR2^Δ2-77, and heterodimer (IX), SH3CYP73A5^Δ2-28/SH3ligATR2^Δ2-77. When heterodimer (VIII) or heterodimer (IX) was introduced into AthPAL1-expressing E. coli, the titer of p-coumaric acid was 143.82 or 94.83 mg L⁻¹ at 48 hpi, respectively (Fig. 4 panel 4). Thus, the biosynthetic performance of N-termini-bridged heterodimer (VIII) and (IX) with SH3/SH3lig pair were 1.91- and 1.26-fold of the free-floating NTA-truncated CYP73A5 and ATR2, respectively (Fig. 4 panel 3 and 4). The results further confirmed that the N-termini-bridged architecture improved the biosynthetic performance of plant P450 system in E. coli, and also indicated that the architectural configuration of the N-termini-bridged CYP73A5^Δ2-28-ATR2^Δ2-77 heterodimer based on the SH3/SH3lig system effected the biosynthetic performance of the reconstructed eukaryotic P450 systems in E. coli. Whereas compared to the biosynthetic performance of N-termini-bridged heterodimer (IV) based on the covalent SpySystem, those of N-termini-bridged heterodimer (VIII) and (IX) with noncovalent SH3/SH3lig system were 84.67% and 55.83%, respectively (Figs. 3b and 4 panel 4). The results suggested that covalent tethering could improve the in vivo biosynthetic performance of N-termini-bridged P450-CPR heterodimer in E. coli.

And then, we tested the role of the covalent isopeptide bond of the SpySystem in improving the biosynthetic performance of N-termini-bridged heterodimer (IV). To abrogate the isopeptide bond, the participant amino acids in SpyCatcher (E80Q) and/or SpyTag (D7A)³⁰ were substituted via site-directed mutation, resulting in the absence of the covalent heterodimer (IV) (Supplementary Fig. 8 lane 9). As shown in Fig. 4 panel 5, the titer of p-coumaric acid dropped to 65.71 mg L⁻¹ (heterodimer X with SpyCatcher^E80Q), 114.38 mg L⁻¹ (heterodimer XI with SpyTag^D7A) and 101.99 mg L⁻¹ (heterodimer XII with both SpyCatcher^E80Q and SpyTag^D7A), which were 38.69%, 67.34% and 60.05% of heterodimer (IV), respectively. Among these heterodimers with N-terminus-appended deficient SpySystem, the heterodimer with SpyCatcher and mutated SpyTag^D7A showed the highest titer and biomass-specific productivity (Fig. 4), probably due to the high affinity (K_d = 0.2 μM) between SpyCatcher and SpyTag^D7A³⁰.

These results further confirmed that N-terminal tethering of plant P450 and the redox partner was beneficial to improve the biosynthetic performance of the eukaryotic P450 enzyme in E. coli, and also indicated that the reconstructed plant P450 system with N-terminal covalent joint, such as the heterodimers (IV) and (VII), possessed superior performance, suggesting that the mechanical stability of the self-assembled peptide bio-machinery, employed for spatially organizing a multienzyme cascade bioreactor, could have a considerable effect on the efficiency of bioproduction.

N-termini-bridged protein self-assembly adaptable to improve the biosynthetic performance of human P450 system in E. coli

Furthermore, we examined the effect of architectural organization on the biosynthetic performance of animal P450 system in E. coli. Human CYP1A2, one of the major oxidative drug-metabolizing enzymes in liver^41,42,43, was chosen for the drug metabolite bioproduction. According to the spatial organization of the plant CYP73A5^Δ2-28 and ATR2^Δ2-77 described above, the NTA-truncated CYP1A2^Δ2-37 and HsCPR^Δ2-52 were architecturally organized with the self-assembled peptide bio-machinery SpySystem (Supplementary Fig. 13a−d). Meanwhile, CYP1A2^Δ2-37 was also expressed in a tandem pattern at N-terminus of HsCPR^Δ2-52 (Supplementary Fig. 13e), or co-expressed with HsCPR^Δ2-52 individually (Supplementary Fig. 13f).

To assess the biosynthetic performance of CYP1A2 variants, the bioproduction of the analgesic drug acetaminophen from phenacetin was carried out via whole-cell biotransformation (Fig. 5a). The product of CYP1A2 variant was confirmed to be authentic acetaminophen, whereas E. coli strain with empty vector was of inability to biosynthesize acetaminophen from phenacetin. As shown in Fig. 5b, the strain with the CYP102A1-like tandem chimera, CYP1A2^Δ2-37-L-HsCPR^Δ2-52, produced 3.47 mg L⁻¹ acetaminophen, while the strain with the free-floating individuals, CYP1A2^Δ2-37 and HsCPR^Δ2-52, produced 4.84 mg L⁻¹ acetaminophen. Regarding the SpySystem-organized heterodimers, the strain with the CYP102A1-like heterodimer, CYP1A2^Δ2-37SpyCatcher-SpyTagHsCPR^Δ2-52, produced 3.73 mg L⁻¹ acetaminophen, which was comparable to the output of the strain with the CYP102A1-like chimera. Obviously, the strain harboring the N-termini-bridged heterodimer, SpyCatcherCYP1A2^Δ2-37-SpyTagHsCPR^Δ2-52, produced the most acetaminophen, which was 21.61 mg L⁻¹. The results indicated that the SpySystem-based counterpart of human CYP1A2 and CPR system showed 6.23-fold higher biosynthetic performance than the CYP102A1-like chimera in E. coli, and also confirmed that the N-termini-bridged architecture favored human P450 system for bioproduction in space. Therefore, these results indicated that the N-termini-bridged protein self-assembly strategy was feasible and adaptable to organize human P450 system to improve the bioproduction of drug metabolites in E. coli.

**Fig. 5: Spatial organization of human P450 system for acetaminophen bioproduction in *E. coli*.**

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Source: https://www.nature.com/articles/s41467-024-54259-1

Tuning architectural organization of eukaryotic P450 system to boost bioproduction in Escherichia coli

The biosynthetic performance of plant CYP73A5 affected by the tandem architecture

Architectural alternatives designed for plant CYP73A5 and ATR2 in E. coli

Enzyme kinetic assays of SpySystem-mediated CYP73A5-ATR2 heterodimers

Configurational modulation of N-termini-bridged CYP73A5-ATR2 heterodimer

Robust performance of N-termini-bridged CYP73A5-ATR2 heterodimer

Superior performance of N-termini-bridged CYP73A5-ATR2 heterodimer

N-termini-bridged protein self-assembly adaptable to improve the biosynthetic performance of human P450 system in E. coli

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